diff --git "a/deduped/dedup_0950.jsonl" "b/deduped/dedup_0950.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0950.jsonl" @@ -0,0 +1,56 @@ +{"text": "Yellowfin and skipjack tuna are globally distributed in the world's tropical and sub-tropical oceans. Since little, if any, migration of these fishes occurs between the Atlantic and Indo-Pacific Oceans, one might expect to see genetic differences between sub-populations in these ocean basins. However, yellowfin and skipjack tuna have extremely large population sizes. Thus, the rate of genetic drift should be slower than that observed for other tunas.Low levels of genetic differentiation were observed between Atlantic and Pacific samples of yellowfin tuna. In contrast, no genetic differentiation was observed between Atlantic and Pacific samples of skipjack tuna.Much lower levels of genetic differentiation were found among sub-populations of yellowfin tuna compared to those observed for other large tunas, probably due to the large population size of yellowfin tuna. Since skipjack tuna appear to have even larger population sizes, it is not surprising that no genetic differentiation was detected between Atlantic and Pacific samples of these fish. Thunnus albacares, has a global distribution in tropical and sub-tropical oceans. Annual catches of yellowfin tuna have averaged 1.2 million metric tons since 1998 with sizes ranging from 5 to 20 kg [The yellowfin tuna, Thunnus albacares) from the Atlantic (n = 20) and Pacific Oceans (n = 100) using an RFLP analysis of whole mitochondrial DNA (mtDNA). Subsequently, Ward et al. [In an earlier study, Scoles and Graves were unad et al. found sid et al. ,5.Katsuwonus pelamis) are found in tropical and warm temperate waters of the world's oceans. They are present in the three major oceans in large numbers and comprise approximately 40% of the annual catch of the world's tunas. Annual catches are on the order of 2 million metric tons or approximately 670 million individuals per year [Thunnus. Tagging studies have demonstrated limited seasonal movements, but not much transoceanic movement. Thus, they probably do not spawn at discrete locations [Skipjack tuna were estimated for both species, which may explain the observed levels of inter-oceanic genetic partitioning.To measure the degree of genetic differentiation between the Atlantic and Pacific sub-populations of either yellowfin or skipjack tuna, we examined the hypervariable control region I (CR-I) and a segment of a coding region gene of the respective mitochondrial DNAs. Genetic differences were observed between the Atlantic and Pacific yellowfin tuna samples with PCR-RFLP data of the ATCO gene region, but not with CR-I sequence data. In contrast, no differences between the Atlantic and Pacific skipjack tuna samples were detected with either type of data. Information contained in the CR-I reveals very different demographic histories for yellowfin tuna and skipjack tuna. However, very large long-term female effective population sizes (Nh = 0.997) and a nucleotide diversity (\u03c0) of 3.5%. Diversity indices for each locality sampled were also high, although values of \u03c0 and h were slightly lower for the NW Atlantic yellowfin sample where five haplotypes were repeated twice. For skipjack tuna, a total of 394 bp was determined for 115 individuals (Table h = 0.999). Nucleotide diversity (\u03c0 = 8.4%) was more than twice as high in skipjack tuna as in yellowfin tuna. All of the sampling localities of skipjack tuna had very high diversity values . For both species, the high haplotypic diversity values are consistent with the observed large census population sizes (Nc). Phylogenetic analyses resulted in very different trees for each species. The CR-I gene-tree topology of skipjack tuna is much larger and better structured than the yellowfin tuna phylogeny, and contains multiple branches with high bootstrap proportion support control region was determined for 148 yellowfin tuna gene by Terol et al. [cytB gene for each DNA in the Atlantic and Pacific skipjack tuna samples. Little additional variation was observed, and allele frequencies were similar in the two samples whereas the effective number of yellowfin tuna females prior to expansion was small (Ne0 = 823). However, the values of \u03b81 suggest large long-term numbers of effective female breeders (Ne1) of about 98 million for skipjack tuna and about 10 million for yellowfin tuna.The unimodal mismatch distribution for the pooled sample of yellowfin tuna Figure and a sist Table both sugst Table and the st Table , suggestCompared to other scombroid species with cosmopolitan distributions, we found substantially less genetic differentiation between Atlantic and Pacific sub-populations of yellowfin tuna and no inter-oceanic genetic differentiation of skipjack tuna. For instance, in bigeye tuna, there are two highly divergent mtDNA clades, one is cosmopolitan and the other is endemic to the Atlantic ,13. ThisAn alternative explanation to account for the lack of inter-oceanic differentiation in skipjack tuna and yellowfin tuna, is that the time since population expansion began has not been sufficient to allow for the populations to become differentiated. Assuming the very conservative mutation rate for CR-I of 4.9% per million years, a generation time 3.5 years, and the tau value 8.52, expansion of yellowfin tuna occurred about 522 Ky ago. By comparison, the estimated time for genetic differentiation of the Atlantic and Pacific populations of swordfish and of the Atlantic and Pacific bluefin tunas, using the same mutation rate, is very similar (450\u2013470 Ky), and an even shorter time (170 Ky) is sufficient to explain the substantial genetic differentiation between the Atlantic and Mediterranean swordfish populations . Thus, te estimates are 900,000 for bluefin tuna and 800,000 for swordfish [ef values. The observed correspondence does not take into consideration dramatic changes in abundance of some of these species over the last 20 years, nor sex ratio differences, or the age distribution of the catch . However, the comparison supports the hypothesis that skipjack tuna and yellowfin tuna, which are the two most abundant species, also have the largest effective population sizes, and the lowest amounts of genetic partitioning compared to other scombroid fishes.Among large pelagic species, the female effective population size can be estimated from the demographic estimates obtained from the CR-I sequences of Atlantic bluefin tuna and swordfish. Assuming a mutation rate of 4.9% per million years and a generation time of 6 years, the female Nwordfish . Effectiwordfish ,31, suggBigeye tuna, and bluefin tuna have much greater levels of genetic differentiation between Atlantic and Pacific subpopulations when mitochondrial DNA control region sequences are compared ,31. HoweOne exception to the patterns described above is albacore tuna. The distribution of albacore tuna mitochondrial DNAs does not appear to fit the patterns described above for the other temperate tuna species. Albacore abundance is similar to that of bigeye tuna and both species have two mtDNA clades. However, in albacore the two clades are not as well differentiated nor do they display the phylogeographic association observed for the bigeye tuna clades . InsteadMuch lower levels of genetic differentiation were found among sub-populations of yellowfin tuna compared to those observed for other large tunas, probably due to the large population size of yellowfin tuna. Since skipjack tuna appear to have even larger population sizes than yellowfin tuna, it is not surprising that no genetic differentiation was observed between Atlantic and Pacific samples of these fish.Samples of yellowfin tuna were obtained from the eastern Pacific Ocean , the Indian Ocean (n = 63), the Gulf of Mexico and the East Coast of Florida n = 38) and the Gulf of Guinea (n = 100). Samples of skipjack tuna were obtained from the Northwest Atlantic (n = 31), off the coast of Brazil (n = 19), the eastern Pacific Ocean (n = 43), and the south Pacific near the Solomon Islands n = 37). DNA isolation, mitochondrial DNA D-loop region amplification, and nucleotide sequence analyses have been described previously ,32. The . DNA iso and the 0 and \u03b81 and their probabilities [The demographic history contained in the mtDNA CR-I sequence data was inferred using two approaches. First, the null hypothesis of neutrality may be rejected when a population has experienced population expansion . Accordibilities were estbilities .DdeI or HpyCH4III and the digestion products were resolved by electrophoresis in a 1.2% agarose gel.For the analysis of the mitochondrial cytochrome b gene, a 650 bp fragment of the skipjack tuna cytochrome b gene was amplified using primers CB3 (GGCAAATAGGAARTATCATTC) and GLUDG (TGACTTGAARAACCAYCGTTG) . The ampBE, JV, and JAB conceived the study, supervised the genetic studies, analyzed the data and wrote the manuscript. JAB, AL, and ET performed the DNA sequence analyses. BE, DB, ET, LL, and KC performed the DNA amplifications and RFLP analyses."} +{"text": "LTR retrotransposons are one of the main causes for plant genome size and structure evolution, along with polyploidy. The characterization of their amplification and subsequent elimination of the genomes is therefore a major goal in plant evolutionary genomics. To address the extent and timing of these forces, we performed a detailed analysis of 41 LTR retrotransposon families in rice.Using a new method to estimate the insertion date of both truncated and complete copies, we estimated these two forces more accurately than previous studies based on other methods. We show that LTR retrotransposons have undergone bursts of amplification within the past 5 My. These bursts vary both in date and copy number among families, revealing that each family has a particular amplification history. The number of solo LTR varies among families and seems to correlate with LTR size, suggesting that solo LTR formation is a family-dependent process. The deletion rate estimate leads to the prediction that the half-life of LTR retrotransposon sequences evolving neutrally is about 19 My in rice, suggesting that other processes than the formation of small deletions are prevalent in rice DNA removal.Our work provides insights into the dynamics of LTR retrotransposons in the rice genome. We show that transposable element families have distinct amplification patterns, and that the turn-over of LTR retrotransposons sequences is rapid in the rice genome. Transposable elements (TEs) make up a large part of eukaryotic genomes. They represent a genomic fraction of 3% in baker's yeast , ~20% inIn plants, Long Terminal Repeat (LTR) retrotransposons are the most common type of TE: they are ubiquitous in the plant kingdom and are Ty1/copia-like and Ty3/gypsy-like elements.LTR retrotransposons are class I TEs and thus replicate using a RNA intermediate, through a \"copy-and-paste\" mechanism. They are related to retroviruses with which they share their structure: the complete copies consist of two LTRs that flank an internal region. LTR sequences contain the signals for transcription initiation and termination, while the internal region encodes the proteins that are necessary for the retrotransposition cycle. LTR retrotransposons are classified into two major families: the i.e. within the past 15 million years) [Plant LTR retrotransposons vary in size from 2 to 18 kb and harbor LTRs that vary in size from a few hundreds bases to several kilobases ,24. LTRsn years) ,25-34. LArabidopsis thaliana [Oryza sativa L.) [The distribution and structure of LTR retrotransposons have been studied in several species, in particular in the two model plants for which a nearly complete genomic sequence is available: thaliana -27,30,31tiva L.) ,32,33. Itiva L.) ,32 or smgypsy-like and 25 copia-like families, Table We extracted the paralogous copies of 41 LTR retrotransposons families of the current rice genome. If all the copies mined correspond to real paralogous copies of the families and if their size upon insertion was similar to that of their corresponding reference copy, they represented 136.8 Mb upon insertion, suggesting that 106.4 Mb (77.8%) of the DNA conferred by their insertion has been removed.The analysis of the mined copies revealed that some reference copies harbor insertions, most of them smaller than 200 bp, and a few larger than 200 bp. It also revealed that one family shares LTR with a LArge Retrotransposon Derivative (LARD). To correct for the possible detection of false paralogous copies, we therefore applied a filter discarding any fragment smaller than 200 bp, and did not take into account families with larger inserts or other problematic features . With this new filter, we estimate that 5154 copies from 19 families, which represented 52.3 Mb upon insertion, remain today in the rice genome as 20.2 Mb, leading to a loss of 32.1 Mb (61.4%).We believe that the first data are an over-estimate and that the latter may be an under-estimate of the total percentage of DNA eliminated from the rice genome. Hence, we estimate that the percentage of DNA that has been eliminated from the rice genome since the insertion of the detected copies is comprised between 61% and 78%.To get an overview of the deletion process for each family, we computed the distribution of the copy size relative to the reference copy size Table . This anTo provide a global overview of the LTR retrotransposon sequence turn-over process in the rice genome, we first wanted to estimate the insertion dates of the largest set of copies possible, for each family.i.e., truncated copies of LTR retrotransposons or copies of other types of TEs such as LINEs or DNA transposons), the method currently used is based on pairwise nucleotide identity between elements that are closely related at the phylogenetic level or between genomic copies and a consensus of the element [For copies with 2 LTRs, the best method to estimate insertion date is based on the divergence between the two LTRs of each copy . For cop element -38. Howegypsy-like LTR retrotransposons [To overcome these caveats, we designed a new method , for which an insertion date could be estimated for more than 15 copies. The histograms of these data, presented in Figure Hopi/Osr27, Osr17, Houba/Osr13 and Osr7 have undergone very recent amplification , whereas that of other families, such as Osr15, Osr29, Osr30, Osr8, RIRE1 is more ancient. Some families, such as Dagul or Houba/Osr13, seem to have undergone several bursts of amplification, since several peaks are noticeable. The dates of these peaks vary between families from around 0.02 and 0.05 for Dagul to 0.01 and 0.04\u20130.06 for Houba/Osr13. Some of the older peaks, such as the second peak for Dagul, could only be observed through the addition of the dated truncated copies, illustrating that estimating the insertion date of truncated copies is important to describe the amplification process LTR retrotransposons in rice as accurately as possible.By applying this method on the complete dataset of all genomic copies detected (after filtering by length), we could analyze the amplification timing of 10 LTR retrotransposon families cloning technique between rice and foxtail millet [Hopi/Osr27 was large and rapid, and took place in a very recent past [In terms of magnitude, some families, such as l millet , and werl millet ,39. Thisl millet . In partent past or may s-9 substitutions per site per year versus 1.3 \u00d7 10-8 in our case). After adjusting their results with the substitution rate we used, our results on a large sample are in agreement with a half-life of 3 My. In both studies large deletions could not be taken into account in the elimination process. Since such deletions could induce the elimination of one or more complete elements, we believe that the elimination of LTR retrotransposons sequences in the rice genome is even more efficient than a 3 My half-life.As shown above, we estimate the amount of LTR retrotransposon DNA removed from the rice genome around 61\u201378%. Considering that most of this removal occurred within the last 5 My (most of the copies have inserted in this time lap), we estimate the half-life of a LTR retrotransposon sequence to be comprised between 3 My and 4 My. In a recent paper, Ma and collaborators estimateOsr2 and Osr12 , the copies harboring two LTRs represent less than 50% of the total number, with a median at 18.9%.To complete our analysis of the elimination process, we classified the copies according to their truncation level, into copies that harbored two LTRs, one LTR and the internal region, only one LTR, and only the internal region . Results of this analysis, shown in Table Hopi/Osr27, which has amplified mainly within the last 0.4 My (LTR divergence < 0.01) shows 40% of complete copies, whereas for Osr8, which has amplified around 1.5\u20132 My ago (LTR divergence mainly within the range 0.04\u20130.05), these copies represent only 18.7%. These results are in agreement with previous studies, in which the same correlation was found [Using these data along with the timing data obtained for the families Figure , we founas found . This feDagul, Hopi/Osr27, Osr17 and Houba/Osr13), revealing that the elimination of LTR retrotransposon sequences is very efficient in the rice genome.In addition, the proportion of the different types of truncated copies over time scale increments of 0.01 (~0.4 My), presented in Figure The global analysis of our data showed that the elimination of rice LTR retrotransposon sequences is efficient in rice . But what are the mechanisms responsible for the elimination of the copies? As shown in previous studies ,29,33, tDagul, Osr29 or Osr30 display a large number of \"LTRs\" compared to the other types of truncated sequences, whereas Houba/Osr13 or Osr17 do not show as many , and a larger LTR (1103 vs. 466 bp) than Osr17. Its LTR frequency is higher (27% vs. 8%). Osr8 and Osr29 have similar internal region size, but Osr8 has bigger LTRs (1220 vs. 656 bp) and its LTR frequency is higher (58% vs. 30%). Therefore, LTR size seems to have an impact on solo LTR formation, but not the size of the internal region, suggesting that formation of solo LTRs is a family-dependent process. More data are however needed to confirm this pattern.Our analysis of the \"LTR\" fragments per 0.01 time scale increment Figure revealedThe accumulation of small deletions has been proposed to occur through illegitimate recombination, as shown by the observation of small patches of micro-homology flanking the deleted sequences ,33,40. TD. melanogaster [D. melanogaster, both mean deletion size (60.7 bp) and deletion rate (0.114) are greater, resulting in a greater DNA loss estimate of 6.9 bp per substitution. These results suggest that the removal of transposable element sequences through neutral accumulation of small deletions is more efficient in D. melanogaster than in rice, probably partially accounting for the genome size difference between these two species.Figure nogaster . In D. mThis half-life of 19 My is very different from the one we calculated based on the global size of the copies because it does not take into account selection against TE insertions and has to be considered as a quasi-neutral deletion rate estimate. Homologous recombination and selection will accelerate the elimination of TEs and thus increase the global DNA loss observed for these sequences. Moreover, with this method, we detect only the accumulation of small deletions , and do not take into account larger rearrangements . In particular, because it is based on the analysis of the sequence pairs in the terminal tree forks, it takes into account only the newer deletions. If small deletions appear faster than large rearrangements in the rice genome, some large rearrangements may not have been taken into account by this method.These results reveal that, even though the neutral accumulation of small deletions plays a role in LTR retrotransposons DNA removal in rice, this force is not the predominant force shaping genome size.gypsy-like and 25 copia-like families. As revealed by our study, these families are low, \u2013 middle- and high-copy number families (Table i.e. 93.6% of the entire genome) allows a particularly extensive characterization of the evolutionary dynamics of LTR retrotransposons in rice. To our knowledge, this is the first time that such an extensive study is made.Any comprehensive analysis of the structure and evolution of LTR retrotransposons in a given species requires a large LTR retrotransposon database and a large genomic sequence as starting point. For our search to be as representative as possible of the whole genome, we analyzed 41 LTR retrotransposons families, including 16 The second key point is the quality of the detection. In particular, estimating how fast copies are eliminated from a genome requires to accurately mine complete copies (which can be fragmented by other insertions), as well as small fragments that have been highly truncated. The use of Blaster, a program suite based on the BLAST program and imprBecause of these features, we believe that our method detects accurately both complete copies and truncated fragments. Since an accurate detection and estimate of the copy size is a critical point in any study on LTR retrotransposons evolutionary dynamics, we analyzed the distribution of copy size for each family.Hopi/Osr27, Osr2, Osr8, Osr20, Osr34 and Osr44), the slight difference in size between the longest copy and the reference copy (99.3 to 99.9% of the reference copy size) is due to the homogenization of the two LTRs in the reference copy . For four other families , however, the longest copy detected is more than 5% shorter than the reference copy, which suggests that this copy was not detected by our search. Osr22, Osr31 and Osr38 were originally characterized by searching part of the rice genome sequence [RIRE1, however, the reference copy could not be detected because this sequence is not derived from a genomic copy, but is a chimeric sequence that originated from the cloning of several genomic copies [First, we expected that the longest copy detected and the reference copy would have the same size because insertions in genomic copies (as compared to the reference copy) were not taken into account in the computation of the copy size. Results of the Blaster output, presented in Table sequence . Since tin silico cross-hybridization between two different families). To check for these possibilities, we looked at the position of the mined copies on the reference copy, using the alignment that we had generated. This analysis revealed that some reference copies harbor insertions, most of them smaller than 200 bp, and a few larger than 200 bp. It also revealed that one family shares LTR with a LARD element.Second, we expected that some short copies could be false paralogous copies of our targeted families. Mainly, we suspected that some of them could correspond to (i) uncharacterized repeated sequences inserted in the reference copy, and (ii) fragments corresponding to coding regions of uncharacterized elements sharing a high sequence identity with the element analyzed . This conservative strategy had mainly two draw-backs: first, some real paralogous copies with a size below 200 bp would be missed due to the filter, leading to an under-estimate of copy number and total fraction of the genome occupied by these elements. However, since the raw data most probably over-estimate these numbers, we believe that the comparison of the two outputs (with and without filter) give a good estimate of these parameters. Second, it decreased the number of families that could be used to perform a detailed analysis of the deletion process. However, by starting with a large number of 41 families, we could estimate the deletion process for 19 families, a large enough number to get an estimate that is representative of the genome.Finally, the use of a new method to date truncated copies allowed us to get a more detailed appreciation of the integration and elimination processes than with complete copies alone, and, therefore, a more accurate estimate of temporal parameters of the deletion processes. We showed in earlier studies (29) that three rice gypsy-like LTR retrotransposons amplify through burst of amplification. Here, the use of complete copies harboring 2 LTRs revealed recent amplification bursts for several other families, suggesting that this is a general feature of rice LTR retrotransposons. Therefore, we developed a method to date LTR retrotransposon fragments based on sequence identity between truncated fragments and a complete copy that comes for the same amplification burst. This method requires the conservation of at least one complete copy per burst. Therefore, we did not expect to estimate the insertion date of all truncated copies, particularly for ancient ones, because copies of ancient burst were expected to be all truncated. It is thus possible that we missed some ancient bursts. However, this should not have any impact on the detection of the more recent bursts that we detect.Because old copies are more likely to be deleted than new ones, this technique revealed ancient amplification events, thus improving our characterization of the amplification process. Moreover, the estimation of the insertion date for all types of copies allowed us to analyze the number of truncated copies per time period, leading to a more accurate characterization of the deletion process. However, due to the stringent parameters that we used to ensure a robust association between a truncated copy and a dated copy harboring two LTRs , the insertion date could not be estimated for all truncated copies Table .Finally, our method allows an extensive characterization of LTR retrotransposon copies in today's rice genome, as well as their temporal dynamics of insertion and deletion, leading to a comprehensive analysis of the processes involved in their evolutionary dynamics in the rice genome.Our results are based on the assumption that the copies extracted are derived from the insertion of complete copies. This assumes that the mechanism of retrotransposition do not lead to truncated copies upon integration. This mechanism has been well studied in retroviruses, and it is commonly accepted that it also applies to LTR retrotransposons. To our knowledge, insertion of truncated copies has not been described, meaning that if this is possible, it would occur rarely. Consequently, we believe that the frequency of such events is low, and should not have a large impact on our results.Hopi/Osr27 and Osr8 the presence of truncated copies sharing a deleted region compared to the reference copy. These two subsets of copies also share similar insertion times, suggesting that they correspond to the amplification of truncated copies. Since this was observed for 2 elements only, we believe that this process does not affect tremendously our conclusions on the overall deletion process. Moreover, since the formation of small deletions is a cellular process and should therefore affect similarly all types of elements, the conclusions drawn on the deletion rate are not affected by this observation.During our analysis, we revealed for O. sativa L.). We show that most of the copies were inserted within the last 2 My, and that the amplification process varies both in timing and extent between families. The copies are subsequently efficiently eliminated from the genome, through both solo LTR formation and accumulation of deletions. We estimate the half-life of LTR retrotransposon sequences in the rice genome to be less than 3 My. However, if only the neutral accumulation of small deletions is taken into account, this half-life would be close to 19 My, revealing that this process is not a major force of LTR retrotransposon removal in the rice genome. Rather, negative selection of these sequences or larger rearrangements may be involved. Altogether, these results reveal a high turn-over of LTR retrotransposon sequences in the rice genome and therefore provide an explanation for the rapid differentiation of intergenic regions in grass genomes. To our knowledge, our study is the first to analyze rice LTR retrotransposons in such an extensive way.Through the analysis of 41 rice LTR retrotransposon families, and the use of a new method to estimate the insertion date of both truncated and complete copies, we could precisely describe the amplification and elimination of LTR retrotransposon sequences in the rice genome and Osr11, between Osr27 and RIRE9 (from Genbank AB033547) or between Tos17 and Osr21, as stated by other authors (28). Since this paper did not clearly states how the \"pre-existing names\" were associated with the \"Osr\" elements, we decided to follow our annotation, not the one published (28), and kept the Osr nomenclature for the elements that we did not annotate. We then carefully analyzed the boundaries of each retrotransposon, and corrected then when needed. For each LTR retrotransposon reference copy where LTRs were detected, the two LTRs were homogenized by replacing the 3' LTR by the sequence of the 5' LTR, to avoid differences in match between the two LTRs. A fasta file of the sequences of the 41 LTR retrotransposon reference copies contained in this database is available in additional file We started by compiling a dataset containing all the rice LTR retrotransposon sequences available from the literature ,44,45 anOsr25/Dasheng, Spip and Squiq) and their autonomous counterparts sequences and partial LTR retrotransposon sequences that were available in the public databanks ,47. SimiE2/Osr26 , RIRE3 aectively ) containOryza sativa cv. Nipponbare) pseudomolecules as query and our transposable elements dataset as subject. We then filtered the resulting High Scoring Pairs (HSPs), using a 1 \u00d7 10-10 E-value threshold. Finally, the selected HSPs were connected by means of a dynamic programming algorithm based on HSP scores and the physical distance that separated them or because our analysis did not confirm the LTR positions described in the literature . In Table The precise localization of the LTR was a key step in our study, both for the classification of the copies based on their truncation level and the estimation of their insertion date. Therefore, we carefully checked the position of the LTRs of the 41 non redundant reference copies. This was performed as follows: we first extracted the data concerning LTR position in the literature. We then annotated the LTR position by (1) aligning each reference sequence with itself, using a \"BLASTN 2 sequences\" analysis and (2) Osr4, presence of a LARD element related to Osr14, as well as insertions in some reference copies. Two types of inserts were found: inserts with a size over 200 bp in reference copies Osr1, Osr5, Osr22, Osr34, Osr37, Osr39, Osr40 and smaller inserts in many other copies.Because the reference copies are derived from genomic copies, we expected some of them to contain uncharacterized insertions, such as unknown MITEs or pieces of unknown transposable elements. For this reason, certain short copies retrieved by our Blaster search could correspond to paralogous copies of these TEs, and not to the LTR retrotransposons of interest. To check for such possible insertions in the reference copies, we aligned all the mined genomic copies on their corresponding reference copies, and looked for subsets of copies that were a complete match with the same region of the reference copy. This allowed us to spot sub-regions of the reference copies that were highly repeated thus revealing inaccurate boundaries for Osr19, Osr44, Tos17, Osr20, Osr24, Osr43) were also discarded, because the filter could lead to an under-estimation of LTR copy number. Finally, we thus conducted a precise analysis of the deletion process for 19 families (10 of copia type and 9 of gypsy type).To discard these false-positive genomic copies that would lead to an over-estimate of the deletion rate, we filtered out every fragment with a size below 200 bp, and did not further analyze the 9 families for which insertions larger than 200 bp, or a related LARD element, were found. Families which LTR size is shorter or close to 200 bp showing the coordinate correspondence between each genomic copy and the reference sequence. Therefore, if the position of the LTR is annotated in the reference copy, the position of the LTR (if it exists) can be determined for each genomic fragment. Using the LTR position of our reference sequences, and the alignments generated for each genomic copy by Blaster, we thus classified the genomic copies as \"2 LTRs\", \"LTR-IR\", \"LTR\", and \"IR\" . Note that the presence of a LTR or an internal region does not necessarily mean that it is complete, since it can harbor internal deletions.To estimate the global genomic part corresponding to the 41 families, we extracted the length of each copy as given by the Blaster search. For subsequent analysis, we needed to estimate the copy length more accurately and thus did not want to take into account small stretches of mismatch that could have been excluded in the Blaster length computation. For our detailed analysis of the deletion process, we therefore computed the copy length by extracting the sequence of each copy, the sequence from Blaster start position to Blaster end position, and then aligning each copy with its corresponding reference copy, using the gap global alignment program , with th-8 substitutions per site per year, as proposed by Ma and Bennetzen [adh genes [i.e., the age) of the oldest and youngest of these matching complete copies did not differ of more than 1%, to ascertain that these complete copies originated from the same amplification burst. This procedure allowed us to purge any fragments that originated from recombination events involving two copies with different insertion times. In this case, the age taken into account was the one of the youngest copy.We estimated the insertion date of the complete copies using the method proposed by SanMiguel and co-workers , which iennetzen after cadh genes to rice The insertion date of the truncated copies was computed as follows: the nucleotide divergence between the truncated copy and the best matching complete copy was computed, and compared to the nucleotide divergence existing between the two LTRs of the complete copy. If these two copies originated from the same amplification burst, these two numbers should be similar Figure . They weet al. [2 approximation of log-likelihood ratio. A modified version of this estimator, that corrects for the sizes of the deletions and the sizes of the sequences in which they occurred, has subsequently been proposed [To estimate the deletion rate, we used the maximum likelihood approach proposed by Petrov et al. ,42. Thisproposed . To compMultiple alignments for each family were obtained by first computing the pairwise global alignment with the reference sequence using the gap program , and theFrom these phylogenies, we extracted only the terminal forks and not all the terminal branches as in ,42. ThisL = L0exp(-rt), where L is the length of the sequence at time t, L0 is the length at time 0, and r the product of the average size of a deletion by the rate of deletions per substitution or per year.The a half-life of a sequence was calculated using a continuous decay formula, as in : L = L0eHSP: High Scoring PairIR: Internal RegionTE: Transposable elementLARD: LArge Retrotransposon DerivativeLTR: Long Terminal RepeatMITE: Miniature Inverted Transposable ElementRDA: Representational Difference AnalysisCV participated in the design of the study, carried out the mining of the genome and the characterization of the LTR retrotransposon copies , analyzed the corresponding data, and drafted the manuscript. OP participated in the design of the study, in the analysis of the data and helped to draft the manuscript. HQ participated in the design of the study, performed the phylogenetics and statistical analyses for the deletion rate estimate, analyzed the data and helped to draft the manuscript.Sequences of the 41 LTR retrotransposon reference copies. Sequences can be uploaded in fasta format from the \"Vitte_et_al_reference_copies.txt\" text file. Note that reference copies are derived from genomic copies. Their LTR sequences have been homogenized by replacing the two LTR sequences by the sequence of the 5' LTR, to avoid detection problems, except for Osr9, Osr10, Osr11, Osr16, Osr18, Osr23, and Osr31 for which the position of the LTR was ambiguous (see text and Table Click here for file"} +{"text": "In 2003, it became legal in the UK for syringe exchange programmes (SEPs) to provide citrate to injecting drug users to solubilise heroin. Little work has been undertaken on the effect of policy change on SEP function. Here, we examine whether the introduction of citrate in Cheshire and Merseyside SEPs has altered the number of heroin/crack injectors accessing SEPs, the frequency at which heroin/crack injectors visited SEPs and the number of syringes dispensed.Eleven SEPs in Cheshire and Merseyside commenced citrate provision in 2003. SEP-specific data for the six months before and six months after citrate was introduced were extracted from routine monitoring systems relating to heroin and crack injectors. Analyses compared all individuals attending pre and post citrate and matched analyses only those individuals attending in both periods . Non-parametric tests were used throughout.Neither new (first seen in either six months period) nor established clients visited SEPs more frequently post citrate. New clients collected significantly less syringes per visit post citrate, than pre citrate . Matched pair analysis showed that the median number of visits for 'longitudinal attenders' (i.e. those who attended in both pre and post citrate periods) increased from four pre citrate to five post citrate but the number of syringes collected remained unchanged. These changes were not due to seasonal variation or other changes in service configuration.The introduction of citrate did not negatively affect SEP attendance. 'Longitudinal attenders' visited SEPs more frequently post citrate, providing staff with greater opportunity for intervention and referral. As the number of syringes they collected each visit remained unchanged the total number of clean syringes made available to this group of injectors increased very slightly between the pre and post citrate periods. However, new clients collected significantly less syringes post citrate than pre citrate, possibly due to staff concerns regarding the amount of citrate (and thus syringes) to dispense safely to new clients. These concerns should not be allowed to negatively impact on the number of syringes dispensed. Such policies were driven by a public health perspective which regarded the prevention of the spread of HIV infection to be more important than preventing any potential drug users from injecting [The majority of drug users who use SEPs in England to obtain clean injecting equipment are injectors of heroin and the majority of these will inject 'brown' heroin . Brown hNo acidifier can be considered safe, but citrate is believed to be the safest to use for the preparation of brown heroin . Whilst Cheshire and Merseyside has a population of 2,345,077 (4.7% of England) and its drug users are served by 15 SEPs based within drug services . Of these, 11 were identified as introducing citrate between May 2003 and October 2004 and consequently were included in the study.Details of the SEP monitoring system, established in Cheshire and Merseyside in 1991, are reported elsewhere . This weEach of the 11 participating SEPs were contacted to identify the exact date that citrate provision commenced. Monitoring data for the six months pre citrate and post citrate were extracted individually for each of the 11 SEPs. Within each six months (i.e. pre citrate and post citrate separately), attributor codes were used to aggregate an individual's transactions into a single client profile for people who had attended SEPs to collect clean syringes. Again, within each six-month period, individuals were identified as either a 'new' client (no previous contact with the SEP) or an 'established' client (previous contact with the SEP between 1991 and the six months in question). Final person-specific pre and/or post citrate profiles for each SEP included age at most recent SEP contact, gender, the clients main injected drug, their number of visits and the median number of syringes collected per visit.Individuals whose main injected drug was heroin or crack cocaine and who were recorded as having collected clean syringes on at least one occasion during either six month period were included in the analyses. Final SEP-specific profiles for pre and post citrate periods comprised the number of individuals, median number of visits per client and median syringes collected per visit for both new and established clients. In addition to comparing pre and post citrate profiles, individuals who were identified in both pre and post citrate periods were included in matched pair analyses, where an individual's pre citrate profile was compared with their own profile for the post citrate period. For the purpose of this study, individuals included in matched analyses are termed 'longitudinal attenders' because they were recorded in both pre and post citrate periods. It is worth noting, however, that these longitudinal attenders may only have visited a SEP once in each six month period.Two additional analyses were necessary to assess whether any observed changes pre versus post citrate were related to natural seasonal variation or other changes in service configuration. Therefore all heroin/crack SEP injectors recorded in the post citrate period were matched with their own SEP profiles for the corresponding time period 12 months previously (n = 314 matched pairs) and the median number of visits and syringes collected compared. Finally, pre and post citrate profiles for anabolic steroid injectors were extracted using the same six month pre and six month post citrate protocol detailed for heroin and crack injectors (n = 295 matched pairs). It was hypothesised that, because steroid users do not use an acidifier, there would be no significant difference in the median visit rate or median number of syringes collected pre versus post citrate.Kolmogorov-Smirnov tests showed that age, the number of visits and the number of syringes collected were all significantly, positively skewed, with the exception of the age of new clients. Non-parametric tests have therefore been used throughout with Wilcoxon sign rank tests used for matched, and Mann Whitney U for unmatched, data. Chi square analyses were used to compare categorical data and all analyses were undertaken using SPSS version 12 or EpiInComparing pre and post citrate periods, there was no significant difference in the age and gender of either new or established SEP clients Table . The medTable Staphlococcus aureus, resulting in considerable levels of morbidity and mortality [Worldwide there are an estimated 13.2 million injecting drug users . In addiortality . GrowingIt was envisaged that the introduction of citrate would increase both the number of injectors attending SEPs and the number of visits each person made . The recMonitoring data also showed no significant increase in the frequency with which heroin/crack injectors attended SEPs following citrate's introduction. The median number of visits made by established clients was three in both pre and post citrate periods while new clients made, on average, two visits within each of the two six month periods. However, matched pair analyses of longitudinal attenders of SEPs, comparing an individual's post citrate profile with their own behaviour pre citrate, showed that this cohort of injectors made significantly more visits post citrate (median = 5) than pre citrate . Further to this, matched pair analyses showed the median visits per person post citrate (median = 5) was significantly greater than the median visits for the corresponding six months in the previous year , for those injectors who were recorded in these two six month periods. We can therefore discount the possibility that the increase in visit rate between pre and post citrate was due to seasonal variation because the increased visit rate following the introduction of citrate occurred across years as well as within the year. Additionally, again using matched pair analysis, we observed no difference in the median number of visits pre and post citrate for steroid injectors (median number of visits being one in both the pre and post citrate periods). Steroid users do not use an acidifier so their behaviour should not be affected by the introduction of citrate. That no change in the behaviour of steroid injectors was observed supports the conclusion that the increased visit rate post citrate of heroin/crack injectors who attended SEPs in both pre and post citrate periods was due to the introduction of citrate. It is important to note, however, that the legal changes that permitted the distribution of citrate also sanctioned the distribution of other injecting paraphernalia , although the distribution of other paraphernalia in SEPs in Cheshire and Merseyside occurred less consistently than the introduction of citrate. Despite this, it is possible that the distribution of other injecting paraphernalia also affected the behaviour of SEP attenders within this geographical area.From these findings, we can conclude that the introduction of citrate did not encourage more clients to contact SEPs to collect clean injecting equipment in the first six months of its introduction, nor can we conclude that its introduction negatively affected attendance. Furthermore, we cannot conclude that overall, people visited SEPs more frequently following the introduction of citrate but that its introduction has encouraged longitudinal attenders of SEPs (i.e. those who were recorded in both the pre and post citrate six month periods) to visit more frequently. Therefore, at SEPs included in this study, the introduction of citrate has resulted in a change in service use amongst certain SEP clients, with less impact on those injectors who visit SEPs infrequently. Any increase in visit frequency should be welcomed as it provides SEP staff greater opportunity to engage with injectors to discuss a range of harm reduction measures and, where appropriate, to refer into other services. It is not clear from this study whether further changes will be observed once information about the availability of citrate at SEPs becomes universal amongst the injecting community.With respect to the number of syringes collected per visit, no difference was evident pre versus post citrate for established SEP clients. Matched pair analysis showed comparable findings. Similarly, no difference was observed in the number of syringes collected for heroin/crack injectors post citrate compared to the corresponding six month period in the previous year or for steroid injectors pre versus post citrate. Established SEP clients are therefore continuing to receive the same number of syringes per visit and, presumably, sufficient citrate for the number of syringes dispensed. Injectors who are classified as 'longitudinal attenders' for the purpose of this study, are thus attending SEPs more frequently post citrate but collecting the same number of syringes per visit, increasing, very slightly, the total number of syringes dispensed to this cohort of injectors from 71,495 in the pre citrate six month period to 71,743 in the post citrate six month period (data not shown). In light of evidence to suggest that clean syringes are used in only 25% of injections , from a Despite the benefits of citrate over other acidifiers , all mayWhile citrate may be the safest acidifier for drug users to prepare heroin for injection, hopes that its introduction would increase the number of injectors accessing SEPs were not supported by this study. Importantly, however, the introduction of citrate did not negatively affect attendance either, indicating that citrate can be added to the spectrum of interventions offered by SEPs without any apparent negative consequences. Furthermore, injectors who already attended SEPs relatively frequently on a longitudinal basis (i.e. in both pre and post citrate periods), attended SEPs more frequently, providing evidence to support a positive change in service use among these particular individuals. Greater levels of engagement provide increased opportunities for interaction between the injecting drug user and the practitioner. This can facilitate a range of harm reduction interventions relating to the prevention of blood borne infections and improvements in injecting techniques. In addition, increased contact rates can provide opportunities for appropriate referral to both specific drug-related interventions and generic health and welfare support. Furthermore, the increased number of syringe exchange visits, without a reduction in the number of syringes provided at each visit, within this client group, has slightly increased the number of clean syringes in circulation. Increasing the number of clean syringes distributed, to enable the use of sterile equipment for each injection, should remain a public health target and developments to facilitate this should be supported. Staff concerns regarding the amount of citrate to dispense to new clients must not impact on the number of syringes given out. It is not clear from this study whether further changes have been observed at SEPs once information about the availability of citrate became ubiquitous among drug injectors. Finally, this study demonstrates the value of utilising routinely collected monitoring data to assess the impact of harm reduction interventions, with further analyses planned to evaluate the longer-term impact of citrate provision at SEPs.The author(s) declare that they have no competing interests.CMB carried out data extraction, performed the statistical analyses and wrote the manuscript. JM conceived of the study, participated in its design and was involved in writing the manuscript. MC coordinated acquisition of data and assisted in writing the manuscript. MW participated in writing the manuscript. MAB provided assistance with the statistical analyses and interpretation and helped to revise the manuscript. All authors read and approved the final manuscript."} +{"text": "NPHP1, 3, 4, 5, 6, 7, 8, and 9) responsible for the juvenile NPH and one gene NPHP2 for the infantile form. NPH and associated disorders are considered as ciliopathies, as all NPHP gene products are expressed in the primary cilia, similarly to the polycystic kidney disease (PKD) proteins.Nephronophthisis (NPH) is an autosomal recessive disease characterized by a chronic tubulointerstitial nephritis that progress to terminal renal failure during the second decade (juvenile form) or before the age of 5\u00a0years (infantile form). In the juvenile form, a urine concentration defect starts during the first decade, and a progressive deterioration of renal function is observed in the following years. Kidney size may be normal, but loss of corticomedullary differentiation is often observed, and cysts occur usually after patients have progressed to end-stage renal failure. Histologic lesions are characterized by tubular basement membrane anomalies, tubular atrophy, and interstitial fibrosis. The infantile form is characterized by cortical microcysts and progression to end-stage renal failure before 5\u00a0years of age. Some children present with extrarenal symptoms: retinitis pigmentosa (Senior-L\u00f8ken syndrome), mental retardation, cerebellar ataxia, bone anomalies, or liver fibrosis. Positional cloning and candidate gene approaches led to the identification of eight causative genes ( Nephronophthisis (NPH), an autosomal recessive disorder initially described in 1945 by Smith and Graham and in 1951 by Fanconi, is a chronic tubulointerstitial nephritis that uniformly progresses to end-stage renal disease (ESRD) . With reJuvenile NPH is an uncommon condition that affects girls and boys equally. The incidence is approximately 0.13 for 10,000 live births in Finland, whereas in Canada, it is 1 per 50,000 live births and in United States 9 per 8.3 million . DecreasRenal insufficiency is often present when the diagnosis is made. Late symptoms are related to the progressive renal insufficiency and include anemia, metabolic acidosis, nausea, anorexia, and weakness. ESRD develops at a mean age of about 13\u00a0years but can also occurs in some rare cases much later during adulthood , 13.Renal ultrasound may be normal, with normal-sized kidneys, but renal parenchymal hyperechogenicity and loss of corticomedullary differentiation are often observed. At later stages, small cysts are present in the medulla , 15. RenNPHP3 gene in a large Venezuelan family in which ESRD occurred at a mean age of 19\u00a0years , cerebellar ataxia [Joubert syndrome (JS)], oculomotor apraxia type Cogan, mental retardation, bone anomalies and hepatic fibrosis. Situs inversus and ventricular cardiac septal defect are associated in some patients with the infantile form (Table\u00a0NPHP genes (except NPHP7), but whereas RP is always present and severe in patients with NPHP5 and NPHP6 mutations, the symptoms are in general mild in patients with mutations in the other NPHP genes. Georges et al. reported on four patients, from three different families, with RP responsible for severe visual impairment during childhood who developed chronic interstitial nephritis with histological lesions characteristic of NPH and renal failure only between 42 and 56\u00a0years of age , a component of cone and rod photoreceptors that is mutated in patients with Leber amaurosis [The oretinin . Nephrocoretinin . Interes apraxia . Twenty-maurosis .NPHP genes, mutations in the IQCB1 gene, now referred to as NPHP5, were reported only in patients with NPH in combination with severe retinal degeneration and early blindness\u2014SLS [NPHP5 mutations, involving both alleles in all cases, were found in 16 of 92 patients with early onset RP [In contrast to the previous ness\u2014SLS . NPHP5 monset RP . NPHP5 eonset RP .NPHP6 gene, also known as CEP290, encodes a centrosomal protein that activates ATF4, a transcription factor involved in the control of the cell cycle. Thirteen different mutations in the NPHP6 gene were initially reported in 12 families with JS [NPHP6 gene was thereafter reported to be mutated in more than 20% of patients with severe congenital blindness but no renal involvement [CEP290 ribonucleic acid (RNA) was detected in 16 (21%) of 76 unrelated patients with blindness but without clinical signs of renal disease. Moreover, these patients had no neurological symptoms typical of JS and had normal cognitive function [NPHP6 gene is the most common cause of Leber amaurosis [NPHP6 mutations, Helou et al. screened this gene in 99 patients with cerebellar ataxia (JS), 75 patients with RP, and 21 patients with isolated NPH and found mutations in seven, two, and one case, respectively [NPHP4 missense mutation was present, arguing for a digenic inheritance. A genome-wide linkage scan in families with MKS led to the identification of NPHP6 mutations in some patients [The with JS . Most ofaurosis) , 70. A hfunction . Anotherectively . In fourpatients .NPHP7 gene, also known as the GLIS2 gene, contains six coding exons and encodes a Kruppel-like zinc-finger transcription factor, which has been found mutated in one consanguineous Oji-Cree Canadian family with isolated NPH in three children who developed ESRD by 8\u00a0years of age. This gene seems to be very rarely involved, as no other mutation was found in a cohort of 470 individuals with NPH-like phenotypes [Glis2 gene are atrophic, with fibrosis starting at 8\u00a0weeks of age. Apoptosis is increased in renal tubular cells, whereas cell proliferation is not. Interestingly, the genes promoting epithelial\u2013mesenchymal transition and fibrosis are up-regulated in the absence of Glis2.The enotypes . The kidRPGRIP1L, have been found in patients with JS and in fetuses with MKS [NPHP4 missense mutations known to cause NPH with RP also disrupt this interaction [RPGRIP1L gene was thereafter analyzed in a cohort of 56 patients with JS. RPGRIP1L mutations were identified in five kindreds, including six individuals (8%). Of note, patients with RPGRIP1L mutations had normal retina, except two patients out of 12 with moderate visual impairment [RPGRIP1L mutations, such as liver fibrosis, postaxial polydactyly, pituitary agenesis, and partial growth hormone deficiency. These last findings indicate a possible overlap with RHYNS syndrome [Mutations in a novel gene on chromosome 16, with MKS . Intereswith MKS . RPGRIP1with MKS . Intereswith MKS , 34. In eraction . Recentleraction . The RPGpairment . Additiosyndrome .Nek8 gene [NEK8 gene in a cohort of 588 patients with NPH led to the identification of three missense mutations in patients with isolated NPH [NPHP5 gene was also present. Interestingly, mutant forms of NEK8 showed defects in ciliary localization.The jck cystic kidney mouse model is associated with a mutation in the ek8 gene . Analysiated NPH . In one NPHP1 to NPHP9 mutations have been reported in cases of juvenile NPH with or without extrarenal symptoms, except for mutations in NPHP2 that have been found only in patients with infantile NPH. NPHP1 mutations were found in ~20% to 40% of cases [NPHP1, NPHP3, and NPHP4 genes in a cohort of 94 different families have shown that a mutation or a deletion in one of these three genes was identified in 44 (47%) patients [NPHP genes were found, whereas two mutations in two NPHP genes (NPHP3 and NPHP4) were found in another kindred. Finally, a single mutation in one of these three genes was discovered in nine other patients [NPHP1 deletion and either a heterozygous NPHP6 or AHI1 mutation [Overall, of cases , whereaspatients . Interespatients . In the mutation . These fCilia are present in almost all cells in the organism and act as sensory organelles that connect visual, mechanosensory, odorant or other stimuli to cell-cycle, control of epithelial architecture, or other yet unknown processes. The finding that polycystin-1 and polycystin-2\u2014the proteins responsible for autosomal dominant polycystic kidney disease and protNPHP1 deletion, which is found in 20\u201340% of cases. In the absence of such deletion, renal biopsy may be proposed to confirm the diagnosis. At present, screening for mutation in all the other NPHP genes is not routinely performed due to the low frequency of detected mutations and the high cost of the procedure. Patients with associated disorders should be considered separately. In case of severe RP, the NPHP5 gene may be screened for mutations, whereas in patients with neurological symptoms such as cerebellar ataxia, the NPHP6 and NPHP8 genes should be analyzed first. In case of early onset tubulointerstitial nephritis with cortical microcysts, the NPHP2 gene should be screened for mutation (Fig.\u00a0In conclusion, and from a practical point of view, the diagnosis of NPH should be considered if a child presents with polyuria, urinary sodium loss, growth failure, renal insufficiency without hematuria or proteinuria, normal blood pressure, and normal-sized kidneys without dilatation of the urinary tract. These patients should be screened for homozygous or heterozygous"} +{"text": "Acute trauma of the wrist is one of the most frequent reasons for visiting the Emergency Department. These patients are routinely referred for radiological examination. Most X-rays however, do not reveal any fractures. A clinical decision rule determining the need for X-rays in patients with acute wrist trauma may help to percolate and select patients with fractures.This study will be a multi-center observational diagnostic study in which the data will be collected cross-sectionally. The study population will consist of all consecutive adult patients (\u226518 years) presenting with acute wrist trauma at the Emergency Department in the participating hospitals.This research comprises two components: one study will be conducted to determine which clinical parameters are predictive for the presence of a distal radius fracture in adult patients presenting to the Emergency Department following acute wrist trauma. These clinical parameters are defined by trauma-mechanism, physical examination, and functional testing. This data will be collected in two of the three participating hospitals and will be assessed by using logistic regression modelling to estimate the regression coefficients after which a reduced model will be created by means of a log likelihood ratio test. The accuracy of the model will be estimated by a goodness of fit test and an ROC curve. The final model will be validated internally through bootstrapping and by shrinking it, an adjusted model will be generated.In the second component of this study, the developed prediction model will be validated in a new dataset consisting of a population of patients from the third hospital. If necessary, the model will be calibrated using the data from the validation study.Wrist trauma is frequently encountered at the Emergency Department. However, to this date, no decision rule regarding this type of trauma has been created. Ideally, radiographs are obtained of all patients entering one of the participating hospitals with trauma to the wrist. However, this is ethically and logistically not feasible and one could argue that patients, for whom no radiography is required according to their physician, are not suspected of having a distal radius fracture and thus are not part of the domain.This study is registered at the Netherlands Trial Register (NTR 2544) and was granted permission by the Medical Ethical Committee of the Academic Medical Center Amsterdam on 06-01-2011. An acute trauma of the wrist is one of the most frequent reasons for visiting the Emergency Department, and fractures of the distal radius account for an estimated 17% of all fractures diagnosed . The incA clinical guideline concerning the X-ray referral policy may help to select and percolate patients with fractures. In 1987, Gleadhill et al. already concluded that: Clinical guidelines on selecting patients for radiography for certain injuries and emergencies reduced the overall X ray referral rate by 18,5% [In 1991, Stiell et al. conducted a study which showed that the vast majority of ankle trauma patients was radiographically examined upon presentation . They esIn 2003, Cevik et al. evaluated the value of physical findings to predict fractures in patients with acute wrist trauma . They inRecently, Calco-Lorenzo et al. analyzed the possibility of creating a clinical decision rule for the assessment of conventional X-rays in acute wrist trauma . They emTherefore, the primary aim of this study is to formulate a clinical decision rule to determine when an X-ray of the distal radius is necessary in patients presenting to the Emergency Department following wrist trauma.The second aim of this study is to validate this decision rule in a new patient population and to present it as simplified risk score.This study consists of two components in which the data will be collected cross-sectionally. The data will be collected in three different hospitals during a period of six months. The participating hospitals include one university hospital, one non-academic teaching hospital, and one non-teaching hospital.One component of this research comprises a study which will be performed to determine which combination of clinical parameters has a predictive value for the presence of a distal radius fracture in adult patients presenting to the Emergency Department following acute wrist trauma. This will be done by creating a model from the data collected in the university hospital and the non-academic teaching hospital. This model will be internally validated by bootstrapping. In the second component of this study, the developed prediction model will be validated in a new dataset. This dataset will be collected simultaneously in the third hospital, the non- teaching hospital, using the same approach as in the other hospitals.The study population is defined as all consecutive adult patients presenting at the Emergency Departments in the participating hospitals following wrist trauma and who are suspected to have sustained a distal radius fracture. Patients are suspected of having sustained a distal radius fracture when pain on pressure in the wrist area is indicated. The wrist is defined as the proximal segment of the hand consisting of the carpal bones and the associated soft parts and the distal segment of the ulnar and radial bone. A traumatic wrist injury is defined as a high or low energetic accident involving the wrist, e.g. a fall on the outstretched hand or a motor vehicle accident.\u2022 Patient aged 18 years and older\u2022 An acute trauma of the wrist (< 72 hrs following trauma)\u2022 Pain on pressure in wrist area\u2022 Pre-existent neurological pathology in the affected limb\u2022 Previous fracture of distal radius <3 months\u2022 Patients referred from another hospital where X-rays of the wrist were performed\u2022 Multi trauma patient\u2022 Patients who are not considered to be competent or capable by their treating physician to answer any questions regarding pain or mechanism of trauma as listed on the Case Report Form and one lateral view with the elbow in 24 degrees of flexion. A fracture is defined as the presence or disruption of one or more of the cortices of the distal radius. Malalignment in the distal radial ulnar joint and radial carpal joint is regarded as a potential fracture. Small avulsions at bony attachments sides of ligaments are considered to be a fracture as well.Data collection will take place from February 2011 till August 2011. During this period, patients presenting following an acute traumatic injury of the wrist will receive care as usual. The difference is that, upon presentation with wrist pain following trauma, an additional Case Report Form (CRF) containing multiple clinical parameters will be completed by the treating physician. These clinical parameters are defined by trauma-mechanism, physical examination and functional testing and include; mechanism of trauma; swelling of the distal radius, swelling of the Anatomical Snuffbox; visible deformity of the wrist; palpability of radial artery pulsations; pain on palpation of the distal radius, distal ulna, Anatomical Snuffbox, radial styloid, ulnar syloid and Lister's tubercle; pain on dorsal flexion, palmar flexion, supination, pronation ulnar deviation, radial deviation; difference in prehensile strength test between affected and unaffected side; outcome distal radial drawer test and pain on axial compression (see appendix). Radiographs of the distal radius will be performed according to Dutch guidelines; at least one posterior anterior (PA) and one lateral view with the elbow in 24 degrees of flexion. Additional imaging for injuries associated with wrist trauma, e.g. suspected fracture of the scaphoid, can be performed selectively and will be on account of the treating physician. The independent radiologist assessing the outcome using the reference standard (X-rays) will be blinded from the results of the CRF to prevent incorporation bias. The dichotomous nature (yes/no) of the CRF and the fact that it is a standardized way of history taking and examination, will decrease interobserver variability and thus minimize underestimation of the potential diagnostic value of the parameters.Upon completion of the data collection in all three participating medical centers, a prediction rule will be developed based on data from two of the participating hospitals; the academic hospital and the non-academic teaching hospital. Since prediction rules always perform perfectly in the patient population they were generated from, a validation study in a new study population is required to assess its predictive quality. To ensure the most stringent form of validation, the rule will be validated in data from the third hospital, the non-teaching hospital. This method is also known as domain validation. The method of data collection in the third hospital will be similar and done simultaneously. This dataset will be comparable to the first regarding size and informative parameters and the model will be tested to verify its predictive value of the primary outcome: distal radius fracture. If necessary, the model will be updated and finally it will be presented as a simplified risk score.Because this is an observational cohort study, no informed consent will be obtained and therefore withdrawal is unlikely to occur. The treating physician can decide to withdraw a subject from the study for urgent medical reasons.The data from the academic hospital and the non-academic teaching hospital will be used to develop the model. The data will be assessed by using a multivariable analysis. However, to prevent too optimistic estimates of the accuracy of the diagnostic model, a more liberal p-value for statistical significance will be employed (p = 0.15). Logistic regression modeling will be used to estimate the regression coefficients and thus the log odds of a fracture versus no fracture after which a reduced model will be created by means of a log likelihood ratio test.The accuracy of the model will be estimated by a goodness of fit test and a ROC curve. If applicable, different models can be compared using the tests mentioned above. The final model will be validated internally through bootstrapping to estimate overfitting in the calibration and discrimination stated above. By averaging the optimism an adjusted model will be generated.Henceforth, the prediction rule will be validated in a new data set, which will be collected simultaneously in the third hospital; the non-teaching hospital. If necessary, the model will be updated using the data from the validation study, by adjusting the intercept.Finally, the prediction model will be presented as a simplified risk score which can be used by physicians to determine the need for an X-ray in patients presenting with wrist trauma at the Emergency Department.The study will be conducted according to the principles of the Declaration of Helsinki and in accordance with the Medical Research Involving Human Subjects Act (WMO).Patients with acute traumatic wrist injury will be treated by the physician on call in the Emergency Department. The only difference is that clinical parameters, as recorded during the physical examination will be more extensive and will be recorded more precise by the treating physician. Because of the observational character of this study, no informed consent was deemed necessary by the Medical Ethical Review Committee of the Academic Medical Center of the University of Amsterdam. This committee stated on March 17, 2011 that The Medical Research Involving Human Act (WMO) does not apply to this study and that an official approval by the committee is not required, reference number; project 2011_078.The data will be coded by patient number. Research data will be stored in a database (PASW statistics 18 and Microsoft Excel), and will be handled confidentially and anonymously. Research data that can be traced to individual persons can only be viewed by authorized personnel. These persons are the members of the research team, members of the health care inspection, and members of the Medical Ethics Committee of the Academic Medical Center Amsterdam. Review of the data may be necessary to ensure the reliability and quality of the research. The handling of personal data is in compliance with the Dutch Data Protection Act and the privacy regulation of the Academic Medical Center Amsterdam.As stated above, wrist trauma is frequently encountered at the Emergency Department. However, to this date, no decision rule regarding this type of trauma has been created.Ideally, we would like to obtain radiographs of every patient entering one of the participating hospitals with trauma to the wrist. This however, is ethically and logistically not feasible. Therefore we should acquiesce to the fact that we obtain the informative parameters listed on the Case Report Form of each patient presenting with wrist pain post injury, but only outcome data on patients sent for X-ray by their treating physician. Any comments on this approach can be invalidated by stating that patients, for whom no radiography is required according to their physician, are not suspected of having a distal radius fracture and thus are not part of our domain.The authors declare that they have no competing interests. No external funding was received for this study.All authors participated in the design of the study and the drafting of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/238/prepubCase Report Form. the case report form containing the variables which will be filled out by the treating physician.Click here for file"} +{"text": "Background: Arsenic exposure from drinking water has been associated with heart disease; however, underlying mechanisms are uncertain.Objective: We evaluated the association between a history of arsenic exposure from drinking water and the prolongation of heart rate\u2013corrected QT (QTc), PR, and QRS intervals.Method: We conducted a study of 1,715 participants enrolled at baseline from the Health Effects of Arsenic Longitudinal Study. We assessed the relationship of arsenic exposure in well water and urine samples at baseline with parameters of electrocardiogram (ECG) performed during 2005\u20132010, 5.9 years on average since baseline.Results: The adjusted odds ratio (OR) for QTc prolongation, defined as a QTc \u2265 450 msec in men and \u2265 460 msec in women, was 1.17 for a 1-SD increase in well-water arsenic (108.7 \u00b5g/L). The positive association appeared to be limited to women, with adjusted ORs of 1.24 and 1.24 for a 1-SD increase in baseline well-water and urinary arsenic, respectively, compared with 0.99 and 0.86 in men. There were no apparent associations of baseline well-water arsenic or urinary arsenic with PR or QRS prolongation in women or men.Conclusions: Long-term arsenic exposure from drinking water was associated with subsequent QT-interval prolongation in women. Future longitudinal studies with repeated ECG measurements would be valuable in assessing the influence of changes in exposure. Epidemiologic studies have linked arsenic exposure from drinking water to cardiovascular disease (CVD) is defined as the time from the onset of the QRS complex to the end of the T wave. It represents the duration of ventricular electrical systole, including depolarization and repolarization. Its prolongation indicates nonuniform recovery of myocardial excitability and has been shown to lower the ventricular fibrillation threshold and increase susceptibility to ventricular arrhythmia and sudden cardiac death . QT-inteIt has been estimated that 13 million Americans have been exposed to drinking water with arsenic concentrations of 10\u201350 \u00b5g/L . In BangStudy population. The Health Effects of Arsenic Longitudinal Study (HEALS) is an ongoing population-based, prospective cohort study in Araihazar, Bangladesh. Details of the study methodologies have been presented elsewhere for at least 48 hr before analysis. Total arsenic concentration was analyzed by high-resolution inductively-coupled plasma mass spectrometry, with a detection limit of < 0.2 \u00b5g/L. Further details on field sampling and laboratory analysis procedures are described elsewhere with a detection limit of 2 \u03bcg/L, as previously described . UrinaryECG measurements and evaluation. Participants in the present analysis were 1,715 cohort members referred for ECG examinations because of high blood pressure or symptoms of heart disease identified during a biennial follow-up study visit, an interim health survey, or a visit to the field clinic for medical treatment. These participants were referred to one of three trained field clinic physicians, who were blinded to arsenic-exposure information, for further evaluation and diagnostic confirmation followed by treatment and referral to the only local hospital in the study area as appropriate. Standard 12-lead resting ECGs were performed using a Bionet Cardiocare 2000 device . All ECGs were processed using the Dalhousie ECG program (1/2) ( program . The QT am (1/2) . The PR am (1/2) , a PR inam (1/2) , and a Qam (1/2) . ECGs weStatistical analyses. We first conducted descriptive analyses to compare arsenic exposure data and demographic and lifestyle characteristics between the present study population and the overall cohort, and between participants with and without QTc prolongation in the study population.n = 273) who did not have follow-up measurements because of the recency of their recruitment . For subjects in the original cohort, we adjusted for the difference in urinary arsenic between baseline and the first follow-up, and between the first and second follow-up, in 1,402 participants from the original cohort whose ECGs were performed after the second follow-up or within the 6 months before the second follow-up. For participants whose ECG was performed > 6 months before the second follow-up (n = 38), we adjusted for the difference between baseline and the first follow-up only. Separate dummy variables for changes in urinary arsenic were created for participants who did not have urine arsenic concentrations measured at the first (n = 15) or second follow-up visit (n = 28) under a \u201cmissing at random\u201d assumption. Linear regression models were also conducted to estimate associations between arsenic exposure and QTc as a continuous dependent variable, with the same adjustments as in the logistic regression models.We used unconditional logistic regression to estimate the odds ratios (ORs) for QTc prolongation in relation to quartiles of baseline well-water and urinary arsenic. We first adjusted for sex and age (years) (model 1); we then additionally adjusted for educational attainment (years), baseline body mass index , and baseline smoking status (model 2) . Data foThe literature suggests that women have longer average QTc intervals compared with men, and therefore we conducted stratified analysis to estimate associations of arsenic exposure with prolongation of QTc, PR, and QRS intervals separately in men and women, similar to previous studies . ORs forn = 1,142), analyses of associations with urinary arsenic in the sample collected closest to the ECG measurement, analyses excluding those with QRS of \u2265 120 msec .Arsenic exposures and demographic and lifestyle characteristics were in general similar between the study population and the overall HEALS cohort, although the study population had a slightly higher proportion of women . In stratified analyses by sex, the prevalence of QTc prolongation was positively associated with baseline arsenic exposure in women, but not in men. The OR for QTc prolongation in association with the highest versus lowest quartile of baseline well-water arsenic was 1.61 in women and 0.76 in men. The adjusted ORs for a 1-SD increase in baseline well-water arsenic were 1.24 , and 0.99 . The ORs for QTc prolongation comparing the highest and lowest quartiles of baseline urinary arsenic, and for continuous baseline urinary arsenic, were also positive in women (ptrend = 0.04), but not in men (ptrend = 0.60).In the overall analysis, there was a positive association between continuous baseline well-water arsenic and QTc prolongation , but thehttp://dx.doi.org/10.1289/ehp.1205197)]. For example, a 1-SD increase in well-water arsenic was associated with a 2.2-msec increase in mean QTc in the total population, and with 3.0-msec and 0.7-msec increases in mean QTc in women and men, respectively.Results from linear regression of QTc as a continuous dependent variable were consistent with the findings based on dichotomized QTc . Exclusion of participants with QRS \u2265 120 msec did not materially change the results . The positive associations remained similar in the subpopulation with \u2265 5 years of arsenic exposure . In the analyses of associations with urinary arsenic concentrations in the samples collected closest to the ECG measurement, effect estimates were consistent with, but weaker than, estimates from the main analysis . For instance, the adjusted OR for QTc prolongation in relation to a 1-SD increase in urinary arsenic concentration (216.8 \u00b5g/g creatinine) was 0.90 in men and 1.10 in women, respectively.Adjusted estimates based on SG-adjusted urinary arsenic were consistent with those for creatinine-adjusted urinary arsenic. For example, the OR for QTc prolongation in relation to a 1-SD increase in baseline urinary arsenic was 0.94 in men chronically exposed to high levels of arsenic in drinking water compared with men (n = 40) not exposed to arsenic . However, in the present study, QTc prolongation was significantly associated with the highest quartile of exposure in women only [OR = 1.61 ]. Given that CVD is a broad set of diseases and condition, QTc prolongation might not be a main driver of the overall CVD mortality. QTc prolongation may be a more critical intermediate endpoint for higher levels of exposure and for subtypes of CVD, such as coronary heart disease. The data on different end points support the idea that the cardiovascular effects of arsenic exposure may differ according to dose and subtypes of CVD, and may be increased in susceptible subsets of the population.in vitro and in vivo studies have shown that female sex is a strong risk factor for drug-induced long QT interval and cardiac arrhythmias and KCNQ1 . Arsenicin vitro . Other pin vitro .The study has several limitations. First, our study population consisted of relatively few overweight individuals, possessing a mean BMI of 20.8. Thus, the findings may not be generalizable to other populations with a different nutritional profile. However, our data were consistent with those of We found a positive association between past arsenic exposure in a population with long-term exposures at moderate levels and QT-interval prolongation measured on average 5.9 years after baseline and the association was present in women but not in men. Our finding may help explain the increased mortality from CVD in humans exposed to arsenic from drinking water. ECG analysis of the QT interval may be useful for early detection of cardiac toxicity induced by arsenic exposure and for evaluation of populations at high risk for cardiovascular events.(389 KB) PDFClick here for additional data file."} +{"text": "Echinococcus infections in canine definitive hosts.Cystic echinococcosis is highly prevalent in northwest China. A cost-effective, easy to operate diagnostic tool with high sensitivity and specificity would greatly facilitate the monitoring of nad5 gene of E. granulosus sensu stricto and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR method, copro-ELISA and microscopy, using the faeces of dogs experimentally infected with E.g.s.s., and field-collected faeces of domestic dogs including 190 from Qinghai province highly endemic for E.g.s.s. and 30 controls from an area in Gansu, where a domestic dog de-worming program was in operation.The primers used in the LAMP assay were based on the mitochondrial E. granulsous s.s. four days earlier than the PCR method. Three copro-samples shown positive by the commercial copro-ELISA were all negative by LAMP, PCR and microscopy, which suggests these samples may have originated from another infection rather than E. granulsous s.s., possibly E. shiquicus or E. Canadensis, which is also present in China.The positivity rates obtained for the field-collected faecal samples were 12.6%, 1.6% and 2.1% by the LAMP, PCR and copro-ELISA assays, respectively. All samples obtained from the control dogs were negative. Compared with the conventional PCR, the LAMP assay provided 88.8% specificity and 100% sensitivity. The higher sensitivity of the LAMP method was also shown by the fact that it could detect the presence of laboratory challenge dog infections of E. granulosus s.s. infections in the field. The LAMP assay may lead to a more cost-effective and practicable way of tracking Echinococcus infections in canids, especially when combined with the copro-ELISA.We have developed a potentially useful surveillance tool for determining the prevalence of canine Echinococcus granulosus (E.g.), is of considerable importance from the public health perspective and also has a significant socio-economic impact. There are an estimated 2\u20133 million cases of human CE Cystic echinococcosis (CE), caused by E.g. is the fact that it comprises a number of intra-specific variants or strains that exhibit considerable variation at the genetic level E.g. sensu lato (E.g.s.l.) comprises previous 10 genotypes (G1 to G10) with the G1 (sheep strain) genotype being the prototypical species, infecting humans and livestock extensively E. granulosus sensu stricto, E.g.s.s.) and G6 genotypes have been reported within China E.g. are canids - dogs, dingoes, foxes, wolves and jackals - which harbour the adult stage in the small intestine. Eggs and gravid proglottides are released periodically in faeces, into the external environment. Humans and herbivorous animals become infected with the metacestode of E.g. following the ingestion of viable eggs. Transmission is completed when the viscera of infected intermediate host animals are consumed by canids E.g., efforts should be directed towards building an effective surveillance system for identifying infected canine hosts, an important component for establishing the epidemiological parameters of CE and for preventing human and livestock infection E.g. in canids is difficult as the infection is generally asymptomatic and the small proglottides spontaneously discharged in faeces are usually overlooked E.g. from other Taenia species An important feature of the biology of E.g. infection in definitive hosts using defined parasite copro-antigens or DNA sequences E.g.-positive canine faecal samples collected in the field. LAMP-based assays E.g. infections E.g. in faecal samples from naturally infected dogs. Here we describe the development of an alternative LAMP assay, based on detecting a specific partial sequence of the mitochondrial nad5 gene of E.g.s.s. in the faeces of dogs experimentally infected with metacestodes of the E.g.s.s. We have assessed its value on canine faecal samples collected in the field and compared its practicability and diagnostic performance with conventional PCR, ELISA and microscopy approaches.The application of immunological approaches and polymerase chain reaction (PCR)-based procedures has proven of value in the detection of The institutional ethical committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province approved the study . The experiments using dogs were undertaken under very strict adherence to the institutional and Chinese national guidelines for animal husbandry. For any locations and activities in the field studies, no specific permissions were required, or no endangered/protected species were involved in.E.g.s.s. isolate cox1 and nad1 sequencing (data not shown)] was obtained from a large unilocular hydatid cyst of E.g.s.s. in a sheep liver at an abattoir in Xining city, Qinghai province in 2010. The hydatid fluid was aspirated into a 500-ml conical flask and protoscoleces were obtained by sedimentation. The sediment was washed four times with sterilized phosphate-buffered saline (PBS). The protoscoleces were then immediately immersed in Dulbecco's Modified Eagle Media (DMEM) containing 5% (v/v) fetal bovine serum (FBS) at 37\u00b0C and maintained for two hours in order to determine their viability by observing their movement microscopically. Samples having 95% viable protoscoleces were used for challenging dogs or stored at \u221270\u00b0C for DNA isolation.An E.g.s.s. isolate with a meal, and then they were fed a heat-treated meal once daily. Faecal samples were collected daily and examined carefully for the E.g. proglottides with macroscopic and stereo microscopic observation after sufficiently homogenizing, until day 70 post challenge, from the bottom of individual cages. They were placed into sterilized 50-ml containers with tight fitting lids and stored at \u221270\u00b0C. The dogs were euthanized to check the small intestine for the determination of the number of the E.g.s.s. worms on day 76 post-challenge infection.Four 6-month old non-pedigree dogs were purchased from a dog-market in Lanzhou city, Gansu province. They were treated with praziquantel (10 mg/kg body weight given on three consecutive days per month) for two months prior to the study commencement, and kept in individual cages at the experimental facility, Lanzhou Veterinary Research Institute, to allow them to adapt to the living environment and diet. After verification that they were helminth worm-free, by microscopic examination of their faeces, each dog (average weight 10 kg) received orally about 10,000 viable protoscoleces of the E.g.-endemic areas; Zhiduo county and Dari county , Qinghai province Faecal samples (n\u200a=\u200a190) were collected from individual domestic dogs in two Faecal samples from all naturally infected dogs were fully homogenized and then subjected to a conventional saturated sodium chloride (NaCl) flotation method E.g.s.s.-positive control to assess the sensitivity of the LAMP assay. E.g.s.s. genomic DNA isolated from faecal samples (f-DNA) was obtained from the faeces of the experimentally infected dogs and faeces from naturally infected domestic dogs. Parasite DNA (f-DNA) was extracted from the faecal samples (200 mg) using Axyprep kits and QIAamp DNA stool mini kits . Genomic DNA samples (g-DNAs) from E.g.s.l. including G4 (named E. equinus), G6 and G6/G7 genotypes (named E. canadensis), E. multilocularis, E. shiquicus, T. hydatigena, T. pisiformis, T. taeniaeformis, T. multiceps and Dipylidium sp. were used to determine the specificity of the E.g.s.s. LAMP assay. The T. taeniaeformis g-DNA was provided by Viktor Dyachenko, Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-University of Munich, Munich, Germany, the E. shiquicus g-DNA was extracted from a cyst collected from a naturally infected plateau pika (Onchotona curzoniae) from Shiqu in 2011, and the g-DNAs of the E. equinus and E. canadensis isolates were provided by Antonio Varcasia from Dipartimento di Biologia Animale, Universit\u00e0 degli Studi di Sassari, Italy. The other cestode g-DNAs, isolated using Axyprep kits, were obtained from worms from experimentally infected dogs at Lanzhou Veterinary Research Institute. In addition, intestinal contents (200 mg) and negative faecal samples (200 mg) (n-f-DNA) from uninfected dogs were obtained from newly born pups and the DNAs were extracted (Axyprep kits) to serve as negative controls. The concentrations of the DNA samples were measured using a Nanodrop 2000 spectrophotometer .Genomic DNA (g-DNA), obtained from protoscoleces of the G1 isolate using an Axyprep multisource genomic DNA miniprep kit , acted as an E.g.A conventional PCR assay was carried out for comparative purposes. The PCR primers were used to amplify a fragment of the mitochondrial 12S rRNA gene of nad5 gene of E.g.s.s. (GenBank accession no. AF297617 or NC_008075) http://primerexplorer.jp/elamp4.0.0/index.html). The sequences for the F3 and B3 primers are located outside of the two other primers (FIP and BIP) in the E.g.s.s. mt-nad5 gene region. Primers were validated using BLAST software (http://www.ncbi.nlm.nih.gov/BLAST), and their sequences are listed in LAMP primers were designed based on the amplification of a specific sequence within the mt To determine the optimal reaction temperature and time, the reaction mixture was incubated at 60\u00b0C, 61\u00b0C, 62\u00b0C, 63\u00b0C, 64\u00b0C and 65\u00b0C, respectively, for 30 min, then heated at 80\u00b0C for 5 min to terminate the reaction; then six different reaction time periods were compared at the optimal reaction temperature.E.g.s.s. DNA, the LAMP primers were tested using g-DNAs from other E.g.s.l., E. multilocularis, E. shiquicus, T. hydatigena, T. pisiformis, T. taeniaeformis, T. multiceps, D. caninum, n-f-DNA and dog intestinal tissue as negative controls. To further confirm the specificity of the LAMP amplifications, the sequences of the LAMP amplicons were determined using a modification of the method described by Nkouawa et al E.g.s.s. mt-nad5 were sequenced by Sangon Biotech Co., Ltd. . In order to determine the sensitivity of the LAMP assay, E.g.s.s. g-DNA was diluted to 10 ng/\u00b5l, then successively diluted 10 times by the addition of 1 \u00b5l of a 1/10 dilution of the previous concentration. The same dilution procedure was also performed on DNA samples from dog faeces (f-g-DNA) obtained at different days post-E.g.s.s. metacestode challenge. In addition, the minimum number of eggs detected by the LAMP assay was determined in the experiments with faeces spiked with eggs obtained from E.g.s.s. adults collected from one of the experimentally infected dogs. The eggs were counted, mixed with faeces from an uninfected dog and the faecal samples were then frozen until use.To verify the specificity of the LAMP assay for detection of E.g.s.s. was used as positive control. The LAMP and PCR products were electrophoresed on a 1.5% (w/v) agarose gel with ethidium bromide and photographed using a gel documentation system. Also, the LAMP products were characterized by visual inspection after the addition of a 1/10 dilution of 1/10000 concentration SYBR Green I (Invitrogen) to the reaction tube.The 190 field-obtained faecal samples collected from dogs, which included samples with taeniid eggs present confirmed by microscopy, were all subjected to the LAMP and PCR assays. The f-g-DNA extracted from the faeces of one of the dogs experimentally infected with E.g. antigens and incubated at 37\u00b0C for one hour. One positive, one negative and three cut-off controls, provided in the kit, were placed in wells of each plate. The plates were washed three times (3 \u00d7 3 min) with washing buffer (0.05% PBS-Tween 20); then, 50 \u00b5l anti-E.g. specific antibodies conjugated with horseradish peroxidase (HRP) (provided in the kit) were added to each well and incubated at 37\u00b0C for 30 min. The wells were washed three times in the washing buffer, 50 \u00b5l each of colour reagent A and B were added, the solution was incubated at 37\u00b0C for 30 min in the dark and then the reaction was stopped with 50 \u00b5l stop solution. Absorbance values were read on a Multiscan ELISA reader at 450 nm. The sample-positive OD-value was used as the average OD-value of the three cut-off controls. The sensitivity and specificity of the ELISA was determined using the conventional PCR assay as reference standard.Two grams of faecal sample were mixed (1\u22362) with phosphate buffered saline (pH 7.2) containing 0.3% (v/v) Tween 20 (PBS-T) in a 15 ml centrifuge tube at room temperature; the tube was shaken vigorously and the contents allowed to sediment. The supernatant was collected into a 2 ml screw capped tube, labelled with a reference number and stored at \u221220\u00b0C until analyzed using a commercial copro-antigen sandwich ELISA kit according to the manufacturer's instructions. One hundred \u00b5l of faecal supernatant in 0.15 M PBS-T was added to the wells of polystyrene plates that had been coated with specific antibody prepared against Differences among the LAMP, PCR and ELISA procedures and microscopy for assessing the sensitivity of each test were determined using One-Way ANOVA with post hoc LSD tests and the Chi-square test using the software package SPSS 11.5 Of a range of temperatures tested, 63\u00b0C was chosen as the optimal reaction temperature . The optThe LAMP products demonstrated typical patterns of ladder-like bands on agar gels, and their EcoRI digestion products were as expected . The LAME.g.s.s. infections. Four of the six dogs yielded 420, 321, 302 and 119 E.g.s.s. worms; no worms were recovered from the other two dogs.All six chellanged dogs were euthanized to retrieve the dogs' intestines for the confirmation of the E.g.s.s. g-DNAs and f-g-DNA, as well as g-DNAs extracted from the other parasites and dog host intestinal tissues were tested to determine the specificity of the LAMP assay for E.g.s.s. DNA. Only the target gene fragments in E.g.s.s. g-DNA and f-g-DNA produced the amplified products, by the PCR assay, 4 samples were copro-ELISA-positive (2.1%) and 3 were positive (1.6%) by microscopy for the target croscopy . All 30 E.g. in intermediate hosts and adult worms infections in definitive hosts in endemic areas worldwide E.g. transmission, due both to the very close relationship they have with humans and the fact they are very susceptible to E.g. infection E.g. are based mainly on the treatment of domestic dogs using the highly effective drug praziquantel as a strategy for interrupting the E.g. life cycle E.g. in dogs for large scale surveillance to obtain data on the local infection pressure is urgently required. Several approaches, with varying levels of success, have been used to develop tools for the coprodiagnosis of Echinococcus species in canids Cystic echinococcosis (CE) is endemic in many parts of China E.g.s.s. was evaluated against a variety of cestode species including the E.g.s.s., E. canadensis, E. equinus and other helminth parasites regularly found in the intestines of dogs in the study area. The outcome of the evaluation thus excluded the possibility that amplification occurred of non-E.g.s.s. DNA present in the intestinal contents of field collected dog copro-samples. None of negative controls gave a signal. Only DNA of the E.g.s.s. was amplified specifically in the PCR and LAMP assays we developed. The LAMP assay in the current study showed a sensitivity that was a hundred times greater than conventional PCR detection. The LAMP system developed in this study has overcome the inhibitory components present in copro-samples E.g.s.s. eggs per gram of faeces. However, even in dogs with mature infections, eggs or gravid proglottids are not shed continuously and are not homogeneously distributed within faeces. Some copro-samples were positive in the LAMP assay but no eggs were evident in the faeces by microscopy. These were probably due to pre-patent infections on the basis of morphological analysis of the recovered worms. E.g.s.s. has a pre-patent period of approximately 6 weeks E.g.s.s. as the LAMP and PCR assays were confirmed as being specific for detecting the E.g.s.s. in this study. While ELISA for antigen detection could reveal the wide-range antigens of Echinococcus spp., such as due to E. shiquicus or E. canadensis positive, since these species have been also reported previously either in animals (including dogs) or/and humans in China Taenia/helminth species E. multilocularis (such as EmA9) have been used for the identification of E. granulosus infections, suggesting the shared antigens exist among Echicococcus species E.g.s.s. meets these routine monitoring/surveillance requirements. The operation of the assay is simple and is readily adaptable to field conditions as it can be performed simply with an affordable heating block or water bath. Furthermore, the LAMP reaction results in the precipitation of white magnesium pyrophosphate in the reaction mixture with the turbidity increasing with DNA concentration which can be visualized by the naked eye or quantified with an inexpensive turbidity-meter The specificity of two DNA test systems, involving PCR and LAMP assays, for the E.g.s.s. (the dominant Echinococcus species impacting on both humans and livestock worldwide) provides a useful tool for routine E.g.s.s. surveillance in wild and domestic canine definitive hosts so as to aid in the control of E.g. transmission globally.In summary, considering the advantages of rapid amplification, simple operation and ease of detection, the field-tested LAMP assay developed for the"} +{"text": "Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed.L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively.The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3\u00a0%, 58.6\u00a0%, 40.5\u00a0% and 10.8\u00a0% by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97\u00a0% for both methods.The LAMP assay detected 1\u00a0fg of This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China. Leishmania. The protozoan parasites are transmitted by the bite of infected sandflies and live and multiply intracellularly in macrophages of their mammalian host. Leishmania parasites cause three different clinical forms of the disease in humans, classified as visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). VL is considered to be the most lethal form of the disease causing, annually, an estimated 59,000 deaths and 2.4 million disability-adjusted life years (DALYs) [L. infantum [Leishmaniasis is a vector-borne parasitic disease of humans and other mammals caused by flagellates of the genus (DALYs) . The disPhlebotomus chinensis as the vector for transmission of L. infantum between humans and dogs. Recent epidemiological studies have shown that the prevalence of canine leishmaniasis in western China is generally very high with over 50\u00a0% of dogs infected with L. infantum [L. infantum to humans [VL is an important public health problem in China and is currently endemic in 61 counties in six provinces or autonomous regions in western China, including Xinjiang, Gansu, Sichuan, Shaanxi, Shanxi and Inner Mongolia [infantum . Eliminainfantum , 8. Diaginfantum \u201311. Ideno humans , is critL. infantum infections in dogs [Serological and parasitological tests have limitations in the diagnosis of canine leishmaniasis, especially in early infected and asymptomatic dogs , 14. On in dogs , 14\u201318. in dogs . This meL. infantum infection in dogs was developed and compared with a conventional PCR test using the same samples.Most molecular tests for diagnosis of leishmaniasis use blood, bone marrow, lymph node or skin samples . HoweverThis study was reviewed and approved by the Ethical Review Committee of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention in Shanghai. Oral informed consent was obtained from dog owners.Leishmania species were used in this study . Serum samples were stored at \u221220\u00a0\u00b0C until analyzed. Bone marrow aspirates of each dog were obtained using a sterile biopsy needle and smeared on three slides. After drying, the smears were fixed with methanol and later stained with Giemsa for microscopic examination.L. infantum, For this reason, no bone marrow samples were taken from these dogs in order to avoid unnecessary distress and pain for the animals.For negative controls, conjunctival swabs and serum samples were collected from 33 dogs living in the Zhang Xian County of Gansu province, a non-endemic area of ZVL. As this county is a high altitude area (>2500\u00a0m above sea level) with a yearly average temperature below 10\u00a0\u00b0C, sandflies do not normally occur and, therefore, dogs are usually not infected with et al. [DNA of cultured promastigotes was isolated using phenol/chloroform extraction method as described in Sch\u00f6nian et al. . DNA frohttp://primerexplorer.jp/e/) based on kinetoplast DNA (kDNA) minicircle sequence of the L. infantum isolate MCAN/CN/90/SC (Table\u00a0L. infantum specific LAMP assay was optimized for temperature and time using the strain MCAN/CN/90/SC. Different Leishmania species and strains (listed in Table\u00a0LAMP primers were designed using PrimerExplorer software (4)2SO4, 8\u00a0mM MgSO4, 0.1\u00a0% Triton X-100, 8 units of Bst DNA polymerase large fragment , 1\u00a0\u03bcl calcein, and 1\u00a0\u03bcl DNA sample. The reaction mixture was placed in a Loopamp Real-time Turbidimeter LA-320C and incubated at 64\u00a0\u00b0C for 60\u00a0min. The reaction was terminated by increasing the temperature to 80\u00a0\u00b0C. For confirmation, 3\u00a0\u03bcl of LAMP product was separated by electrophoresis on a 2\u00a0% agarose gel and visualized under UV light after staining with ethidium bromide (5\u00a0\u03bcg/ml).The LAMP assay was carried out in 25\u00a0\u03bcl of reaction mixture containing 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers, 1.4\u00a0mM of each deoxynucleotide triphosphate, 0.8\u00a0M betaine, 20\u00a0mM Tris\u2013HCl (pH\u00a08.8), 10\u00a0mM KCl, 10\u00a0mM isolated from promastigotes of L. infantum infection from Sichuan province served as positive controls. DNA samples extracted from conjunctival swabs from non-infected dogs were used as negative controls.When analyzing field samples, DNA extracted from conjunctival swabs from six dogs with symptomatic VL and confirmed Leishmania genus-specific primers (RV1 (sense): 5\u2019-CTTTTCTGGTCCCGCGGGTAGG-3\u2019; RV2 (antisense): 5\u2019-CCACCTGGCCTATTTTACACCA-3\u2019 [For PCR, CACCA-3\u2019 ) were usCACCA-3\u2019 . PCR proLeishmania amastigotes. Two investigators examined independently at least 2000 microscopic fields of each smear.Methanol-fixed bone marrow smears were stained with Giemsa and examined under 100\u00d7 oil-immersion lens for presence of L. infantum MCAN/CN/90/SC [An enzyme-linked immunosorbent assay (ELISA) was carried out as previously described using promastigote antigen isolated from cultured CN/90/SC .\u03c72-test (http://www.quantpsy.org/chisq/chisq.htm) was used for comparing LAMP, PCR, ELISA and microscopy results. A p value less than 0.05 was considered to be statistically significant.The L. infantum MCAN/CN/90/SC kDNA minicircle amplified the targeted sequences included in the study to determine whether the primers were highly specific for the L. infantum isolate of Chinese origin gave a negative result were analyzed by LAMP and PCR , ELISA (serum samples) and microscopy (bone marrow samples). Only 12 dogs were demonstrated to be positive by all four methods Table\u00a0. These iL. infantum using the LAMP assay, 58.6\u00a0% (65 dogs (63 in left eye and 61 in right eye)) using the PCR test, 40.5\u00a0% (45 dogs) using ELISA and 10.8\u00a0% (12 dogs) using microscopy . On the other hand, the sensitivity of the LAMP assay, the PCR test and the ELISA were all extremely significantly different from that of the microscopic examination . Both LAMP assay and PCR test were statistically significantly different from the ELISA .Of the 111 dogs examined, 61.3\u00a0% (68 dogs (66 in left eye and 65 in right eye)) tested positive for the presence of All of the 33 dogs from the Zhang Xian County tested negative in the ELISA a negative result was obtained. This finding indicates that the LAMP primers were only useful for the detection of Chinese L. infantum strains. Similar geographic restrictions have also been reported for PCR primers. For example, PCR primers amplifying a 204\u00a0bp fragment from L. donovani kDNA minicircles have been shown to be highly specific for L. donovani isolates of Indian origin but not for L. donovani strains from other parts of the world [L. infantum and not many is not a disadvantage per se, as MCAN/CN/90/SC is the only strain in China to infect dogs to cause canine VL. In addition it is very difficult to design universal LAMP primers that can amplify kDNA minicircles from different strains of L. infantum because there are only a few short sequence stretches of kDNA minicircles that are common between different Leishmania strains [The LAMP primers used in this study amplified a specific target sequence from kDNA minicircles of the he world . However strains \u201331.Leishmania spp. in dogs, molecular techniques have typically much greater sensitivity than parasitological or serological tests. The LAMP assay developed in this study had a detection limit of 1\u00a0fg of L. infantum DNA purified from cultured promastigotes; an amount that represents an equivalent of about 0.1 parasites. This high sensitivity was achieved by targeting kDNA minicircles, of which, thousands of copies are present in a trypanosomatid cell [L. infantum LAMP assay targeting the cysteine protease B multi copy gene [Accurate and rapid diagnosis of canine leishmaniasis is essential to reduce the number of infected dogs in endemic areas in order to prevent the transmission of the disease from the animal reservoir to humans. Although various methods are available for detection of tid cell . This deopy gene .Leishmania infections with high sensitivity and specificity when using blood samples [Leishmania infections in sick or subclinically infected dogs [LAMP assays have been previously shown to detect samples \u201336. Howe samples . In addited dogs , 38\u201340. L. infantum. That the LAMP method achieves higher positive detection rates than PCR tests is not uncommon and has been reported for other parasitic diseases [In the absence of a gold standard , thdiseases , 42.The observed true negative rate (specificity) of the newly developed LAMP assay (97\u00a0%) is comparable with the specificities of other LAMP assays designed for the detection of VL in blood samples . OnL. infantum in dogs was developed. As this test is an affordable and easily applied method, it is useful for epidemiological surveillance of L. infantum infections in dogs in order to control ZVL in endemic areas in western China.In summary, a simple and rapid non-invasive conjunctival swab LAMP assay for the sensitive and specific detection of"} +{"text": "Helicobacter pylori infection with peripheral blood neutrophil/lymphocyte ratio (NLR) and mean platelet volume (MPV).To determine the correlation of The NLR, MPV, platelets, leukocytes, neutrophils, and lymphocytes were calculated and the differences between groups were investigated.H. pylori-positive patients than in H. pylori-negative patients . There was no significant difference between H. pylori-negative patients and H. pylori-positive patients of severe intensity in terms of MPV. However, peripheral blood lymphocytes and platelets were statistically significantly higher in H. pylori-positive patients of severe intensity compared with H. pylori-negative patients.A total of 199 patients were included in the study. Neutrophil/lymphocyte ratio was statistically lower in H. pylori does not lead to a significant change in MPV as measured by hemogram; however, it gives rise to a statistically significant fall in NLR. Presence of severe H. pylori-positive intensity leads to a statistically significant increase in peripheral blood lymphocytes and platelets compared with H. pylori-negative patients.A moderate increase in the intensity of How to cite this article: Guclu M, Agan AF. Association of Severity of Helicobacter pylori Infection with Peripheral Blood Neutrophil to Lymphocyte Ratio and Mean Platelet Volume. Euroasian J Hepato-Gastroenterol 2017;7(1):11-16. H. pylori is the most common infectious disease in the world.1 More than half of the world\u2019s population has H. pylori colonization.2 It is a widespread microorganism encountered at a frequency of 80% in developing countries and 20 to 50% in Western societies.1H. pylori causes local inflammation in the stomach and systemic humoral immune response. The majority of the cases have an asymptomatic, chronic inflammation.3H. pylori infection, which has a high rate of morbidity, is recognized as a worldwide problem and the most frequent cause of chronic gastritis.4 The International Agency for Research on Cancer reported H. pylori as a class 1 carcinogen in humans in 1994.5H. pylori is inclined to settle irregularly in the gastric antrum as this is a less acidic medium. H. pylori attracts neutrophils and lymphocytes with several chemotactic proteins released in the stomach. Some substances secreted by mononuclear cells and neutrophils induce mucosal inflammation and thus cause gastritis. In conclusion, the gastric mucosa is infiltrated by neutrophils, macrophages, and lymphocytes in addition to several signal cytokines, and a subclinical systemic, low-grade inflammation occurs. H. pylori is particularly associated with severe gastric diseases like chronic gastritis, peptic ulcer, gastric lymphoma, and gastric cancer.23 In Japan, it was discovered that serum interleukin-6 (IL-6) was significantly high in patients with high serum anti-H. pylori levels.6 Interleukin-6 is a multifunctional cytokine secreted from numerous cells, including notably, mono-cytes, lymphocytes, mesangial cells, and endothelial cells. Some epidemiological studies have shown that there is a direct correlation between serum IL-6 level and severity of coronary artery disease.78 For this reason, they emphasized that the presence of H. pylori has a very important role in other extragastroenterologic diseases like cardiovascular diseases, and induces systemic inflammation due to elevation of IL-6. Due to chronic inflammation that it stimulates, H. pylori is able to create persistent antigenic stimulation, thus causing a systemic inflammatory reaction. On the contrary, the increase in C-reactive protein (CRP) is correlated with endothelial dysfunction, and it has been demonstrated that the presence of H. pylori infection elevates CRP in blood.9 C-reactive protein is a hepatic acute-phase reactant whose synthesis is regulated by IL-6.10H. pylori stimulates production of proinflam-matory cytokines like tumor necrosis factor (TNF), IL-1, IL-6, and IL-8.11 In persons infected with H. pylori, TNF, IL-1, IL-6, and IL-8 serum levels are found to be high.12 C-reactive protein production is influenced by hormones, such as cortisol, mainly ILs.13 Therefore, there is interaction and correlation between H. pylori and acute-phase reactants like CRP and ILs.H. pylori reacting with platelet glycoproteins, and, as a result, idiopathic thrombocy-topenic purpura (ITP) develops. In patients with ITP, platelet counts increase due to H. pylori eradication and autoantibodies disappear in majority of the sub-jects.14 Platelet count and functions play a key role in cardiovascular events. Mean platelet volume (MPV) is an index showing platelet volume. High MPV values show larger and very active platelets, which contribute to thrombocytic events. Some studies show that, in cases, such as diabetes mellitus and coronary artery disease, where low-grade inflammation exists, these patients have higher MPV values.15It is asserted that reduction in platelets occurs as a result of antibodies to 16 When evaluated with sepsis scores, such as Acute Physiology and Chronic Health Evaluation II (APACHE 2) and Sepsis-related Organ Failure Assessment (SOFA), this ratio was found to be consistent with the severity and prognosis of the disease, and was named as the neutrophil-lymphocyte stress factor. These NLR and MPV in peripheral blood are used as a parameter that provides information about the correlation between the inflammatory medium and physiological stress.As the physiological response of leukocytes in circulation to stress causes an increase in neutrophil count and a decrease in lymphocyte count, the proportion of these two subgroups to each other is used as a sensitive marker of inflammation. Changes occur in rates of leukocytes in circulation during the inflammatory response. Neutro-philia is accompanied by relative lymphopenia. Neutro-phil to lymphocyte ratio (NLR) is obtained from a simple hemogram and is an inflammatory marker of various diseases. Increased NLR was found to be an indicator of bad prognosis in patients undergoing cardiovascular intervention, and recently, it was shown that mortality rate increased in acute coronary syndromes with the elevation of NLR.This study was performed with the approval of the ethics board of Istanbul Medipol University Faculty of Medicine dated January 23, 2015 Nr.10840098-19 and 15. A total of 199 prospective patients, consistent with the study criteria, who applied to the Istanbul Medipol University, Gastroenterology polyclinic, during the period between January 2015 and September 2015 have been included in the study after having obtained their consent.17 Patients with atrophy and intestinal metaplasia according to Sydney classification were excluded and only patients with antral gastritis were included in the study. Patients with additional pathology other than antral gastritis were excluded. H. pylori charges were identified as mild, moderate, and severe H. pylori intensity. Patients with gastritis were split into four groups as H. pylori-negative and H. pylori-positive according to intensity . Hemogram tests were performed for all patients. The NLR, MPV, platelets, leukocytes, neutrophils, and lymphocytes were calculated by a simple hemogram test and the differences between these four groups were investigated; NLR was calculated by dividing absolute neutrophil count by absolute lymphocyte count as measured by hemogram.In this study, 199 patients with only dyspeptic complaints with no other disease, and with antral gastritis on gastroscopy were enrolled. Only from patients with antral gastritis, two biopsies were endoscopically obtained from the antrum, and all biopsies were evaluated histopatho-logically according to Sydney classification.Patients with (1) gastroscopy findings other than antral gastritis, (2) gastrointestinal hemorrhage, (3) gastric and duodenal ulcer, (4) portal hypertension, (5) diabetes mellitus, (6) systemic diseases and chronic diseases, and (7) nonsteroidal anti-inflammatory, proton pump inhibitors, and cytotoxic medicines were excluded from the study.H. pylori by Sydney classification.Mucosal samples of all patients were stained with hema-toxylin and eosin. Histopathologic parameters were determined according to Sydney system. Hematoxylin-eosin-stained sections were evaluated for the presence of Samples were collected from the antecubital vein into ethylenediaminetetraacetic acid (EDTA)-containing vacuum tubes (15% K3 EDTA 0.054 mL/4.5 mL blood) for hemogram and automated blood count, and the samples obtained were analyzed in 1 hour. Hemogram tests were performed in automatic complete blood count analyzer . Normal laboratory reference values were as follows: MPV: 8 to 12 fL, platelet: 150,000 to 450,000/\u03bcL, leukocyte: 3,500 to 11,000/\u03bcL, neutrophil: 1,800 to 7,920/\u03bcL, lymphocyte: 1,500 to 4,000/\u03bcL.H. pylori patients. Statistical analysis included parametric tests, nonparametric tests of comparison, and one-way analysis of variance. Results were presented as mean \u00b1 standard deviation (SD) and a value of p < 0.05 was considered as statistically significant.The patients who complied with the study criteria and applied to the Istanbul Medipol University Gastroen-terology Polyclinic during the period between January 2015 and September 2015 were consecutively admitted into the study. A total of 199 prospective patients were included in the study after having obtained their consent. This is a prospective and single-center study. Statistical Package for the Social Sciences (SPSS) for Windows 21.0 version was used for statistical study and analysis of data. The differences between the groups were compared using chi-square and Student\u2019s t-test. Subgroup analyses were made among the mild, moderate, and severe H. pylori-positive patients with a mean age of 48 \u00b1 17 years, comprising 54 females and 47 males, and a total of 98 H. pylori-negative patients with a mean age of 45 \u00b1 16 years, comprising 57 females and 41 males, were enrolled in the study. No significant difference was identified between the groups in terms of mean age and gender. The distribution of this parameter, in which 199 patients enrolled in the study of H. pylori-negative and H. pylori-positive patients are compared, is presented in A total 199 patients, 88 males and 111 females, who had only antral gastritis on endoscopy, were included in the study. A total of 101 H. pylori-negative patients and H. pylori-positive patients. There were no differences in terms of NLR and MPV between H. pylori-negative patients and 57 H. pylori-positive patients of mild intensity. There was no difference between H. pylori-negative patients and 26 H. pylori-positive patients of moderate intensity in terms of MPV , whereas NLR was statistically lower in H. pylori-positive patients than in H. pylori-negative patients . While there was no significant difference between H. pylori-negative patients and 18 H. pylori-positive patients of severe intensity in terms of MPV and NLR, peripheral blood lymphocytes were statistically significantly higher in H. pylori-positive patients of severe intensity . There was no statistically significant difference between the groups in terms of peripheral blood leukocytes and neutrophil counts; other parameters tested in hemogram and compared. No significant difference was noted between the groups in terms of MPV. However, in the comparison of these four groups, statistically higher platelet counts were found in H. pylori-positive patients of severe intensity than in H. pylori-negative patients on hemogram (p = 0.02).There were no differences in terms of NLR and MPV between total H. pylori-negative patients and 57 H. pylori-positive patients of mild intensity according to Sydney classification are compared, is shown in The distribution of parameters, in which 98 H. pylori-negative patients and 26 H. pylori-positive patients of moderate intensity according to Sydney classification are compared, is shown in The distribution of parameters, in which 98 H. pylori-negative patients and 18 H. pylori-positive patients of severe intensity according to Sydney classification are compared, is shown in The distribution of parameters, in which 98 H. pylori is a microorganism causing gastritis and gastric functional changes virtually in all infected persons; peptic ulcer in 15 to 20%, ulcer complication in 2 to 12%, and serious gastric diseases like stomach cancer in 1 to 3%, and B-cell lymphoma (mucosa associated lymphoid tissue lymphoma), a primary gastric lymphoma, in 0.1% of infected persons.1 The results of a meta-analysis on three prospective epidemiologic studies have shown that H. pylori-positive patients are exposed to four times higher cancer development risk than normal patients, and the recent decline in antrum and corpus cancer is parallel to the decline in the prevalence of 5H. pylori. In a study, sedimentation fibrinogen and CRP, acute-phase reactants, were found to be high in H. pylori-positive patients, and a significant relationship was shown between H. pylori and systemic inflammatory response.18 The purpose of our study was to determine the changes of H. pylori, the most common infectious agent in the world and recognized as a carcinogen, on automated hemogram test, a simple blood test. A simple, inexpensive, and quickly concluded hemogram does not only give us values, such as hemoglobin and leukocyte but also MPV, absolute lymphocyte, and absolute neutrophil values. Moreover, NLR is a value calculated by dividing absolute neutrophil count by absolute lymphocyte count. Although studies related to platelet and MPV were present as they are closely related to the cardiovascular system, H. pylori causes a chronic inflammation in the gastric mucosa that prevails for years, though in varying severity, and chronic infection depends upon several factors, including bacterial resistance and host response.19H. pylori-positive patients and H. pylori-negative patients, we could not find any significant difference between them in our study in terms of NLR and MPV values. However, Farah and Khamisy-Farah20 found in their 2014 study that the H. pylori-positive patients had higher NLR values than H. pylori-negative patients. They also identified a parallel increase between the severity of the H. pylori gastritis and NLR values.20 In another study conducted by Jafarzadeh et al,21 it was established that the H. pylori-positive patients, independent of the bacterial cytotoxin-associated gene A (CagA) status and regardless of peptic ulcer, had more leukocyte, neutrophil, and NLR ratios in their blood than that of the H. pylori-negative patients. It is interesting that, in the subgroup analyses of our study, we have found lymphocyte and thrombocyte values of the H. pylori-positive patients within the normal range but significantly higher than that of the H. pylori-negative patients. Such an increase in the absolute lymphocyte values has probably avoided the finding of a higher NLR ratio. Having a limited number of severe H. pylori gastritis cases in our study has a restrictive effect on our study in that particular respect. In this respect, more studies should be conducted with higher number of severe H. pylori gastritis patients.Comparing the H. pylori increased, and this finding appears to support the study carried out by Karttunen et al.22 There is a very tight correlation between H. pylori count in the mucosa and severity of tissue inflammation.23 As there is no organized lymphoid tissue in the gastric mucosa, any increase in cell count associated with H. pylori bacteria at this site will undoubtedly be correlated with peripheral blood cells. Since H. pylori is not an invasive microorganism and causes low chronic antigenic stimulation, we could not determine NLR values in H. pylori-positive patients; however, mean NLR value was higher in H. pylori-positive patients than in H. pylori-negative patients, but was not statistically significant. We found that the reason for absence of significantly high NLR, as expected in H. pylori-positive patients, was the fact that absolute lymphocyte counts increased as the intensity of H. pylori increased and that NLR value did not rise. The increase in absolute lymphoid count in peripheral blood must be evaluated as a very important finding.In our study, we found that absolute lymphocyte count in peripheral blood increased significantly as intensity of 24 Furthermore, the correlation between increases in MPV and myocardial infarction and mortality has been shown.15 The intensity of systemic inflammation determines size and volume of platelets. Some epidemiologic studies have shown the correlation between H. pylori and coronary artery disease.15 In a study that we previously carried out in relation to MPV in the past, we found statistically lower MPV values in ulcerative colitis (UC), and construed that lower MPV values might reflect UC disease activity.25 In this study, we did not find any change in MPV in H. pylori-positive patients, and this may be due to absence of a significant inflammatory effect like active UC and presence of a chronic low-grade inflammatory event. In light of this information, there is no consensus in the literature about MPV and inflammation. There are studies on ITP in H. pylori-positive patients, and a decline in platelets is expected in H. pylori-positive patients. There are studies performed with 9.25 and 9.70 fL for MPV, indicating that it predicts cardiovascular disease.26 In our study, only patients with antral gastritis on endoscopy were divided into four groups as H. pylori-negative and mild, moderate, and severe H. pylori-positive according to intensity, and no significant difference was found between these four groups in terms of MPV. This is consistent with the previous studies in the literature.14 However, in the comparison of these four groups, statistically higher platelet counts were found in severe H. pylori-positive patients than in H. pylori-negative patients on hemogram. This finding strengthens the correlation between cardiovascular, notably coronary artery disease, and presence of H. pylori infection.Mean platelet volume can be obtained from hemo-gram, which is a routine and easy-to-obtain indicator showing systemic inflammation. High MPV values show larger and active platelets that contribute to thrombocytic events. The MPV is a marker showing platelet volume and can be easily determined by hemogram. Larger platelets mean much more active platelets, both metabolically and enzymatically, and have been shown to contribute to proinflammatory and prothrombotic situation.H. pylori-negative patients and H. pylori-positive patients. A moderate increase in the intensity of H. pylori does not lead to a significant change in MPV measured by hemogram; however, it gives rise to a statistically significant fall in NLR, which, we estimate, is due to the increase in absolute lymphocyte count on hemogram. Presence of severe H. pylori-positive intensity leads to a statistically significant increase in peripheral blood lymphocytes and platelets compared with H. pylori-negative patients.There were no differences in terms of NLR and MPV between total"} +{"text": "Scientific Reports7: Article number: 4055910.1038/srep40559; published online: 01132017; updated: 03142017The original version of this Article contained an error in the spelling of the author Xiaoxing Xi, which was incorrectly given as Xiaoxing X. Xi.In addition, the Article contained a typographical error in the Results section. The subheading,\u201cX-ray photoemission spectromicroscopy and\\mu-ARPES\u201dnow reads:\u201cX-ray photoemission spectromicroscopy and \u03bc -ARPES\u201d.These errors have now been corrected in the PDF and HTML versions of the Article."} +{"text": "P\u2009<\u20090.001) repression of a full-length CASP3 reporter. Further, three EBV miRNAs, including BART22, exhibited repression of endogenous CASP3 protein. These data confirm that CASP3 is a direct target of specific EBV BART miRNAs.The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that transforms B cells and causes several malignancies including Burkitt\u2019s lymphoma. EBV differentially expresses at least 49 mature microRNAs (miRNAs) during latency in various infected epithelial and B cells. Recent high-throughput studies and functional assays have begun to reveal the function of the EBV miRNAs suggesting roles in latency, cell cycle control, and apoptosis. In particular, the central executioner of apoptosis, Caspase 3 (CASP3), was proposed as a target of select EBV miRNAs. However, whether CASP3 is truly a target of EBV miRNAs, and if so, which specific miRNAs target CASP3 is still under debate. Based on previously published high-throughput biochemical data and a bioinformatic analysis of the entire CASP3 3\u2032-UTR, we identified 12 EBV miRNAs that have one or more seed binding sites in the CASP3 3\u2032-UTR. We individually tested all 12 miRNAs for repression of CASP3 in luciferase reporter assays, and nine showed statistically significant (The online version of this article (doi:10.1186/s12985-016-0602-7) contains supplementary material, which is available to authorized users. Epstein-Barr virus persists in more than 95 % of the adult human population. Initial EBV infection is either asymptomatic or manifests as self-limiting mononucleosis. However, in a subset of cases, EBV is also associated with the oncogenic transformation of cells, resulting in malignancies including Burkitt\u2019s lymphoma (BL) and several epithelial cell cancers (reviewed in ). After BamHI A rightward transcripts; BARTs1-22) [EBV precursor miRNAs arise from two genomic clusters: three surrounding the BHRF1 gene , and 22 within the BART transcripts (RTs1-22) . Most huRTs1-22) . Key to RTs1-22) ; Table\u00a01RTs1-22) . DiffereRTs1-22) . BecauseRTs1-22) , 17, 18.Three types of studies have demonstrated the important and complex role of EBV miRNAs in apoptosis. Phenotypic experiments implicated the BHRF1 miRNAs in apoptotic inhibition and promotion of the cell cycle during the early phase of infection of human primary B cells , 19. ExpPerhaps the most compelling mechanism for EBV miRNA inhibition of apoptosis is the potential repression of Caspase 3 (CASP3), the central proteolytic executioner of apoptosis , first iAn individual study of each EBV miRNA with a potential binding site in CASP3 has not yet been undertaken, so it is unclear if single EBV miRNAs directly target CASP3. If single miRNAs are used as therapeutic targets, it is important to identify the unique targets/function of each. We employed systematic testing with standard reporter assays and Western blots to address our initial observation that CASP3 is a target of multiple EBV BART miRNAs and clarhttps://genome.ucsc.edu) was used to generate an image from our BED file to overlay with the 3\u2032-UTR of CASP3 experiment identified CASP3 as a putative target of one or more EBV miRNAs, but we had not examined the CASP3 3\u2032-UTR in single nucleotide resolution . High-coSP3 Fig.\u00a0. The CASSP3 Fig.\u00a0, Table\u00a01The full length 3\u2032-UTR of CASP3 was PCR amplified from genomic DNA and inserted downstream of firefly luciferase between NheI and XhoI in the pmiRGLO dual luciferase vector in 24-well plates were co-transfected with 40 nM synthetic miRNA duplex and 2.5 ng pmiRGLO reporter plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer\u2019s instructions. Cells were lysed 24 h post-transfection, and the Dual-Luciferase Assay Reporter System (Promega) manufacturer\u2019s protocol was performed with the GloMax Junior Luminometer (Promega). Firefly luciferase activity was normalized to the control Renilla luciferase activity in each well, and this ratio was then normalized to the control construct appropriate for each experiment. At least four independent transfections for each condition were measured. Excel was used to calculate the standard deviation for each data point, and two-tailed Student\u2019s HEK293T cells were transfected in triplicate with the denoted synthetic miRNAs (40 nM) in the figures using Lipofectamine 2000 (Invitrogen). Protein lysates were prepared from cells, and Western blot analyses were performed as described using NuPAGE 4-12 % gels and associated buffers (Invitrogen). Primary antibodies were anti-Caspase 3 and anti-alpha-tubulin #2125 . Western blots were quantified using ImageQuant software, with CASP3 levels normalized to the tubulin loading controls and these values normalized to the control transfection condition.Argonaute HITS-CLIP experiments yield genome-wide maps of likely Argonaute and miRNA binding. Our previous HITS-CLIP data revealed potential interactions between EBV miRNAs and 132 host apoptotic mRNAs, including CASP3 . We examIn order to validate the bioinformatic and HITS-CLIP predicted interactions, the full-length 3\u2032-UTR of CASP3 was cloned into a dual-luciferase reporter vector and co-transfected into HEK293T cells with individual synthetic miRNAs. Significant repression of the full-length CASP3 3\u2032-UTR reporter occurred after transfection of synthetic BART 4, 3, 13, 8, 7, 18, 1, 2, or 22 Fig.\u00a0. BART22 The specificity of downregulation by CASP3 at the each of the most statistically significant and repressed (>20 %) binding sites was further confirmed by comparing wild-type 3\u2032-UTR repression to the repression of a corresponding reporter with 2-3 point mutations in each seed binding region Fig.\u00a0, Table\u00a01Given that these miRNAs significantly repress protein made from a reporter construct, we hypothesized that we might also see an effect on endogenous levels of CASP3 protein. Thus, we transfected individual synthetic EBV miRNAs into HEK293T cells, which lack endogenous EBV miRNAs, to confirm downregulation of endogenous CASP3 protein by Western blotting. CASP3 was downregulated most significantly by BART22 but also weakly by BARTs 8, 7, 1, and 2 recently conducted a reporter assay experiment similar to ours but only tested five miRNAs that had exerted an anti-apoptotic phenotype when expressed alone in their epithelial (gastric carcinoma) cell model . The authors concluded that none of these miRNAs significantly repressed their CASP3 luciferase reporter in HEK293T cells. This conclusion may be explained by two key differences in our experimental designs: (1) the differing identity of our negative controls, and (2) the origin of our miRNAs. Cognizant that ectopic expression/transfection of both miRNAs and luciferase reporters skews the natural stoichiometry within a cell , and synThe BART miRNAs are expressed together in large clusters and likely operate in tandem , 19, 33.A previous study showed downregulation of CASP3 by BARTs 1-3p and 16 . While oKaposi\u2019s sarcoma herpesvirus (KSHV), a human gamma-herpesvirus responsible for primary effusion lymphoma (PEL) and Kaposi\u2019s sarcoma (KS), also expresses miRNAs that repress apoptotic targets including CASP3 , 38. ThoA decrease in apoptotic activity of EBV-positive cells could promote malignancy in conjunction with the deactivation of other important cellular pathways by EBV miRNAs. The 100+ other apoptotic transcripts with putative EBV miRNA binding sites should be studied to elucidate the mechanism by which EBV controls host cell apoptosis and further our understanding of how the virus causes malignancy. While the EBV BART miRNAs work in tandem to repress CASP3, therapeutically targeting a large number of miRNAs is likely more challenging and may have more off-target consequences. Knowing that single miRNAs can repress a key target may help us better conduct future rational drug design regarding EBV and BL."} +{"text": "Smoking during pregnancy has adverse health consequences for the mother and fetus. E-cigarettes could aid with smoking cessation but there is limited research on the prevalence and patterns of e-cigarette use, and their association with smoking cessation among pregnant smokers.n\u2009=\u2009428). Outcomes assessed were trajectories of e-cigarettes use from baseline to one-month follow-up, and longitudinal association between e-cigarette use at baseline and smoking cessation at one-month follow-up.We conducted a secondary analysis of a randomized controlled trial of a text-messaging program for smoking cessation among a U.S. national cohort of pregnant smokers pregnant smokers used e-cigarettes in the past 30\u2009days and 36 (8.41%) used e-cigarettes in the past 7\u2009days. The primary reason\u00a0stated for using e-cigarettes during pregnancy was for quitting. E-cigarette use between baseline and 1-month was inconsistent. Of 36 dual-users at baseline, 20 (55.56%) stopped using e-cigarettes by the\u00a01-month follow-up and 14 initiated e-cigarette use. There was no evidence of an association between e-cigarette use at baseline and the\u00a0primary smoking cessation outcome, 7-day point prevalence abstinence .A secondary analysis of a national sample of pregnant smokers indicates that use of e-cigarettes is inconsistent and is not associated with improved smoking cessation outcomes. There is an urgent need to further examine the risk and benefits of e-cigarette use, especially during pregnancy.The online version of this article (10.1186/s12889-019-7299-7) contains supplementary material, which is available to authorized users. Electronic cigarettes (e-cigarettes), battery-operated devices that vaporize nicotine and produce an inhaled aerosol, have burgeoned into a billion-dollar industry in the United States (U.S.) with over $10 billion (US dollars) in revenues by 2017 , and havDue to the high urgency to quit cigarette smoking during pregnancy and the potential efficacy of e-cigarettes as a smoking cessation device \u201312, pregThere is a dearth of research conducted to date focusing on the effects of e-cigarette use among pregnant women and few estimates exist on the prevalence of e-cigarette use among pregnant women. A recent systematic review found that the prevalence of e-cigarette use during pregnancy ranges from 0.6 to 15% . A 2017 Given the potential link between nicotine exposure and adverse fetal and pregnancy outcomes , 27, undN\u2009=\u2009508) were recruited from enrollees in Text4baby, the largest text messaging service for pregnant women and mothers in the United States. As such, all participants were receiving Text4baby text messages at the time of enrollment. Some participants were randomized to also receive Quit4baby [The sample for this analysis comes from the Quit4baby trial, a randomized controlled trial of text messaging for smoking cessation in pregnant women in the U.S. Recruitment for the study occurred between August, 2015 and February, 2016 , 29. Paruit4baby . This stn\u2009=\u2009428 participants including 4 minors; 13 were removed because they were no longer pregnant and 67 were removed because they did not complete the 1-month survey).To be eligible, participants had to have a cell phone for their personal use, be willing to receive text messages on their phone, be 14\u2009years old or older, be pregnant at the time of enrollment and report having smoked at least one puff of cigarette in the past 2\u2009weeks at the time of assessment . Per IRBn\u2009=\u20091). Secondary smoking outcomes included self-reported attempt to quit for longer than 1\u2009day and changes in average cigarettes smoked per day (CPD) between 1-month and baseline. Changes in CPD was calculated by subtracting CPD at 1\u2009month from CPD at baseline.The primary cigarette smoking outcome for these analyses was self-reported 7-day point prevalence abstinence (PPA) at 1-month follow-up using the question, \u201cHave you smoked a cigarette, even a puff, in the last 7 days?\u201d , body mass index (BMI), race/ethnicity, employment, education, health insurance type, marital status, income, geographic region and intervention allocation . Smoking variables included CPD at baseline, the Fagerstrom Test for Cigarette Dependence (FTCD) , 31 and Analyses for this study were conducted using SAS 9.4. Fisher Exact and Mann-Whitney U tests were used to compare groups of interest \u2013 cigarette/e-cigarette dual-users and cigarette-only users, with respect to baseline demographic and smoking characteristics of the sample. To assess the association between the use of e-cigarettes at baseline and smoking cessation outcomes at 1-month, three logistic and linear regression models were constructed. Model 1 was an unadjusted bivariate logistic/linear regression. Model 2 was a multivariable logistic/linear regression that controlled for intervention allocation. Despite no differences in e-cigarette use observed between groups for intervention allocation, Model 2 was constructed because the intervention was shown to have positive smoking cessation outcomes . Model 3n\u2009=\u2009428) was on average 26.39\u2009years old (Standard Deviation (SD)\u2009=\u20095.80), 18.07\u2009weeks pregnant (SD\u2009=\u20097.75) and had an average BMI of 27.73 (SD\u2009=\u20097.77). They were primarily non-Hispanic White (63.00%), unemployed (67.53%), with Medicaid or Medicare insurance (80.52%), with a high school degree or less (60.28%) and from the south (55.61%). At baseline, the sample smoked an average of 7.50 cigarettes per day (SD\u2009=\u20096.32), had an average FTCD score of 2.85 (SD\u2009=\u20092.28), and had a motivation to quit score of 6.04 out of 7 (SD\u2009=\u20091.31). There were no significant differences in demographic and smoking characteristics between participants lost to follow up and participants who completed the 1-month follow-up.Table\u00a0n\u2009=\u200936) reported using e-cigarettes in the past 7\u2009days and 17.29% (n\u2009=\u200974) reported using e-cigarettes in the past 30\u2009days. At 1\u2009month, 7.00% of participants (n\u2009=\u200930) reported using e-cigarette in the past 7\u2009days and 11.92% (n\u2009=\u200951) reported using e-cigarettes in the past 30\u2009days. No differences in demographic variables were observed between e-cigarette dual users (past 7-day) and cigarette-only users except race/ethnicity, where a greater percentage of dual users were non-Hispanic White . There was no significant difference in the proportion of e-cigarette dual users between the 67 participants lost to follow up (12.9%) and participants who completed the 1-month follow-up (8.41%), p\u2009>\u20090.05.At baseline, 8.41% of participants (n\u2009=\u200929) of women reported that they were using e-cigarettes to \u201chelp me quit\u201d, followed by \u201cthey are safer for me than regular cigarettes\u201d (41.67% or 15/36), \u201ce-cigarette tastes good and does not smell\u201d (38.89% or 14/36) and \u201csafer for my baby than regular cigarettes\u201d (36.11% or 13/36). A small number also reported they were using e-cigarettes due to \u201ccost\u201d (13.89% or 5/36) or because \u201cfriends and family use them\u201d (5.56% or 2/36).When asked about reasons for using e-cigarettes at baseline, 80.56% reporting 7-day PPA abstinence at 1-month, followed by \u201cstopped users\u201d (25.00% or 5/20), \u201ccontinued users\u201d (12.50% or 2/16) and \u201cnew users\u201d (7.14% or 1/14).ns). Additionally, no differences were observed between use of e-cigarette and attempt to quit for more than 1\u2009day and differences in CPD . In the multivariable models (Models 2 and 3), the association between e-cigarette usage and smoking outcomes were not statistically significant. A sensitivity analysis that included the 67 participants lost to follow up (coded as smokers) yielded similar results for the association between e-cigarette use at baseline and 7-day PPA at 1-month follow-up.Table\u00a0The current study is among the first to examine the use of e-cigarettes among pregnant smokers prospectively. In this national sample of pregnant smokers recruited in 2015 and 2016, 8.41% of pregnant cigarette smokers were found to also be concurrent e-cigarette users. E-cigarette users were found to be similar to non-users with the exception of being more likely to be of white ethnicity. More than half of those who used e-cigarettes at baseline had stopped using by 1-month follow-up. After adjusting for intervention assignment and demographic factors, no association was observed between e-cigarette use and cigarette smoking related outcomes.The most prevalent reasons for using e-cigarette in our sample were to \u201chelp me quit smoking\u201d, followed by \u201ce-cigarettes are safer for me compared to regular cigarettes.\u201d This is similar to findings from previous studies that examined perceptions of e-cigarette use among pregnant women , 24. GivSimilar to other U.S. based studies , 14, 24,Although e-cigarettes could benefit adult smokers if used as a complete substitute for combustible tobacco smoking \u201312, evidA strength of this study is the use of a large, national sample and therefore the findings can be generalized to pregnant women in the U.S. Our participants\u2019 demographic characteristics \u2013 based on their predominantly White ethnicity, on public insurance (i.e. Medicaid), and having a household income of $30,000 or less per year- were found to match those of previous national samples of pregnant smokers in the U.S. .Several limitations should be considered in this study. This study is a secondary analysis from an intervention study on quitting smoking. All participants were receiving health related text messages and therefore it is likely that the participants studied are more health-oriented than the general population of pregnant smokers. Additionally, as the number of e-cigarette users in our sample was relatively small, estimates may be unreliable. As with all self-reported measures, social desirability bias could be a limitation. Future studies may benefit from more targeted outreach of cigarette and e-cigarette dual users to examine the association between e-cigarette use and tobacco cigarette cessation. This study also did not collect information about the extent of e-cigarette use, frequency and duration of use, and the concentration of nicotine fluid in e-cigarettes. This information is critical to accurately classify participants as dual users as well as to have a comprehensive understanding of the safety and efficacy of e-cigarettes as a smoking cessation aid for this particular population. As a result, these findings should be interpreted with caution. Future research with appropriate measures is needed to further determine whether and how e-cigarettes can be an effective tobacco cigarette cessation or reduction aid for pregnant smokers.These data are among the first to provide estimates for the prevalence of e-cigarette use during pregnancy, their trajectory of use and their association with smoking cessation for pregnant smokers. This secondary analysis of a national trial of pregnant smokers provides some indication that use of e-cigarettes to quit smoking may be common in pregnant smokers but found no association with improved smoking cessation outcomes. There is an urgent need to further examine the risk and benefits of e-cigarette use, especially during pregnancy.Additional file 1:Questionnaires. Baseline and 1-month follow-up questionnaires. (PDF 144 kb)"} +{"text": "Little is known about the risk and addiction perceptions of e-cigarettes among Asian populations. We examined e-cigarette perceptions among young adults in Hong Kong and the association between the perceptions and e-cigarette use patterns.N\u2009=\u20091186). Measures of e-cigarette perceptions included perceived harm and addictiveness of e-cigarettes, perceived harm of secondhand e-cigarette aerosol, and perceived popularity of e-cigarette use among peers. Separate multinomial logistic regression models were conducted to examine the associations between the four perceptions and former and current use of e-cigarettes relative to never use, controlling for demographics and current cigarette smoking status. Interactions of e-cigarette perceptions and current cigarette smoking were assessed in all models. Among current e-cigarette users, bivariate exact logistic regression models were used to examine the relationships between each of the perceptions and frequent e-cigarette use (\u22653\u2009days in past 30-day vs. 1\u20132\u2009days). Among participants who had never used e-cigarettes, separate multivariable logistic regression models were conducted to examine the associations between e-cigarette perceptions and susceptibility to e-cigarette use.An online survey was administered to a convenience sample of Hong Kong residents aged 18\u201335 . Young adults perceived e-cigarettes (and aerosol) as less harmful, less addictive, and less popular than cigarettes. Current cigarette smokers reported significantly lower perceived harmfulness and addictiveness of e-cigarettes, lower perceived harmfulness of e-cigarette aerosol, and higher perceived popularity than nonsmokers. The lower degree of harm and addiction perceptions, and higher levels of popularity perceptions were associated with greater odds of e-cigarette use, and these relationships were generally stronger among nonsmokers compared to current cigarette smokers. E-cigarette perceptions were not associated with frequent e-cigarette use. Perceiving e-cigarettes (and aerosol) as less harmful and less addictive were associated with greater susceptibility to e-cigarette use. Compared to nonsmokers, current smokers were more likely to report e-cigarette use and susceptibility.Continued monitoring of e-cigarette use and perceptions is needed. Educational programs should emphasize the potential harmful and addictive properties of e-cigarettes and the risks of secondhand exposure to e-cigarette aerosol.The online version of this article (10.1186/s12889-019-7464-z) contains supplementary material, which is available to authorized users. E-cigarette use has surged in popularity worldwide, particularly among young people , 2. In tIt is well established that the low degree of perceived harm of e-cigarettes is associated with e-cigarette use. According to the Population Assessment of Tobacco and Health (PATH), the top reasons for e-cigarette use among US adults center around perceptions that e-cigarettes are less harmful than cigarettes to users and other people . In MalaThe low addiction perception of e-cigarettes and its relationship with e-cigarette use have also been documented in the literature. In a nationally representative sample of US adults, Wiseman et al. found thResearch on social norms around e-cigarette use is nascent and findings have been mixed. Measures of social norms have focused on either descriptive norms or injunctive norms . Texan aUnderstanding young adults\u2019 perceptions of e-cigarettes can help understand the mechanisms through which young people become interested in e-cigarettes. It can help inform regulatory policies on e-cigarettes and educational interventions that address e-cigarette use. In Hong Kong, e-cigarettes entered the marketplace around 2008. Awareness of e-cigarettes is high, with 84% of residents reporting having heard of e-cigarettes in 2015 . HoweverPublished data on perceptions of e-cigarettes among Asian populations is limited. How young adults in Asian countries think about e-cigarettes, and how their perceptions affect e-cigarette use behaviors remain unknown. Furthermore, there is a paucity of research on the link between e-cigarette perceptions and the frequency and susceptibility of e-cigarette use. This study examined the perceptions of e-cigarettes among young adults in Hong Kong, and the association between these perceptions and e-cigarette use behaviors .We collected data through an online survey , POP sent an invitation email to a panel of 5237 Hong Kong residents who were recruited for POP\u2019s previous and other ongoing research projects. The invitation email provided information on study purpose and eligibility criteria , and included a link to an online consent form. People who confirmed their eligibility and completed the online consent form were directed to a 22-item online survey that was developed for this study. Participants could choose survey language based on their preference . As an incentive for participation, the first 200 respondents who submitted a completed survey received a HK$50 gift card . A total of 1049 surveys were received, yielding a crude response rate of 20% in Stage 1.In Stage 2 (December 2016\u2013March 2017), POP added one eligibility criterion and sent the invitation email to another 37,050 Hong Kong residents, and 315 current cigarette smokers completed the survey, yielding a crude response rate of 0.9% in Stage 2. We were not able to determine the number of young adult current smokers, the true denominator for the response rate. Therefore the adjusted response rate was unknown. As an incentive, the first 50 respondents who completed the survey in Stage 2 received a HK$50 gift card.In total, 1364 surveys were received. We excluded 178 surveys due to incomplete submissions (with more than 50% missing items) and missing cigarette and e-cigarette use questions. Thus, 1186 responses were included in the analysis. The study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 16\u2013349).Demographics included age, gender, education, and place of birth. Awareness of e-cigarettes was assessed by asking, \u201cHave you ever heard of electronic cigarettes prior to the survey?\u201d with a brief description of e-cigarettes and a picture showing different types of e-cigarettes (yes/no).E-cigarette use was derived from two items assessing ever use and current use . Participants were categorized as \u201cnever users\u201d, \u201cformer users\u201d (ever use but no current use), and \u201ccurrent users\u201d (ever and current use). Following the previous research on e-cigarettes among youth , 26, curMeasures on e-cigarette perceptions were adapted from PATH surveys. Three items assessed harm and addiction perceptions . One item assessed the descriptive norms by asking \u201cHow popular among your peers do you think each of the following tobacco products is?\u201d For each item, participants were asked to rate on a 0\u201310 visual analog scale for e-cigarette, cigarette, and waterpipe, respectively . Participants could choose \u201cDon\u2019t know\u201d as an answer option for each item.Susceptibility to e-cigarette use was measured by two items with response options of \u201cdefinitely yes\u201d, \u201cprobably yes\u201d, \u201cprobably no\u201d, \u201cdefinitely no\u201d, and \u201cDon\u2019t know\u201d. Participants who answered \u201cdefinitely no\u201d to both items were considered unsusceptible to e-cigarette use, and those who answered otherwise were considered susceptible.Stata version 14.2 was used for data analyses. Sample characteristics were summarized using descriptive statistics. Fisher\u2019s exact tests and t-tests (for continuous variables) were conducted to compare e-cigarette awareness, perceptions, and use patterns by current cigarette smoking status. Among respondents who were aware of e-cigarettes, paired sample t-tests were used to compare perceptions of e-cigarettes and cigarettes. Separate multinomial logistic regression models were conducted to examine whether the four perceptions of e-cigarettes were associated with former and current e-cigarette use (relative to never use), controlling for age, gender, education, place of birth, and current cigarette smoking status. Interactions between e-cigarette perceptions and current cigarette smoking status were tested in all models. When the interaction was significant, we reported estimates by current cigarette smoking status; when the interaction was not significant, we reported the overall estimates only.Next, subgroup analyses were conducted. Among current e-cigarette users, bivariate exact logistic regression models were used to assess the association between each of the four perceptions and frequent e-cigarette use by current cigarette smoking status. Among e-cigarette never users who were aware of e-cigarettes, separate multivariable logistic regression models were conducted to examine the association between e-cigarette perceptions and susceptibility to e-cigarette use, controlling for demographics and current cigarette smoking. Interactions between e-cigarette perceptions and current cigarette smoking were tested in all of the models.p\u2009=\u2009.051). About 16.1% of respondents reported having used e-cigarettes, including 11.3% former users and 4.8% current users. E-cigarette use was more prevalent among current cigarette smokers than nonsmokers . Current e-cigarette use was more prevalent in males than females, although the difference was not significant . But no difference was detected in former e-cigarette use by gender .The respondents included 811 nonsmokers and 375 current cigarette smokers (Table\u00a0p\u2009=\u2009.97). Among e-cigarette never users, 34.0% were susceptible to e-cigarette use, and the susceptibility among current cigarette smokers was 3.5-fold that for nonsmokers .Among current e-cigarette users, 36.8% reported frequent e-cigarette use. There was no difference in frequent e-cigarette use between current cigarette smokers and nonsmokers ; less addictive with 7.4% \u201cDon\u2019t know\u201d ; and less popular with 5.3% \u201cDon\u2019t know\u201d . E-cigarette aerosol was perceived as less harmful than secondhand cigarette smoke with 7.7% reporting \u201cDon\u2019t know\u201d . Compared to nonsmokers, current cigarette smokers reported significantly lower perceived harm of e-cigarettes and aerosol, lower perceived addictiveness of e-cigarettes, and higher perceived popularity of e-cigarette use among their peers (p\u2009<\u2009.001).E-cigarettes were perceived as less harmful than cigarettes with 4.1% reporting \u201cDon\u2019t know\u201d than current cigarette smokers . Similarly, respondents who perceived more harm from e-cigarette aerosol and greater addictiveness of e-cigarettes had decreased odds of e-cigarette use, and these relationships were stronger among nonsmokers than current cigarette smokers. Respondents who perceived higher popularity of e-cigarettes had increased odds of e-cigarette use, and the relationship was similar for current cigarette smokers and nonsmokers. In all of the models with combined smokers and nonsmokers, current cigarette smokers had increased odds of former and current e-cigarette use than nonsmokers . Gender was not related to e-cigarettes use (p\u2009=\u2009.14\u2013.74).Respondents who perceived more harm from e-cigarettes had decreased odds of both former and current use of e-cigarettes, relative to never use Table\u00a0, and thip\u2009=\u2009.03). Current cigarette smoking status was unrelated to frequent e-cigarette use .In general, e-cigarette perceptions were not related to frequent e-cigarette use Table\u00a0. But amop\u2009<\u2009.001 in all models).Among e-cigarette never users, those who perceived more harm from e-cigarettes and aerosol and greater addictiveness of e-cigarettes had lower odds of e-cigarette susceptibility Table\u00a0, and theThis is the first study to comprehensively assess e-cigarette perceptions in a sample of Asian young adults. The majority (97.2%) of young adults were aware of e-cigarettes. E-cigarette use was more prevalent in our sample than the general adult population in Hong Kong. It might be that we oversampled current cigarette smokers and our sample was composed of young adults only.Young adults, particularly current cigarette smokers, perceived e-cigarettes as less harmful and less addictive than cigarettes, and perceived e-cigarette aerosol as less hazardous than secondhand cigarette smoke. Our findings are consistent with earlier studies on e-cigarette perceptions among young adults , 27, 28.Both cigarette smokers and nonsmokers perceived e-cigarettes as less popular among peers than cigarettes. Compared to nonsmokers, current cigarette smokers perceived e-cigarettes as more popular. Findings reflect the tobacco landscape in Hong Kong, where e-cigarettes are less common than cigarettes. In 2015, 0.2% of Hong Kong residents reported current e-cigarette use , whereasConsistent with previous studies among young adults , 19, 31,Interestingly, most current e-cigarette users were infrequent users, and e-cigarette perceptions were generally unrelated to frequent e-cigarette use. Our finding contrasts with a prior longitudinal study of US young adults which found that the low harm perception of e-cigarettes predicted more frequent e-cigarette use . It is pOne-third (34%) of never users of e-cigarettes were susceptible to using e-cigarettes. It is of concern as a longitudinal study has found that e-cigarette susceptibility predicts the initiation of e-cigarette use among US adolescents . Future The perceived popularity of e-cigarette use was unrelated to e-cigarette susceptibility. Our finding is inconsistent with a study of Mexican adolescents which found that perceived higher popularity of e-cigarette use was associated with greater susceptibility to e-cigarette use . The difn\u2009=\u200957). Future research with larger sample sizes of e-cigarette users would be desirable to examine the effect of cigarette smoking on e-cigarette use trajectories among Asian populations.Not surprisingly, current cigarette smokers were more likely to report e-cigarette use and susceptibility than nonsmokers, which is consistent with the literature , 31, 37.Gender was found to be unrelated to e-cigarette use. It contrasts with our previous study conducted in a representative sample of Hong Kong adults which concluded that ever e-cigarette use was more prevalent among males . The incThis study has certain limitations. First, owing to the cross-sectional study nature, causal inferences cannot be drawn regarding the association between e-cigarette perceptions and use behaviors. Second, due to the convenience sample of young adults who were recruited online, findings may not be generalizable to the entire young adult population in Hong Kong. Third, the low crude response rates, particularly among current cigarette smokers, might suggest response bias. However, we could not ascertain the true number of eligible people and therefore, we could not determine the adjusted response rates. Fourth, the small size of current e-cigarette users made our estimate of this subgroup unstable. Lastly, self-reports were subject to recall and reporting errors.This study contributes important knowledge on perceptions of e-cigarettes in a sample of Asian young adults. Findings suggest that strategic interventions need to communicate with young adults about the harmful and addictive properties of e-cigarettes and the adverse health consequences of secondhand exposure to e-cigarette aerosol. Messages emphasizing the uncertainty of long-term effects of e-cigarette use may deter young people from using e-cigarettes.Additional file 1:Survey on the Perceptions and Use of Electronic Cigarettes and Waterpipe among Young Adults in Hong Kong. Survey of the study (English version). (DOCX 42 kb)"} +{"text": "What is the association between electronic cigarette (e-cigarette) use and depression?In this cross-sectional study of 892\u2009394 participants in the Behavioral Risk Factor Surveillance System from 2016 to 2017, e-cigarette users had higher odds of reporting a history of clinical diagnosis of depression compared with participants who never used e-cigarettes. In addition, increased frequency of e-cigarette use was associated with incrementally higher odds of reporting depression.These findings highlight the need for longitudinal studies to examine the association between e-cigarette use and depression, which may be bidirectional. This cross-sectional study examines the association between electronic cigarette (e-cigarette) use and depression in a nationally representative sample of the adult population in the United States. The prevalence of the use of electronic cigarettes (e-cigarettes) in the United States has grown rapidly since their introduction to the market more than a decade ago. While several studies have demonstrated an association between combustible cigarette smoking and depression, the association between e-cigarette use and depression has not been thoroughly studied.To examine the association between e-cigarette use and depression in a nationally representative sample of the adult population in the United States.Cross-sectional study of the Behavioral Risk Factor Surveillance System database, 2016 to 2017. The Behavioral Risk Factor Surveillance System is the largest national telephone-based survey of randomly sampled adults in the United States. A total of 892\u2009394 participants with information on e-cigarette use and depression were included. Data analysis was conducted in May 2019.Electronic cigarette use status defined by self-report as never, former, or current use.Self-reported history of a clinical diagnosis of depression.Of the 892\u2009394 participants , there were 28\u2009736 (4.4%) current e-cigarette users, of whom 13\u2009071 (62.1%) were aged between 18 and 39 years. Compared with never e-cigarette users, current e-cigarette users were more likely to be single, male, younger than 40 years, and current combustible cigarette smokers . In multivariable adjusted models, former e-cigarette users had 1.60-fold higher odds of reporting a history of clinical diagnosis of depression than never users, whereas current e-cigarette users had 2.10 times higher odds. Additionally, higher odds of reporting depression were observed with increased frequency of use among current e-cigarette users compared with never users . Similar results were seen in subgroup analyses by sex, race/ethnicity, smoking status, and student status.This study found a significant cross-sectional association between e-cigarette use and depression, which highlights the need for prospective studies analyzing the longitudinal risk of depression with e-cigarette use. If confirmed by other study designs, the potential mental health consequences may have regulatory implications for novel tobacco products. When used, e-cigarettes may generate volatile organic compounds, including acetaldehyde and formaldehyde, especially at high temperatures.6Electronic cigarettes (e-cigarettes) were introduced into the US market more than a decade ago as an alternative to combustible cigarettes.1 Among current e-cigarette users, 3.6 million (33.5%) reported daily use, with 54.6% also reporting traditional cigarette smoking.1In 2016, 10.8 million adults (4.5%) in the United States reported current e-cigarette use, more than half of whom were younger than 35 years.7 smoking rates are estimated to be 70% greater than in the general US population,8 with these individuals consuming approximately 56.4% of all cigarettes sold in the United States.7 Cigarette smokers with MHCs also tend to smoke more heavily and find it harder to quit smoking.9 The prevalence of depression in the population varies widely, from 6.7% to 16.6%,11 and a recent study found that prevalence of current e-cigarette use among those who reported having depression was 9.1%, compared with a prevalence of 4.5% in the general population.1Among individuals with mental health conditions (MHCs), who make up 36.4% of the US population,12 To test our hypotheses that e-cigarette users might be more likely to have depression and that depressed individuals might be more likely to use e-cigarettes, we sought to examine the cross-sectional association between the use of e-cigarettes and depression in a large representative sample of adults in the United States.The popularity of e-cigarettes among the general population and their appeal to those who are susceptible to MHCs, including younger adults, raises a question regarding the association between e-cigarette use and mental health.13 The survey includes participants in all 50 states as well as Washington, DC, and 3 US territories, making it the largest dynamic telephone-based health survey in the world.13The Behavioral Risk Factor Surveillance System (BRFSS) is a nationwide, telephone-based survey of randomly sampled US residents older than 18 years, established by the US Centers for Disease Control and Prevention to collect information on health-related risk behaviors, chronic medical conditions, and the use of preventive health services.14 our analysis of the data set was exempt from institutional review board review. Each section of this report was prepared using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.The BRFSS is a publicly available data set containing deidentified data. Thus, based on the code of federal regulations revised common rule,To examine the association between e-cigarette use and depression, we pooled data from the 2016 and 2017 BRFSS surveys, which included 936\u2009319 participants. Of the participants in the data set, we excluded participants with missing data on e-cigarette use (n\u2009=\u200939\u2009729 [4.2%]), self-reported history of clinical diagnosis of depression (n\u2009=\u20093818 [0.4%]), and both (n\u2009=\u2009378 [0.1%]), leaving a total sample size of 892\u2009394 (95.3%).Participants were categorized as ever or never e-cigarette users based on their response to the question, \u201cHave you ever used an e-cigarette or other electronic vaping product, even just 1 time, in your entire life?\u201d Those who answered yes were then further classified into occasional or daily users based on their response to the question, \u201cDo you now use e-cigarettes or other vaping products every day, some days, or not at all?\u201d We classified e-cigarette smokers into 4 statuses, as follows: everyday e-cigarette user, some days e-cigarette user, former e-cigarette user, and non\u2013e-cigarette user.16 In separate supporting analyses, subjective poor mental health was explored as an alternative outcome, defined by evaluating participants\u2019 response to the question, \u201cNow thinking about your mental health, which includes stress, depression, and problems with emotions, for how many days during the past 30 days was your mental health not good?\u201d17The dependent variable of interest was depression, defined as a self-reported history of clinical diagnosis if participants answered yes to the question, \u201cHas a physician ever told you have a depressive disorder ?\u201d18 Data on alcohol and combustible cigarette use were also collected. Heavy alcohol use was defined as more than 14 drinks per week for men and more than 7 drinks per week for women. Combustible cigarette smoking status was categorized as current, former, and never.Baseline demographic factors, including age, sex, race/ethnicity, marital status, education, and employment status, were self-reported according to BRFSS protocol. Income was defined using federal poverty line cutoffs for each state, taking into account the number of adults and children in the household for each state.19 These weights were applied in all analyses done with the data set.The BRFSS uses design weighting and iterative proportional fitting to ensure representativeness of the data.Sociodemographic and risk factor characteristics were described according to e-cigarette use categories by applying sampling design elements and using the svy subpop command. Multivariable logistic models were used to assess the association between e-cigarette use and depression, adjusting for age, sex, race/ethnicity, income, marital status, employment, education, heavy alcohol use, and combustible cigarette smoking. The prevalence of depression in the data set was high at 18.9%; thus, supplemental analyses were also performed using Poisson regression models to test the stability of the estimates. Given the known high prevalence of use of e-cigarettes among the younger population as well as among smokers, we performed subgroup analyses among college students and among combustible cigarette users.Formal effect modification was tested using interaction terms for e-cigarette use and sex, age, race/ethnicity, and cigarette smoking. Stratified analyses were then performed and presented by these variables.To further test our estimates, subjective mental health was explored as an alternative outcome. We compared those reporting 1 or more days of poor mental health with those reporting no days of poor mental health.P\u2009<\u2009.05. Data analysis was conducted in May 2019.All analyses were conducted using Stata statistical software version 14.2 (StataCorp) with the significance level set at a 2-sided Of 892\u2009394 participants , 28\u2009736 (4.4%) were current e-cigarette users, 111\u2009337 (16.5%) were former e-cigarette users, and 752\u2009321 (79.1%) were never e-cigarette users . CompareIn multivariable-adjusted logistic regression analyses, compared with never users, former e-cigarette users had 1.60-fold higher odds of reporting a history of clinical diagnosis of depression, while current e-cigarette users had 2.10-fold higher odds. Among current e-cigarette users, we found graded higher odds of reporting depression among occasional and daily users compared with never users . SimilarIn separate supporting analyses using self-reported days of poor mental health as a binary dependent variable, compared with never users who reported no days of poor mental health, former users had 1.52-fold higher odds of reporting at least 1 day of poor mental health, while current users had 1.67-fold higher odds. Similarly, occasional and daily users were more likely to report at least 1 day of poor mental health compared with never users .Among never cigarette smokers, current e-cigarette users were 2.16-fold more likely to report depression compared with never e-cigarette users. These results were similar when comparing dual users to current smokers who never used e-cigarettes .Both men and women who were current e-cigarette users were twice as likely to report depression compared with never e-cigarette users (eTable 1 in the P\u2009<\u2009.001), sex , and race/ethnicity . However, point estimates of the association between e-cigarette use and depression were qualitatively consistent across all subgroups, with a consistently increased odds of depression with former e-cigarette use and an even stronger association with current e-cigarette use. No statistically significant interactions were observed between e-cigarette use and cigarette smoking .In subgroup analyses, we observed statistically significant interactions between e-cigarette use and age (OR per increase in age category, 0.97; 95% CI, 0.96-0.97; In this cross-sectional analysis of a large nationally representative survey of adults in the United States, we found that former and current e-cigarette users were more likely to report a history of clinical diagnosis of depression compared with never users. In addition, we found a graded association between frequency of use and depression. We further explored subgroups defined by age and race and found no significant differences in the association between e-cigarettes and depression or mental health, suggesting that the association between e-cigarettes and these mental health outcomes was similar across these broad segments of the population.20 conducted among adolescents in Los Angeles, California, which showed that sustained e-cigarette use was associated with an increase in depression symptoms over a 12-month period. While cross-sectional, our sample size was larger, enabling us to conduct important subgroup analyses.Our study provides additional evidence to establish an association between e-cigarette use and depression, which could have potentially significant implications for public health, clinical practice, and health policy. Our results are supported by findings from a small longitudinal study by Lechner et al21 we also found that the association between e-cigarettes and depression did not differ significantly by sex or among current and former cigarette smokers. However, while that study found no association between e-cigarettes and depression in nonsmokers, we found that never cigarette smokers who used e-cigarettes had twice the odds of reporting a clinical diagnosis depression compared with those who did not use e-cigarettes, an estimate similar to that found among dual users.Similar to a study that was conducted in France in 2019,23 Therefore, we explored the association between e-cigarette use and depression in the subpopulation and found that current e-cigarette users also had twice the odds of reporting depression compared with never users in the same category. This highlights the potential susceptibility of e-cigarette users in this group to depression.Previous work has shown that college students are more likely to explore new products, including e-cigarettes, and it is not uncommon for this group to be targeted by tobacco companies\u2019 marketing strategies.20 As many e-cigarettes contain nicotine,24 this could explain the graded association seen with increased frequency of use and depression, suggesting a potential dose-response relationship. In addition to nicotine, e-cigarettes contain varying amounts of trace metals including arsenic, aluminum, and lead,27 many of which are on the priority pollutant list of the Agency for Toxic Substances and Disease registry, with known adverse health effects.26 In particular, lead and aluminum affect the central and peripheral nervous systems and may potentially contribute to the observed association between e-cigarettes and depression.27Prolonged nicotine exposure has been shown to disrupt the cerebral dopamine pathway, amplify stress sensitivity, and distort the coping mechanisms that buffer against depressive symptoms.29 Analyses of e-cigarette contents have shown that they also contain tobacco-specific nitrosamines and some volatile organic compounds, although in less quantity than observed in traditional cigarettes.31 This raises further concern about the use of e-cigarettes among individuals with MHCs, as their use might complicate the treatment of psychiatric conditions.Traditional cigarette smoking affects the metabolism of a number of psychiatric medications, mainly via the induction of metabolic enzymes, leading to reduced blood levels and reduced therapeutic effectiveness.33 and a brief prospective study done in adolescents found smoking to increase the risk of developing an episode of major depressive disorder.34 Among traditional cigarette smokers, patients with depression are less likely to successfully quit smoking, possibly because of worsening depressive symptoms during quitting attempts and poor tolerance of nicotine withdrawal symptoms.21 Smoking has also been shown to be highly predictive of future suicidal behavior, making it particularly concerning in individuals with a history of depression.35 Considering the similarities between e-cigarettes and traditional cigarettes, individuals with depression might be more susceptible to sustained use of e-cigarettes and may find it harder to reduce or quit their e-cigarette use. Furthermore, e-cigarette use might predispose users to developing major depression in the long term.Furthermore, a higher frequency of regular cigarette smoking has been reported among people with major depression compared with individuals who have never had major depression or any psychiatric illness,38 This theory, as well as the well-established addictive nature of nicotine,40 make the rapid uptake of e-cigarettes among teenagers\u2014described as an epidemic by the US Food and Drug Administration41 and the US Surgeon General42\u2014particularly concerning, especially because our findings show no difference in association between e-cigarette use and depression among younger and older adults.Consistent evidence of the gateway theory of an association between e-cigarette use and subsequent cigarette smoking initiation has been reported in a meta-analysis done by Soneji et al.Our findings, if confirmed in other study designs with longitudinal follow-up, may provide data to inform policies that could protect populations susceptible to depression. For example, the association between e-cigarette use and depression might justify further regulation of advertisements and marketing strategies, appropriate warning labels that highlight the potential risk of depression associated with e-cigarette use, and public health education about the potential effects of e-cigarettes, especially among those with MHCs. This association could also inform clinical practice by providing information that physicians could consider in counselling patients seeking information about the use of e-cigarettes, especially those with depression. At the very least, our findings warrant careful and thorough evaluation of e-cigarette use in both youth and adults with depression. Physicians should consider routine collection of information pertaining to e-cigarette use during clinic visits, especially in patients with depression, and routine counseling for those who use e-cigarettes, offering support to those who express willingness to quit.To our knowledge, this study was the first to examine the association between e-cigarette use and depression across several subpopulations in the largest nationally representative database in the United States. However, our study has limitations. It was observational and cross-sectional, and therefore, we can neither infer causality nor ascertain the direction of the association, which we submit might be bidirectional. Furthermore, we lacked granular information on product characteristics, such as the brands of e-cigarettes used, and detailed exposure characterization, including frequency or duration of vaping. We also cannot exclude the possibility of residual confounding. Finally, data on both the exposure and outcome were self-reported, which might lead to nonrandom misclassification.Our results suggest a strong association between e-cigarette use and depression, and this may have potential implications for regulation of e-cigarettes. Our results also highlighted the need for longitudinal studies to investigate the risk of depression associated with e-cigarette use and the potential bidirectionality of the association between e-cigarette use and depression."} +{"text": "Two reviewers screened all titles and abstracts independently, blinded to authors and journal titles (Cohen\u2019s Kappa = 0.83), resulting in 72 eligible articles. Risk perceptions, perceived benefits, and reasons for e-cigarette use were categorized in themes and sub-themes. Risk perceptions included harmfulness in general, and specific health risks. Perceived benefits included improved taste and smell, and safety for bystanders. Reasons for use included benefits, curiosity, smoking cessation, and friends using e-cigarettes. The findings highlight that there is a variety of perceptions and reasons mentioned by adult and youth e-cigarette users, cigarette smokers, dual users, and non-users. As such, this overview provides valuable information for scientists, public health professionals, behavior change experts, and regulators to improve future research, risk communication, and possibilities to effectively regulate e-cigarettes.This paper aims to provide an in-depth understanding of the attractiveness of e-cigarettes for several different groups. For this purpose, perceptions of and reasons for e-cigarette use were systematically reviewed as reported by e-cigarette users, cigarette smokers, dual users, and non-users, among both adults and youth. MEDLINE Electronic Nicotine Delivery Systems (ENDS) are devices that vaporize a solution of nicotine, additives, glycerin, and propylene glycol that is inhaled by the user ,2,3,4. EThe current overview provides scientists, public health professionals, behavior change experts, and regulators with key constructs for the development and validation of measures to assess perceptions of e-cigarettes and reasons for e-cigarette use. Public health professionals are able to use the overview on perceptions and reasons when developing health education and behavior change programs. On a population level, policy makers are able to use this inclusive overview to intensify smoking bans to avoid dual use and to target product characteristics of e-cigarettes attractive for specific user groups.The search strategy developed for the purpose of this narrative review aimed to retrieve articles focusing on perceptions and reasons related to e-cigarette use without any restrictions on location. Databases searched (and interfaces) were MEDLINE (Ovid) and Scopus (without date restrictions) till February 2018. Concepts included in the search were \u201celectronic cigarette\u201d, \u201cperception\u201d, \u201creason\u201d, \u201copinion\u201d, and \u201csmoking cessation\u201d guidelines see . First, Kim A.G.J. Romijnders extracted all relevant findings from the included studies See . Due to After coding, results were stratified by type of user and age. Adults were categorized as eighteen years or older, and youth were categorized as younger than eighteen years old. For each type of user, there was variability in reporting. For example, some studies report current use of e-cigarettes among current cigarette smokers without categorizing them as dual users, whereas other studies reported the current use of e-cigarettes with simultaneous current tobacco cigarette use as dual use ,33. Thern = 49), but studies were also conducted in the UK (n = 11), New Zealand (n = 6), Canada (n = 4), France (n = 2), Switzerland (n = 3), Australia (n = 2), and Belgium (n = 1) see . Due to Perceived risks pertained to risks for individual e-cigarette users , and risks for the social environment of these users ,101,102.Perceived benefits of e-cigarettes mentioned in the literature are summarized in This section reports reasons for use among e-cigarette users, cigarette smokers, dual users, and non-users. Non-users were asked about possible reasons for them to personally initiate e-cigarette use. Reasons for explaining the appeal of e-cigarettes go beyond smoking cessation . In addiThis review provides a comprehensive overview of risk perceptions, perceived benefits, and reasons for use of e-cigarettes, as reported in Current data showed a variety of perceptions about e-cigarettes and reasons for e-cigarette use reported by e-cigarette users, cigarette smokers, dual users, and non-users. For example, e-cigarettes were perceived as being less harmful by e-cigarette users. This perception of reduced harm could lead to use or, vice versa, by initiating e-cigarette use, the perception of harm may decrease. However, research showed that the perceived harm of e-cigarettes as compared to tobacco cigarettes has increased among all types of users over the years , and e-cBased on these findings, tailored communication on risks and benefits of e-cigarette use could increase awareness about risks and benefits of e-cigarette use among user groups. For example, targeted risk communication on risks of e-cigarette use for non-users, and benefits of e-cigarette use compared to smoking for cigarette smokers would increase factual knowledge about risks of e-cigarette use among these user groups. If the latter were to perceive e-cigarettes as less harmful, they may be more inclined to switch to e-cigarettes. Furthermore, if non-users were not to perceive fruit- and candy-flavored e-liquids as harmless, they might be less inclined to initiate e-cigarette use. Summarizing, risks and benefits could be communicated to increase knowledge about e-cigarette use among user groups.E-cigarette users expected (before initiation) and experienced (after continuation of use) benefits from e-cigarette use. In addition, this paper noted that reasons for initiation of e-cigarette use evolved to reasons for continuation of e-cigarette use . When poCigarette smokers and non-users also mentioned expected benefits from e-cigarette use. However, not all cigarette smokers continue with e-cigarette use after initiation or initiate e-cigarette use. In some cases, the expected benefits of e-cigarettes for cigarette smokers\u2014the ability to mimic smoking behavior\u2014did not result in the expected experience. Cigarette smokers who tried e-cigarettes often expressed the inability to mimic smoking behavior with an e-cigarette ,42,43,49Perceptions and reasons regarding e-cigarette use provide additional input for public health education, behavioral change programs, and regulation. Regulation, such as warning labels on tobacco products, is used to target misperceptions regarding tobacco products on a population level. Public health education can use the overview, presented in this study of perceptions on risks and benefits, to highlight factual risks and benefits of e-cigarette use in tailored communication. For example, tailored risk communication on the reduced harmfulness of e-cigarettes compared to cigarettes may reduce misperceptions among cigarette smokers initiating e-cigarette use for smoking cessation purposes. With risk communication tailored to specific personal needs and personal outcome expectancies, behavior change experts are able to target these personal misperceptions, and confirm factual risk perceptions and perceived benefits. Policy makers can also use this overview for product regulation measures. For example, available e-liquid flavors play an important role in the initiation of e-cigarette use for both cigarette smokers looking for an alternative for cigarettes and for curious non-users . From a Heterogeneity in the reporting of types of users made it difficult to classify types of users. For future research, it is therefore of vital importance to formulate standard definitions for ever, current, and dual use of e-cigarettes to assess population effects of e-cigarette use. In defining e-cigarette use, it is important to distinguish between experimental and daily use. For example, asking about e-cigarette use during the previous 30 days does not distinguish between experimental and daily use.This review noticed the lack of reporting on perceptions towards e-cigarettes and reasons for use among adult dual users and non-users, and youth non-users and dual users. Future research needs to identify the rates of dual use among youth. Overall, only perceptions of harm were assessed in extensive cross-sectional, cohort, and longitudinal studies compared to other risk perceptions, and less regarding perceptions in general.E-cigarette use is a complex behavior, and response options in questionnaires assessing perceptions and reasons in general may not be representative for all users, cigarette smokers, dual users, and non-users. Our overview, in addition to the work of Gibson et al. and PearHeterogeneity between the different papers in statistical methods and reporting makes it difficult to generalize findings across countries and study samples. Therefore, the results do not display analyses across countries. For this reason, the current paper was unable to display changes in risk perception over time. Due to the variability in reporting type of users and frequency of use, users were classified according to the classification of original articles. Consequently, this overview was unable to differentiate between former and never e-cigarette users, or to clearly differentiate between cigarette smokers and dual users, as not all cigarette smokers currently using e-cigarettes were classified in original studies as dual users of tobacco and e-cigarettes. In addition to a variety in study designs, more studies were found reporting on adult perceptions and reasons than youth, and cigarette smokers than non-users. This means that some perceptions and reasons regarding e-cigarettes could have been missed among the understudied user groups.This study is an exploratory narrative review into perceptions and reasons regarding e-cigarette use. Different perceptions of risks and benefits, and reasons for e-cigarette use were summarized for different types of users in themes and sub-themes, such as convenience, social environment, and disadvantages. Adults\u2019 perceptions and reasons for e-cigarette use are often related to smoking cessation, while youth like the novelty of the product. Tailored information about e-cigarettes for the different user groups is necessary to correct misperceptions about e-cigarettes and highlight the risks and benefits of e-cigarette use.For public health professionals, behavior change experts, and regulatory science, our overview of risk and benefit perceptions of e-cigarettes, and reasons for e-cigarette use provides insight into the initiation of e-cigarette use."} +{"text": "Evidence indicates that one reason cigarette smokers value e-cigarettes is the ability to use them in places where smoking is not permitted. We sought to: 1) explore adult daily smokers\u2019 experiences using e-cigarettes in the context of smoke-free places; and 2) describe smokers\u2019 perceptions of bystanders\u2019 reactions.Twenty adult daily smokers in Washington, DC initiated e-cigarettes for three weeks and completed in semi-structured interviews at the end of each week. All interviews (n=60) were digitally-recorded, transcribed verbatim, imported into NVivo 10.0, and analyzed using thematic analysis methodology.The sample had a mean age of 37.9 years and 18 participants reported having smoked their first cigarette by age 18. Common themes included descriptions of: 1) uncertainty about whether smoke-free policies included e-cigarettes; 2) using e-cigarettes in smoke-free places ; 3) approaches to e-cigarette use in smoke-free places as part of a complex decision-making process, ranging from testing and establishing the social and spatial boundaries of e-cigarette use, to confining e-cigarette use to inside their home; and 4) favorable, unfavorable, and impartial reactions from bystanders facilitated or impeded e-cigarette use, indicating social approval/social disapproval.Results suggest a continuum of factors, including smoke-free policies and reactions from bystanders may facilitate or impede e-cigarette use among smokers in environments where a smoke-free imperative is well-established. As e-cigarette use evolves, study findings indicate the importance of the social environment and how it could affect those switching from cigarettes to e-cigarettes. Empirical evidence indicates that smokers perceive e-cigarettes as less harmful than cigarettes, as cessation aids, and as alternative products that can be used in \u2018no smoking\u2019 places/situations6. Past studies7 have found that smokers\u2019 perceptions of e-cigarettes have been largely influenced by tobacco industry marketing strategies that promote e-cigarettes as smokeless, odorless, and discreet products that can be used anywhere, anytime.In the United States, electronic cigarette use is most common among adult (\u226518 years) cigarette smokers5 found that current adult smokers reported using e-cigarettes in smoke-free places like bars, restaurants, and places of employment. A recent qualitative study8 found that e-cigarettes were used by dual users (i.e. cigarette smokers and e-cigarette users) in places or situations when cigarette smoking was either not allowed or socially disapproved by family (e.g. home/indoors). This suggests that smokers may choose to use e-cigarettes in places/situations where they cannot smoke cigarettes, and may perceive e-cigarette use as convenient, more socially acceptable than smoking cigarettes, and lawful.We currently know little about e-cigarette use among adult daily smokers in the context of smoke-free environments. A cross-sectional study of national data by Shi et al.9 reported that e-cigarette use in restaurants and workplaces is viewed as more socially acceptable than cigarettes. Similarly, evidence12 indicates that public support for policies to restrict e-cigarette use in public places is considerably lower than for smoking bans. This suggests that e-cigarette use in smoke-free environments may be socially acceptable, which may facilitate use among smokers initiating e-cigarettes. Nevertheless, to our knowledge, no study has investigated how adult daily smokers understand the role of smoking restrictions in e-cigarette use, and how social norms surrounding cigarettes and e-cigarettes affect product use in smoke-free places. Therefore, it is important to identify factors that explain why adult daily smokers may or may not use e-cigarettes in smoke-free places/situations.To date, research investigating the role of social approval/disapproval of e-cigarette use in smoke-free environments is sparse. One studyThrough one-on-one interviews with adult daily smokers in Washington, District of Columbia (DC), we sought to: 1) explore adult daily smokers\u2019 experiences using e-cigarettes in the context of smoke-free places/situations; and 2) describe smokers\u2019 perceptions of bystanders\u2019 reactions to their e-cigarette use in smoke-free places/situations.13. In November 2016, the DC City Council extended smoke-free indoor air policy to cover e-cigarette use, including use in the workplace, public transit, and sporting events14. It is important to note that while data were being collected for the current study, e-cigarettes had not yet been included in the DC smoke-free policy.Washington, DC is an informative setting in which to investigate the use of e-cigarettes in smoke-free environments. In April 2006, the DC City Council enacted a 100% smoke-free indoor air policy, and in January 2007 the policy was implemented in restaurants, bars, and nightclubs15. Briefly, the Moment Study\u2019s design featured concurrent collection of multiple data streams, including: 1) ecological momentary assessment, 2) geotracking, 3) three semi-structured interviews, and 4) biosamples. To elicit participants\u2019 experiences and their views on using e-cigarettes in smoking restricted areas, only data from the semi-structured interviews are included in this paper, as they focus on the subjective perspective of the observed participants.Data were drawn from the parent study (\u2018Moment Study\u2019), a three-week intensive longitudinal mixed-methods study. The Moment Study investigated e-cigarette initiation and cigarette displacement among adult daily smokers in Washington, DC, and is described in detail elsewhere16.Adult daily smokers interested in using e-cigarettes were recruited via public online postings (e.g. Craigslist), paid advertisements (e.g. Washington Post Express), and flyers. Inclusion criteria included: 1) be 18 years or older, 2) reside in the Washington, DC metropolitan area, 3) report daily smoking of at least eight cigarettes a day for the past five years, 4) report no e-cigarette use in the past 30-days, and 5) not currently taking a smoking cessation medication (e.g. varenicline) or nicotine replacement therapy. Once enrolled, in-person procedures consisted of four office visits. All participants provided written informed consent and were compensated with up to $285 if they completed all study activities, including the three interviews. The Chesapeake Institutional Review Board approved all study procedures. For this qualitative analysis of initial understanding of e-cigarette use in smoke-free places/situations, three repeated-measures interviews (n=60) from 20 smokers in our sample of 107 were selected to be part of a subsample. This sample size reflects the \u201815 \u00b1 10\u2019 metrics for qualitative interview studiesFollowing the first office visit (baseline interview), two packs of five disposable NJOY King \u2018cigalike\u2019 e-cigarettes (3.0% nicotine) were provided to participants at the end of the interviews conducted at the second and third office visits. Participants were provided regular or menthol \u2018cigalike\u2019 e-cigarettes, depending on their cigarette brand flavor preference. At the second office visit, participants were instructed to try a minimum of three e-cigarette puffs per day over the course of the week. At the end of the third office visit, participants received two additional five-packs and were instructed to use them as they desired over the course of the week.17 were developed [by authors S.L.S. and J.L.P.], and included questions designed to examine how individuals understand the role of smoking restrictions on their e-cigarette use, and how social norms surrounding cigarettes and e-cigarettes affect product use independently read the transcripts and held debriefing meetings that involved writing preliminary analytical interpretations of the data, and developing a priori codes and emergent codes in relation to the interview guide questions, transcripts, and research questions. Two of the authors (S.L.S. and E.C.K.) independently coded a subset of transcripts line-by-line and compared them, which led to additional codes. The codebook consisted of each code, its complete definition, and an example of a quotation from a participant\u2019s transcript. These authors (S.L.S. and E.C.K.) independently coded the larger set of transcripts, and themes were generated iteratively during review of coded transcripts. After multiple rounds of coding, the first and second authors (S.L.S. and E.C.K.) determined that coding saturation had been reached20, as new themes ceased to emerge. These authors also wrote analytical memos throughout the coding process and used them to confirm coding saturation. Lastly, the first author (S.L.S.) adjusted and re-checked the resulting themes against the transcripts21. All quotes are provided verbatim.Two of the authors (S.L.S. and E.H.) were trained in qualitative interviewing techniques and conducted interviews separately at participants\u2019 second, third, and fourth office visits. Each interview lasted up to 30 minutes and took place in a dedicated interview room at Truth Initiative\u2019s Washington, DC office. The interviews (n=60) were digitally recorded with consent, professionally transcribed verbatim, edited to remove identifiers, cross-checked for accuracy, and imported into NVivo 10.0, a qualitative software package that aids in the organization, coding, and analysis of qualitative dataThe sample compriseThe results that follow are organized in relation to the study\u2019s two research objectives. These include: 1) Explore adult daily cigarette smokers\u2019 experiences using e-cigarettes in the context of smoke-free places; and 2) Describe adult daily cigarette smokers\u2019 perceptions of bystanders\u2019 reactions to them using e-cigarettes in smoke-free places/situations. We expand and discuss these themes in the sections below.\u2018The first time [e-cigarette use]\u2026I was walking down the street when I left here.\u2019 . Another participant reported in her interviews that she was more likely to use e-cigarettes at home. When asked about where she used e-cigarettes, she replied: \u2018I used them [e-cigarettes] mostly in my bedroom.\u2019 . In addition to using e-cigarettes inside his house, a participant reported in his interviews that he used e-cigarettes in his personal vehicle: \u2018I smoked [e-cigarette] in the car. I smoke it in the house. It was okay too because it doesn\u2019t have that smell or nothing like\u2026it\u2019s just smoke.\u2019 . The use of e-cigarettes inside his house and vehicle suggests that indoor places are more conducive to e-cigarette use than cigarette smoking because of the lack of tobacco smell.Throughout the narratives, participants reported using e-cigarettes in various public and private places, such as their homes/indoors, public transportation, private vehicles, workplaces, and bars/restaurants. One participant reported that he first used an e-cigarette after his second interview: . Another participant, who reported being employed at a restaurant, stated: \u2018I\u2019ve just been trying to do it [use e-cigarettes] where I like.\u2019 . He explained:Some participants described how they integrated e-cigarettes into their daily work routine. For example, one participant said: \u2018on my breaks, it\u2019s easier to do it [use e-cigarettes].\u2019 \u2018I\u2019ll just leave it [e-cigarette] on my register or whatever like that and I\u2019ll just grab like a draw or two off of it. Then you know, it might be another hour or two before I even get back to it. I might have like another draw or two off of it, versus having to go take like a five or 10-minute break, have somebody cover my register, then I\u2019ll go smoke a cigarette or two.\u2019 .\u2018It\u2019s [e-cigarette] not dissatisfying to people that be around me, like at work. I told them what it was and everything and they said we don\u2019t smell anything.\u2019 . His viewpoint indicates that e-cigarette use at work is socially acceptable, because unlike cigarettes, e-cigarettes do not produce a smell that is offensive to his co-workers.For these participants, e-cigarette use at work was perceived to be convenient and beneficial, as an alternative to routine smoke breaks, and in turn, an uninterrupted work shift. Similarly, another interviewee stated: \u2018I think because I\u2019ve been smoking the e-cigarettes only inside and I don\u2019t typically smoke inside anywhere, and I don\u2019t know if it\u2019s [e-cigarette use] allowed or whatever, I mean, all these other places. I know that I\u2019m allowed to do that [e-cigarette use] at my house.\u2019 . According to this participant, e-cigarette use inside her home is acceptable and free of unintended consequences. In the exchange given below, a participant conveyed to the interviewer that inside her house was more conducive to e-cigarette use than cigarette smoking, and how e-cigarette use in outside places would feel strange:While some participants used e-cigarettes at work, other participants reported in their interviews that they restricted e-cigarette use to inside their house during the trial period. For example, one participant expressed uncertainty about e-cigarette use in places/situations other than her house: \u2018Yeah, I didn\u2019t have to go out so that was kind of cool. I can\u2019t really ever imagine using it [e-cigarette] outside. That would be weird to me.\u2019 \u2018Why?\u2019 (Interviewer)\u2018Because why would you go outside? I guess\u2026if I\u2019m already outside. I don\u2019t know, it just feels like\u2026I like regular cigarettes more, so that\u2019s what I\u2019m going to do if I\u2019m outside.\u2019 .Her remarks suggest that bans on smoking in enclosed places have normalized cigarette smoking, not e-cigarette use, as typical outside behavior. She also states that she likes cigarettes more than e-cigarettes, and that she\u2019s willing to go outside to smoke cigarettes, suggesting how a smoker might weigh the pros and cons of switching from cigarettes to e-cigarettes. Similarly, when asked if she used e-cigarettes outside, another participant reported that she only used e-cigarettes outside as a cigarette substitute when she ran out of cigarettes.\u2018I remember you said last time you were using them in the house.\u2019 (Interviewer)\u2018Yes.\u2019 \u2018Okay. Did you use it [e-cigarette] at all outside?\u2019 (Interviewer)\u2018Not really. Like if I didn\u2019t have any more cigarettes I just smoked them [e-cigarettes]\u2019 \u2018I think because they\u2019ve become more mainstream no one really reacted. Certainly, they didn\u2019t say anything to me about using them.\u2019 . Similarly, a participant who reported using e-cigarettes in bars to circumvent smoke-free polices, described having no reactions from bystanders. He said: \u2018You see a lot of them [e-cigarettes] out at the bar actually. You kind of get away with it inside.\u2019 .In their second and third interviews, participants described trying to test and establish the social and spatial boundaries of e-cigarette use while in smoke-free places and recounted divergent experiences of their perceptions of how bystanders reacted to them using e-cigarettes in smoke-free environments. Perceived bystanders\u2019 reactions were described as impartial, unfavorable, or favorable. According to one participant, the widespread use of e-cigarettes in public places is the reason bystanders did not react negatively or positively to his e-cigarette use: However, another interviewee modified his e-cigarette use in response to negative reactions. He reflected on bystanders\u2019 reactions to him using e-cigarettes inside the bus:\u2018I got on the bus. I didn\u2019t have no cigarette to put out. I just kept it [e-cigarette] in my mouth while I scanned my card. I just walked on the bus. I took my seat. He didn\u2019t say anything. The bus driver didn\u2019t say nothing. So, I did about three or four puffs and then\u2026they all did look at me like I was losing my mind, but I didn\u2019t say anything. I just stopped dragging on it\u2026put it back in its case\u2026it\u2019s a non-smoking bus.\u2019.\u2018No, nobody said nothing.\u2019 . This suggests that the negative facial expressions from other passengers signaled a social cue that using e-cigarettes, like smoking cigarettes, is unacceptable and prohibited inside the bus. Another interviewee reported that she was very aware of her e-cigarette use inside the train. Recalling how her friend initially thought she was about to smoke a cigarette, she explained that she anticipated bystanders\u2019 possible negative reaction to her e-cigarette use:When asked if bystanders told him it\u2019s \u2018a non-smoking bus\u2019, he remarked: \u2018We sat on the train and we was waiting for it to pull off. I pulled out my e-cigarette right there and I was puffing, and then he said, \u2018you know you can\u2019t\u2019 \u2026then he said, \u2018oh shoot, that\u2019s them fake outs\u2019 [laughs]. People would look for a second, but then I guess maybe they realized it wasn\u2019t, you know, a real cigarette after they would look. But, a couple of people looked at me and you know, they turned like they wasn\u2019t trying to look, but they looked, and I would see their reaction. That\u2019s what I was going for though. I wanted to see people\u2019s reaction. And, I\u2019m going to try that again, you know, somewhere like at a restaurant or something, I\u2019m going to try it again. I want to see their reactions.\u2019 .She went on to describe how being able to satisfy her desire to smoke a cigarette at that moment, motivated her e-cigarette use inside the train:\u2018It felt good to be able to smoke [e-cigarette] because we had like maybe 10-15 minutes before the train pulled off\u2026so I\u2019m like, okay we can\u2019t go back off to smoke a cigarette or we got to pay again, and I wanted something to puff on so I pulled it [e-cigarette] out and I just sat there and puffed on it. And I felt good I was able to do that.\u2019. Another participant also reported e-cigarette use inside the train. Describing himself as a \u2018militant smoker\u2019 in his first interview, he mentioned that an appealing commercial motivated him to try e-cigarettes and counter the cigarette smoking stigma. He explained:\u2018I was kind of a militant smoker. It was like, you know, you\u2019re not going to push me out of society because I smoke\u2026it just happens to be a habit that I have. I understand that it\u2019s uncomfortable for some people and distasteful for some people, but it\u2019s my right to smoke. And, that\u2019s what the sort of gist of the commercial was. You know, regain your independence\u2026because as I said, I do enjoy the ingesting of the smoke. If there was some way that it [e-cigarette] doesn\u2019t bother other people\u2026if it [e-cigarette] doesn\u2019t encroach in sort of their personal space, I think that\u2019s like a brilliant invention, if it [e-cigarette] works.\u2019 .After initiating e-cigarettes, he reported in his second interview that he tested the social and spatial boundaries of e-cigarette use inside the train but not inside other smoke-free places that he frequents, like church and restaurants:\u2018I was with a friend who was fearful that we would get arrested [laughs]. There were people around and there was no reaction. I was thinking I didn\u2019t feel I had the guts enough to really kind of like, do it [use e-cigarette] in church or [laughs]\u2026do it in restaurants, to really sort of test that, but public transportation was enough. I think even though I talked about being a kind of militant smoker, I\u2019m a coward I suppose\u2026when it comes to drawing a line in the sand\u2026 at least in terms of smoking, you know. I exert my rights, but perhaps if I got more comfortable with using it [e-cigarette] you know, I would definitely give it [e-cigarette use in church or restaurants] a shot.\u2019 .Although bystanders did not react negatively or positively to his e-cigarette use inside the train, he described a complex decisional balance influenced by fear, perceived rights as a smoker, and a lack of comfort using e-cigarettes that prevented him from \u2018testing\u2019 other smoke-free places.\u2018I feel like everybody wanted to smoke what I was smoking. Everybody, they seen me with it [e-cigarette] and they wanted to have it. I felt like I was a trendsetter or something.\u2019 . Additionally, in his third interview, he reported that he used e-cigarettes inside a restaurant and recalled the initial reaction from bystanders, including the security guard:However, one participant described himself as a \u2018trendsetter\u2019, and articulated positive reactions from bystanders and social acceptance: \u2018I\u2019m smoking inside\u2026they thought it [e-cigarette] was a regular cigarette. I puffed. Like, I ate, and then I puffed it. The security guard came over, he was like, \u2018You know you can\u2019t smoke in here,\u2019 all this other stuff\u2026\u2019You can get arrested for that\u2019. I was like \u2018This is the e-cigarette\u2019. He said, \u2018Alright,\u2019 and walked off.\u2019.His remark that \u2018this is the e-cigarette\u2019, and the security guard\u2019s consensus, suggests that e-cigarette use inside the restaurant is permissible because e-cigarettes are not cigarettes.This study is an initial attempt to investigate where, why and how adult daily smokers are trying to test and establish the social and spatial boundaries of e-cigarette use. The adult daily smokers in this study: 1) reported e-cigarette use in the context of indoor smoke-free policies and inside their house; 2) described shoehorning themselves back into certain exclusionary spaces; 3) described uncertainty in relation to where they could use e-cigarettes; and 4) perceived that favorable, unfavorable, and impartial reactions from bystanders either facilitated or impeded their e-cigarette use, suggesting social approval/disapproval. These findings are a major step towards understanding the contexts of e-cigarette use from the perspective of smokers initiating e-cigarettes. As mentioned previously, during the time these interviews were conducted, the public smoking ban in Washington, DC had not yet been extended to e-cigarettes. Further research is needed to investigate how smokers initiating e-cigarettes are aware of and interact with public smoking bans that have been extended to e-cigarettes, and how these bans affect cigarette smoking and e-cigarette use.Several participants described how e-cigarettes operated as props that signaled to bystanders that they were attempting to test the social and spatial boundaries of e-cigarette use. While some participants associated favorable reactions with social approval, others associated unfavorable reactions with social disapproval, and described how unfavorable reactions impeded e-cigarette use in smoke-free places/situations. Further, participants who perceived impartial reactions from bystanders described e-cigarette use to be common inside places where smoking is prohibited in Washington, DC, such as bars, where e-cigarettes were used to circumvent smoke-free policies. While state and localities are considering whether to permit or prohibit e-cigarettes in smoke-free public places, our findings suggest that it is important to consider whether smokers initiating e-cigarettes view smoke-free policies as facilitating or impeding e-cigarette use. Some participants reported that smoke-free policies have prompted smokers to switch to using e-cigarettes in bars, without negative reactions or judgment. Other participants primarily limited e-cigarette use to inside their house, a space exclusive to them, making it possible to use the device unaccompanied by negative consequences. These findings suggest that smokers do not know where they can use e-cigarettes, which could affect those switching from cigarettes to e-cigarettes.22 suggesting that e-cigarettes are marketed as devices that can be used anywhere. Our current findings extend earlier studies by not only identifying where smokers use e-cigarettes, but also examining why they do or do not decide to use e-cigarettes in smoke-free places, and how they perceive bystanders\u2019 reactions. The narratives in this study attest that approaches to using e-cigarettes are part of a complex decisional balance influenced by both individual and situational factors that range from testing the social and spatial boundaries reserved for non-smokers, to restricting e-cigarette use to inside the home. Moreover, participants viewed e-cigarette use indoors and outdoors as holding different meanings. While some participants discussed how they used e-cigarettes indoors (e.g. while eating at a restaurant) and outdoors , other participants described how outside/outdoor spaces were only used to smoke cigarettes, suggesting that smoke-free policies influence where smokers use e-cigarettes and that a smoker might weigh the pros and cons of switching from cigarettes to e-cigarettes. For example, some participants explicitly stated a preference for cigarettes and were willing to go outside to use them, but not e-cigarettes. This suggests that if smokers perceive that e-cigarettes cannot outcompete traditional cigarettes on nicotine delivery, taste, and \u2018whole body\u2019 satisfaction, then they may not switch to e-cigarettes23, and they may not comply with indoor smoke-free policies that prohibit e-cigarette use.There have been studies24, an important caveat is the limited generalizability of the study\u2019s findings beyond its 20 interviewees. The study does, however, offer new insights and raise important questions for further investigation. Providing adult smokers e-cigarettes and asking them to report back on their use via repeated individual interviews provides a novel way to collect qualitative data. Studies of the differences in reporting between na\u00efve and established e-cigarette users are needed. Comparisons with adult daily e-cigarette-only users could also extend understanding of how users incorporate these devices into their daily lives. Further, comparisons to dual cigarette and e-cigarette users in rural areas could validate or invalidate the influence of the urban-suburban environment.By employing repeated one-on-one interviews with adult daily cigarette smokers transitioning to e-cigarettes, our findings fill an existing gap in the literature and offer new explanations of why, where, and how e-cigarette use in smoke-free places/situations occurs. While the present study is based on a small and non-random sample that is justifiable for qualitative research25, investigating contexts specific to e-cigarette use among adult daily smokers may propel research and policy developed to evaluate the potential public health risks and benefits of e-cigarettes.This study builds upon existing qualitative research, as we identified several different experiences of adult smokers with e-cigarettes in smoke-free places/situations. The findings of this study provide us with a deeper understanding of environmental and place experiences and meanings that facilitate or impede e-cigarette use in smoke-free places/situations. Given the rapidly changing e-cigarette landscape"} +{"text": "To assess prevalence and predictors of e\u2010cigarettes/cigarettes patterns of use in adolescents in England.Prospective study with 24\u2010month follow\u2010up of e\u2010cigarette/cigarette ever/regular use with data from an intervention evaluation.Forty\u2010five schools in England (Staffordshire and Yorkshire).A total of 3210 adolescents who, at baseline, were aged 13\u201314\u00a0years and had never used e\u2010cigarettes/cigarettes.Based on e\u2010cigarette/cigarette ever use at follow\u2010up, six groups were created: (a) never user, (b) e\u2010cigarette only, (c) cigarette only, (d) dual use\u2014order of use unclear, (e) dual use\u2014e\u2010cigarettes used first and (f) dual use\u2014cigarettes used first. Baseline measures were: gender, ethnicity, socio\u2010economic status, impulsivity, family plus friend smoking and smoking\u2010related beliefs .In groups (a) to (f), there were 71.5, 13.3, 3.3, 5.7, 2.9 and 3.4% adolescents, respectively. Among groups using cigarettes, regular smoking was more prevalent in group (f) than in groups (c), (d) and (e) combined . Among groups using e\u2010cigarettes, regular use was less prevalent in group (b) (e\u2010cigarette only) than in groups (d), (e) and (f) combined . Higher impulsivity plus friends and family smoking were predictive of being in groups (b) to (f) compared with group (a) (never users). Males were more likely to be in group (b) compared to group (a); females were more likely to be in groups (c) to (f) compared to group (a).Regular use of e\u2010cigarettes/cigarettes varies across groups defined by ever use of e\u2010cigarettes/cigarettes. Interventions targeted at tackling impulsivity or adolescents whose friends and family members smoke may represent fruitful avenues for future research. Electronic cigarettes (e\u2010cigarettes) represent a means to deliver inhaled aerosol to the lungs. E\u2010cigarettes have been recognized as a means to reduce harm in adult smokers The present research is novel in examining differences between six groups of 15\u201316\u2010year\u2010old adolescents : never users of cigarettes or e\u2010cigarettes; e\u2010cigarette only users; cigarette only users; dual users\u2014order of first use unclear; dual users\u2014e\u2010cigarettes used first; and dual users\u2014cigarettes used first. Previous reports of the current data focused on examining how e\u2010cigarette use as a 13\u201314\u2010year\u2010old predicted progression to cigarette smoking at age 14\u201315\u00a0years To address the study aims, data from a 4\u2010year cluster randomized controlled trial of a school\u2010based smoking initiation intervention Data from 3210 adolescents from 45 schools in England (Staffordshire and Yorkshire) who self\u2010reported never having used a cigarette or an e\u2010cigarette at age 13\u201314\u00a0years are reported here. Head teachers consented to school participation with parents given the option to withdraw children from the study. Adolescents consented by completing questionnaires matched across time\u2010points using a personally generated code. The University of Leeds, UK (Faculty of Medicine) ethical review committee approved the study (reference 12\u20130155).Cigarette use was assessed using a standardized measure one of the following is closest to describing your experience of e\u2010cigarettes or vapourizers', I have never used them; I have tried them once or twice; I use them sometimes (more than once a month but less than once a week); I use them often (more than once a week)']. Marking the first response versus other responses was coded to indicate never versus ever using e\u2010cigarettes, while marking the last responses was coded to indicate regular use of e\u2010cigarettes.E\u2010cigarettes/vaporizers were described as \u2018a tube that sometimes looks like a normal cigarette and has a glowing tip. They all puff a vapour that looks like smoke but unlike normal cigarettes, they don't burn tobacco'. Use of e\u2010cigarettes was tapped by a single item at both time\u2010points . Five imputed data sets were created and multinomial logistic regressions averaged across the data sets. All analyses were conducted in SPSS version 24. Full data are available from the first author.In order to assess the study aims we: (1) report the frequencies and percentages in each of the six groups; (2) use \u03c7In relation to our first aim (estimating the size of different user groups at 15\u201316\u00a0years), we observed that the majority of the sample remained as never users. Adolescents who initiated e\u2010cigarette use only comprised the second largest group, followed by the dual user (12.0%) and cigarettes only groups. The dual user group split into dual user\u2014order of first use unclear, dual user\u2014e\u2010cigarettes used first and dual user\u2014cigarettes used first (Table\u00a0In relation to our second and third aims (rates of regular use of cigarettes and e\u2010cigarettes across user groups), Table\u00a0Rates of regular use of e\u2010cigarettes across the e\u2010cigarette user groups ranged from 1.9 to 14.8% and also significantly differed across the four groups (Table\u00a0Ps\u00a0<\u00a00.05). In addition, higher levels of family smoking were associated with being significantly more likely to be in four of the five user groups compared to the never user group. Less consistent patterns were observed for attitudes, perceived behavioural control and intervention condition. More negative attitudes towards smoking , less perceived behavioural control over not smoking and being in the intervention condition were found to reduce the likelihood of belonging to some user groups compared to the never user group . Relating to our first aim, never users and e\u2010cigarette only users were found to be the largest groups among adolescents. It is notable that only using e\u2010cigarettes was the largest of the five groups who used cigarettes and/or e\u2010cigarettes confirming other studies showing relatively high rates of e\u2010cigarette use in UK adolescents Related to our fourth aim, the multinomial logistic regression analyses Table\u00a0 indicateHigher levels of impulsivity were also associated with being more likely to be in each of the user groups compared to the never user group. The effects for impulsivity were somewhat smaller for e\u2010cigarette only use. Males were statistically significantly more likely to be in the e\u2010cigarette only group compared to the never user group, while females were statistically significantly more likely to be in each of the other smoking groups compared to the never user group. The fact that e\u2010cigarette use appears to be appealing more to males than females deserves further attention, as it is not clear what is the basis of these differences.Ethnicity, family affluence, perceived behavioural control and intervention condition were generally not predictors of being in one of the user groups compared to the never user group. Finally, positive attitudes towards not smoking were associated with not being a member of each of the user groups compared to the never user group.In general, the data Table\u00a0 did not The most novel aspect of the present research was the examination of differences between dual user\u2014cigarettes used first and dual user\u2014e\u2010cigarettes used first groups. The predictors of these different patterns of dual smoking were very similar across the set of variables examined. Other research has reported that low levels of friends smoking may be associated with a greater likelihood of transitioning between using e\u2010cigarettes and cigarettes Our study has a number of strengths, including a large demographically diverse sample, measurement of e\u2010cigarette and cigarette use over 24\u00a0months, and exploration of a large set of covariates including socio\u2010demographic variables as well as measures of health cognitions about smoking. There are, however, also a number of weaknesses. First, our study focused on self\u2010reported e\u2010cigarette and cigarette use. Secondly, we failed to distinguish types of e\u2010cigarette use (e\u2010cigarettes vary in a number of ways including the delivery method and whether they contain nicotine). Thirdly, our research did not include measures of perceptions of e\u2010cigarettes shown to predict e\u2010cigarette and dual use In summary, this is the first study, to our knowledge, to report longitudinal relationships between different patterns of e\u2010cigarette and/or cigarette use among UK adolescents. A key recommendation would be to focus on preventing the initiation of cigarette use because this, irrespective of subsequent e\u2010cigarette use, may lead to increased regular cigarette use.All authors report receiving grants from the National Prevention Research Initiative during the study. The authors have no conflicts of interest."} +{"text": "E-cigarettes were invented in China, and most of the world\u2019s e-cigarettes have been produced in China. However, awareness and use of e-cigarettes in China are lower than in Europe and America. Against the backdrop of the increasing use of e-cigarettes, the supervision of e-cigarettes in China has been almost non-existent.A literature search was carried out in five popular Chinese and English databases. These databases were PubMed, EMBASE, the Cochrane Library, China national knowledge infrastructure (CNKI), and Wan-fang database.Twenty-one studies were identified, and a comprehensive analysis of e-cigarette awareness ratio was conducted for different regions of China and according to gender, age group, and smoking status. We also examined e-cigarette use and associated risk factors, and the relationship between e-cigarette use and smoking cessation. In 2015, the prevalence of \u2018ever use\u2019 and \u2018current use\u2019 of e-cigarettes in China were 3.1% and 0.5%, respectively. The review indicates that the awareness ratio of e-cigarettes was about 66% in Hong Kong, whereas the ratio for Tianjin was lower (43.6%). Online sales were the main channel for selling e-cigarettes to 80% of the users. Awareness of e-cigarettes has been increasing in China. Awareness was higher in men compared to women in all age groups. Nevertheless, e-cigarette use in China was lower than in developed countries. E-cigarette users were more likely to try to quit smoking, but the relationship between e-cigarette use and smoking cessation is still unclear. Governance is necessary for e-cigarette use and marketing effort.This study investigated the awareness and use of e-cigarettes in China and the existing regulations for e-cigarette use and marketing. The lack of regulations for e-cigarette use and the unrestricted practice encourage the increase in adoption of e-cigarettes and misconceptions of the benefits of using e-cigarettes. Hence, it is crucial that the government of China prioritize the establishment and implementation of regulations for e-cigarette use and marketing. The prevalence of \u2018ever use\u2019, \u2018currently use\u2019 (at least 1 of the last 30 days), and \u2018regular use\u2019 (at least 20 of the last 30 days) of e-cigarettes in the US were 7.7%, 2.1%, and 0.9%, respectively2. In Europe, the prevalence of e-cigarette \u2018current use\u2019 was 1.8%3. The 11.6% prevalence of \u2018ever use\u2019 of e-cigarettes in Europe is higher than in the US3. However, in China where the e-cigarette originated, the prevalence of e-cigarette use is low4. According to the China Adult Tobacco Survey in 2015, 40.5% of adults aged 15 years and older had heard of e-cigarettes and 3.1% had ever tried them5. Hence, in this work we have collected, analyzed, and synthesized the existing literature on the Chinese population\u2019s awareness and use of e-cigarettes. In our study, the \u2018awareness of e-cigarettes\u2019 is defined by the first incidence the individuals heard of e-cigarettes. The awareness ratio characterizes the proportion of the population that has heard of e-cigarettes.Although electronic cigarettes (e-cigarettes) were invented in China, the majority of e-cigarettes users reside in Europe and the United States6. Hon Lik, a Chinese pharmacist, is widely considered to be the inventor of the first generation of e-cigarettes with the first nicotine-based e-cigarette produced in 2003. The first generation of e-cigarettes was introduced to the Chinese market in 2004 and exported since 20057. In 2015, China produced about 80% of the world\u2019s e-cigarettes8, with a 33% increase in sales in 2015 alone9.E-cigarettes, most accurately known as \u2018electronic nicotine delivery systems (ENDS)\u2019, consist of a lithium battery, smoke bomb (containing volatile liquid), pressure sensor, control circuit board, and light emitting diode. When the volatile liquid of the smoke bomb is heated, the device produces smoke fog. Depending on the preference of the users, nicotine and other chemicals may be added into the volatile liquid. The operational principle of e-cigarettes mimics that of traditional cigarettes10. However, the International Trade Center (ITC) China survey revealed that the percentage of smokers who had heard of e-cigarette rose from 29% to 60% . Similarly, the percentage of smokers who had tried e-cigarette surged from 2% to 11% 11. These surveys suggest that both the number of individuals who are aware of e-cigarettes and the number of individuals using e-cigarettes are rising.Compared with people in the European Union, the US and other countries, Chinese know less about the use of e-cigarettes than people of other Asian countries12. In China, the only regulation of e-cigarette use was published by the State Administration of Market Supervision and Administration and the State Tobacco Monopoly Bureau in 2018. This regulation was stated in the \u2018Circular of the State Administration of Market Supervision and Administration and the State Tobacco Monopoly Bureau on the Prohibition of the Sale of Electronic Cigarettes to Minors\u2019. Hang Zhou city formulated local regulations of e-cigarettes to manage the use of e-cigarettes in smoke-free places13. Until today, national law or regulation, published by NPC and Standing Committee of NPC and the State Council, to control e-cigarette use is still lacking. Nevertheless, the government of China realizes the importance and urgency of controlling e-cigarette use nationally14.Despite the rapidly increasing use of e-cigarettes in China, the governance of e-cigarette use is still at an early stage compared to other countries that have enacted relatively clearer regulations related e-cigarette use and supplyA literature search was carried out in five popular electronic Chinese and English databases on 1 December 2018. These databases were PubMed, EMBASE, the Cochrane Library, China national knowledge infrastructure (CNKI), and Wan-fang database. The queries combed through the publications in the databases from their inceptions to 30 November 2018. We used subject terms to search in the databases and manually retrieved the list of references.In the Chinese databases, we used the subject heading search, and the search term was expressed as \u2018\u7535\u5b50\u70df\u2019 (i.e. e-cigarette). In addition, the search field included the title, abstract, keyword, and subject word. In the English databases, we used Medical subject Headings (MeSH), and the search term was \u2018electronic nicotine delivery systems\u2019 and \u2018e-cigarette\u2019.Two investigators screened the literature independently based on the inclusion and exclusion criteria . The purWe searched five Chinese and English databases and obtained 4075 articles initially. We then imported all 4075 citations into EndNote and deleted duplicated documents.After eliminating the duplicates, we read the headings and abstracts and conducted a preliminary screening of the literature based on the inclusion and exclusion criteria . We exclWhen disagreement appeared during the screening process, we discussed and exchanged views with another independent investigator in order to reach an agreement. Finally, we further filtered the papers that failed to meet the inclusion criteria and kept 21 papers .15 because this approach evaluates six general aspects of the 21 papers. These aspects are: 1) clarifying the research purpose clearly; 2) clarifying the research method clearly; 3) explaining the study subjects and their settings clearly; 4) clarifying the source of data clearly; 5) whether the results are measured in a scientifically valid manner; and 6) the logic and conviction between conclusions and analysis.As some of the included literature adopted a combination of several research methods in a single study and some studies had narrowly defined research objectives for the designated disciplines, we could not find an evaluation framework that was suitable to evaluate such a diverse body of literature. Therefore, we adopted the evaluation approach proposed by Moola et al.Papers that matched at least five aspects suggested by Moola et al. were regarded as high-quality literature. Papers that fulfilled three or fewer aspects were considered low-quality documents. The remaining papers were considered as medium-quality literature.According to the inclusion and exclusion criteria , we extrWe extracted the data independently and reached consensus through discussions. The extracted data included author/year, investigation method, study area, sample, statistical method, report result and quality (Appendix 1). For the remaining 8 papers, the extracted data included author/year, study area, literature results, and quality (Appendix 2).We measured the awareness of e-cigarettes among the sample populations in the included literature with a closed-ended question: \u2018Have you heard of e-cigarettes?\u2019. Individuals in the sample population in the included literature who answered \u2018yes\u2019 were considered as being aware of e-cigarettes. The total number of individuals who answered \u2018yes\u2019 divided by the sample population represents the \u2018awareness ratio\u2019.11. Recently, a study in Hong Kong indicated that the awareness ratio of e-cigarettes is about 66% and seems to be rising16, while in Tianjin, the e-cigarette awareness ratio was slightly lower (43.6%) and the awareness ratio of e-cigarettes among the 25\u201344 years age group was the highest among all the age groups17. In China, the awareness ratio of e-cigarettes among adolescents (aged 12\u201318 years) was 89.52% with the e-cigarette awareness ratio of current cigarette smokers (95.93%) and former cigarette smokers (93.23%) both higher than for non-smokers (85.75%)18. The study further suggests that most adolescents knew about e-cigarettes through the internet and television18. About 69.8% of cigarette smokers in Beijing (age \u226520 years) had heard of e-cigarettes from their friends and tobacco retailers besides the advertisements on television and the internet19. In Hong Kong, 82.6% (95% CI: 80.2\u201384.9%) of cigarette smokers had heard of e-cigarettes20.According to the 2015 China Adult Tobacco Survey, 40.5% of adults aged 15 years and older had heard of e-cigarette. Moreover, the International Tobaco Control (ITC) China survey revealed that the percentage of smokers who had ever heard of e-cigarettes rose from 29% to 60% 22. The awareness ratio of e-cigarettes among adolescents in Hong Kong (71.1%) was higher than in mainland China23, and close to the level of awareness among the adults in Hong Kong 16.Several studies focused on the awareness of e-cigarettes among junior high school students because they are susceptible to online advertisements. One survey in Guangxi province showed that the e-cigarette awareness ratio among junior high school students was 46.71%, consistent with the results of the national survey on e-cigarette awareness (45%)22. The survey also indicates that the awareness of e-cigarettes among youth was influenced by social and individual factors. Individuals that have close friends who smoked were more likely to know about e-cigarettes than those who did not have close friends who smoked (OR=1.66). Individuals who had smoking experience (OR=1.72) or individuals who paid attention to tobacco advertisements were also more likely to be aware of e-cigarettes (OR=1.55)22.Several studies found that the awareness of e-cigarettes was higher in men compared to women in all ages, as expected considering that men smoke more often than women and they can learn about e-cigarettes through more sources17. This trend was also similar among cigarette smokers20. Furthermore, younger people and people with higher education were more likely to be aware of e-cigarettes11.A survey carried out in Tianjin showed that the awareness of e-cigarettes was positively related to the level of education. The awareness ratio of e-cigarettes was higher among the population with more educationIn the included literature, the \u2018use\u2019 of e-cigarettes was broken down by \u2018ever use\u2019, \u2018current use\u2019, and \u2018use in past 30 days\u2019. The first category was answered by the question: \u2018Have you ever tried e-cigarettes?\u2019. The number of people who answered \u2018yes\u2019 divided by the sample population represents the prevalence of \u2018ever use\u2019. The second category \u2018current use\u2019 was measured by the question: \u2018Do you use e-cigarettes now?\u2019. The third category was measured by the question: \u2018Did you use e-cigarettes in the past 30 days?\u2019. We distinguished these three categories by seeking the questions and answers in the included literature that contained the phrase \u2018current\u2019 and \u2018in the past 30 days\u2019. The prevalence of \u2018current use\u2019 and \u2018use in past 30 days\u2019 are defined in a similar way.3. According to the 2015 China Adult Tobacco Survey, 3.1% of adults aged 15 years and older had ever tried e-cigarettes and the current use prevalence of e-cigarettes was 0.5% to 11% 11.E-cigarette use in China was lower than that in developed countries such as the US and European countrieswas 0.5% . Althoug17. Similarly, in Hong Kong, e-cigarette use among adults was 2.3% (\u2018ever use\u2019) with men using e-cigarettes more often than women (p=0.03). People between the age of 15 and 29 years were more likely to use e-cigarettes (p=0.002). The current cigarette smokers had the highest prevalence16. The use of e-cigarettes was different among different ages. A study showed that 26.44% of Chinese adolescents had used e-cigarettes (including one-time use)18. Another study conducted in Hong Kong among adolescents found that e-cigarette use was about 8.9% (\u2018ever tried\u2019)23, which was higher than for the adult population (between age 15 and 65 years). The e-cigarette \u2018use in the past 30 days\u2019 among adolescents in Hong Kong was about 1.1%24. Among the younger students in Guang Xi junior high school, the use of e-cigarettes was relatively low, at 8.5% for \u2018ever tried\u2019, and 1.2% for \u2018use in the past 30 days\u201922. Surprisingly, the e-cigarette use \u2018in the past 30 days\u2019 among adolescents in Taiwan increased rapidly to 27.46%25. In general, cigarette smokers had a higher use prevalence of e-cigarettes. In Hong Kong, 13.3% (95% CI: 11.3\u201315.5%) cigarette smokers had ever used e-cigarettes20. In Beijing, 122 (12.8%) cigarette smokers had ever used e-cigarettes19. These findings imply that adolescents used e-cigarettes more often than adults.The survey conducted in Tianjin showed that 2.3% of the respondents had ever used e-cigarettes and only 0.5% of them were still using e-cigarettes. These findings are consistent with the results of the national survey17. Meanwhile, the study in Hong Kong suggests that men used e-cigarettes more often than women (p=0.03)16. In the survey of e-cigarette use among junior high school students in Guangxi, the multivariable logistic regression analysis shows that the prevalence of e-cigarette use was 2.422 times in men than in women (p<0.01). Besides, age, academic performance (such as grades) and peer smoking could also influence e-cigarettes use (p<0.01)21.Research shows that gender and smoking status can influence e-cigarette use19. Among the cigarette smokers, the prevalence of e-cigarette use among urban smokers (18.2%) was significantly higher than among rural smokers (9.3%) (p<0.001). However, the intention of e-cigarette use among rural smokers (25.7%) was higher than that of urban smokers (17.2%) (p=0.022)19. The study also investigated the attitudes of e-cigarette use among cigarette smokers, and the results show that 57.0% of cigarette smokers had heard of e-cigarettes but not used e-cigarettes, 22.6% would like to try, and 12.1% did not give a clear attitude. Amongst the 65.3% cigarette smokers, the percentage who did not want to use e-cigarette because of they did not intend to quit smoking was 40.7%, distrusted of the effects of e-cigarettes was 27.8%, worried about the safety of e-cigarettes was 19.7%, concerned about prices was 3.7% and others (8.1%)19. A similar result was found in the 2015 China Adult Tobacco Survey, in which younger people, male, and urban residents, preferred using e-cigarettes5.In Beijing, 122 (12.8%) cigarette smokers, 114 men and 8 women, had ever used e-cigarettes26.One study about urban male tobacco cigarette smokers in China revealed that the younger, with higher education, those who attempted to quit smoking in the past year, and who smoked more cigarettes per day, were more likely to use e-cigarettes18.Among Chinese adolescents, most used e-cigarettes because they wanted to avoid the harms from directly inhaling the chemicals in traditional cigarettes (44.63%) and indirectly inhaling the chemicals in secondhand smoke (26.30%) produced by other traditional cigarette smokers, while 24.26% wanted to quit smoking by using e-cigarettes. Some respondents used e-cigarettes because they thought it was fashionable (15.37%) or simply because they were curious about e-cigarettes (25.56%). A small number of e-cigarette users thought that e-cigarettes were cheaper than traditional cigarettes and could be used in smoke-free places16.For slightly different purposes, adults in Hong Kong used e-cigarettes for curiosity (47.4%), fashion (25.8%) and quitting smoking (13.6%)We searched for the keyword \u2018e-cigarettes\u2019 on the Taobao website (China\u2019s largest e-commerce platform) and found that more than 10000 e-cigarette products were sold monthly. On the \u2018Sales\u2019 webpage, the following information was presented: 1) e-cigarettes were healthier than traditional cigarettes; 2) e-cigarettes could help smokers quit traditional cigarettes; 3) e-cigarettes had many flavors; and 4) e-cigarette smoking was permitted in non-smoking areas. On the \u2018Ingredients\u2019 webpages, marketers disclosed that e-cigarettes contained propylene glycol, nicotine salt, and essence.27 found that most websites (16 out of 18) claimed that e-cigarettes were healthier and excluded hazardous ingredients such as nicotine. Also, 14 websites indicated that e-cigarette use might prevent secondhand smoking. Secondhand smoking refers to the inhaling of smoke emitted from the burning of tobacco products involuntarily. Twelve websites claimed that e-cigarettes could help cigarette smokers quit smoking and that they were allowed to be used in non-smoking areas. Eight websites indicated that e-cigarettes were cleaner than traditional cigarettes, while 7 websites hinted that e-cigarettes could enhance users\u2019 social status27.Yao et al.28.Similar to Western countries, 22 markets of e-cigarettes in China targeted the young generation. The Chinese marketers promoted their products as fashion accessories with the e-cigarette\u2019s modern and stylish design and multiple flavors. The e-cigarettes were seen as part of the fashion of pop icons among the youth. The marketing companies launched competitions with appealing prizes and hired young, attractive women to boost e-cigarettes sales29.In China, e-cigarette sales reached 4.09 billion RMB (about 589 million USD) in 2017, an increase of 25.3% year-on-year in the consumer market. Online sales of e-cigarettes accounted for around 80% of the consumer market and store sales 14%, while sales from other marketing channels represented a small market share19. The study found that 71.0% of cigarette smokers expected to quit smoking by using e-cigarettes. In fact, 8.3% of cigarette smokers had quit smoke successfully, 47.1% cigarette smokers used fewer cigarettes, and 15.1% cigarette smokers used the first e-cigarette later in the morning compared to the time when they smoked the first traditional cigarette in the morning. In this study, most cigarette smokers (84.0%) believed that e-cigarettes were not addictive or less addictive than traditional cigarettes. More than 70.0% of cigarette smokers believed that e-cigarettes were healthier and less risky than traditional cigarettes19. Chen et al.25 suggest that 71% of e-cigarette users had tried to quit smoking in the past year compared to 69% of non-e-cigarette users, with a statistically significant difference (p<0.05)25. Of the people who used e-cigarettes, 39.47% had attempted to quit smoking in the past 12 months, significantly higher than those who had never used e-cigarettes (13.98%) (p<0.01)17. In the Wang et al.18 study, 36.02% of cigarette smokers had tried to quit smoking by using e-cigarettes. However, only 13.54% of cigarette smokers quit smoking successfully. They used multivariable logistic regression to analyze the relationship between e-cigarette use and smoking cessation behavior. The study indicated that using e-cigarettes was related to smoking cessation attempts . E-cigarette users were more likely to try to quit smoking, but the relationship between e-cigarette use and smoking cessation was still unclear18.Recently, a study in Beijing showed that e-cigarettes could help cigarette smokers quit smoking or reduce smoking25, i.e. the use of e-cigarettes increases the proportion of people who quit smoking, attempt to quit smoking, and smoke less frequently. In contrast, some research indicates an inverse relationship between the use of e-cigarettes and smoking cessation, attempt at smoking cessation, and smoking reduction30.The relationships between the use of e-cigarettes and smoking cessation, attempt at smoking cessation, and smoking reduction, are inconclusive. Some studies report a positive relationship22, users of e-cigarettes were more likely to use tobacco products in the next 12 months (OR=7.0), more likely to use tobacco products provided by good friends (OR=5.1), and more likely to smoke (OR=14.6). One study among adolescents in Hong Kong showed that e-cigarette use was significantly correlated with increased smoking and smoking immediately after waking (p<0.001), but no remarkable correlation with smoking cessation behavior (p>0.05)24. A similar result was presented in a study among the Community-Recruited cigarette Smokers in Hong Kong20. The study showed that e-cigarette use is associated with lower levels of intention to quit and has no association with attempts to quit (p=0.45 for trend)20. Another study among cigarette smokers in Hong Kong showed that using e-cigarettes failed to predict self-reported point prevalence abstinence , biochemically validated quitting , cessation attempt , or smoking reduction 30.In the study of Xiao et al.26 declared that the male respondents who attempted to smoke and smoked more cigarettes per day were more likely to use e-cigarettes. This finding may seem contradictory, but it may imply that the male respondents choose e-cigarettes to help them to quit smoking initially, but unexpectedly, e-cigarettes prompted them to smoke more.Significantly, Zhao et al.31. The control of e-cigarette use by the Chinese government is almost non-existent. There is no law and administration regulation published by NPC and Standing Committee of NPC and the State Council for e-cigarettes29. The only regulation published by State Administration of Market Supervision and Administration and the State Tobacco Monopoly Bureau on 28 August 2018, was in the \u2018Circular of the State Administration of Market Supervision and Administration and the State Tobacco Monopoly Bureau on the Prohibition of the Sale of Electronic Cigarettes to Minors\u201914.In China, e-cigarettes are not medicines, health products, medical devices, or tobacco13.Considering the rapid increase of e-cigarette consumption, some areas in China have already established local e-cigarette use regulations. Hang Zhou city prohibits using e-cigarettes in non-smoking areas32.When searching \u2018electronic cigarettes\u2019 in the \u2018National public service platform for standards information\u2019, we did not find any published guidelines for e-cigarette use. The search results only show two national guidelines that are pending for approval. One was \u2018Electronic Cigarette\u2019 and the other was \u2018E-liquid\u2014Determination of nicotine, propylene glycol and glycerol\u2014Gas chromatographic method\u201933. However, \u2018Guang Ming Daily\u2019, a national, comprehensive and influential daily newspaper directly under the Central Committee of the Communist Party of China with a worldwide readership, reports that some e-cigarettes had high nicotine content, carcinogens, and other toxic chemicals. The high concentration of harmful chemicals in e-cigarettes may cause more harm to humans than traditional cigarettes34. One study in Hong Kong showed that 71.1% of the residents supported the prohibition of e-cigarette promotion and advertisement, 81.5% supported the banning of e-cigarette use in smoke-free venues, 93.9% were in favor of banning the sale of e-cigarettes to minors, and 80.9% were in favor of sale restriction on nicotine-free e-cigarettes. Over half (57.8%) supported all four regulations35. From this study, we learned that most of the residents were in favor of some form of legislation targeting e-cigarette use and sales.Additionally, some studies report that the nicotine concentration in the e-cigarette products was inconsistent with the label36. According to the \u2018Attitudes of Europeans towards tobacco and electronic cigarettes\u2019 report issued by the European Commission in 2017, the awareness ratio of e-cigarettes in European Union (EU) countries increased from 69% (2012) to 84% (2017)37.A meta-analysis of 28 studies from 2009 to 2014 showed that the global average e-cigarette awareness ratio was about 61.2%5. The review of e-cigarette studies at the local level, such as Tianjin and Beijing, demonstrates that the Chinese e-cigarette awareness ratio is lower than the global and average European Union awareness ratios. The awareness ratio of e-cigarettes is slightly different amongst the various regions of China. Differences were predominantly caused by the diversity of economic development and culture in the different regions. For example, the awareness ratio of e-cigarettes among adolescents and adults in Hong Kong16 is about 70%, which is significantly higher than in mainland China (40.5%)5.According to the China Adult Tobacco Survey in 2015, 40.5% of adults aged 15 years and older had heard of e-cigarettes22; the ratio for males is higher than for females22; the ratio of online surveys18 is higher than of offline surveys22. These distribution characteristics are consistent with the conclusions of other international studies. In a study of e-cigarettes among adults in the US, awareness ratio was higher among adults aged 18\u201334 years, males, Whites, participants with some college education, individuals with household incomes above $75000, and those living in the Midwest38. In an Italian study, the awareness ratio was lowest among women, the elderly, those with low education, and non-smokers39. Our results illustrate that the above might correlate with the hypothesis that populations with higher awareness ratio are more accessible to the advertisement of tobacco.The awareness of e-cigarette ratio is also affected by the characteristics of the population. The distribution characteristics of awareness ratio can be expressed as: the awareness ratio of cigarette smokers is higher than that of non-smokers36. A survey of the use of e-cigarettes among US high school students showed that the prevalence of the past 30 days use of e-cigarettes increased ten-fold from 1.5% in 2011 to 16.0% in 2015, exceeding the proportion of traditional cigarettes40. By contrast, the use of e-cigarettes in China is still relatively low (3.1%)5, but its growth trend cannot be ignored11. A large rise in prevalence of e-cigarette use11 combined with a significant smoking population4 has the potential for a e-cigarette use to soar in China in coming years.From a meta-analysis of 67 surveys on the use of e-cigarettes, the overall estimate of e-cigarette use prevalence was 16.8%43. These findings are consistent with survey results in Hong Kong16. However, most people, especially cigarette smokers in mainland China, perceived the use of e-cigarettes as less harmful than traditional cigarettes. Also, most people saw e-cigarettes as an alternative to traditional cigarettes that may help them to quit smoking19. As for adolescents and young adults, most were attracted by novel flavors and the fashionable design of e-cigarettes. Some cigarette smokers believed in advertisements that e-cigarettes did no harm their health and might help them quit smoking.Several surveys reported that the main reasons for using e-cigarettes are that it is fun, enjoyable and cool44 mention that the advertisements of e-cigarettes might remind people to smoke, and subsequently they fail to quit smoking. In our survey, one study suggested that the use of e-cigarettes might help adults quit smoking19. Some studies show that using e-cigarettes were correlated to willingness to quit smoking25. Other studies reveal that e-cigarette users are more likely to smoke30. In 2014, the World Health Organization (WHO) revealed that e-cigarettes had health risks to users and non-users. Besides, there was insufficient evidence showing that e-cigarettes could help quit smoking. One major concern is the conflicting interests in maximizing the potential benefits for smokers while increasing the possibility of encouraging the initiation of nicotine consumption among the non-smokers, especially the younger population. Such concerns are referred to as the gateway and renormalization effects45. The alarming fact that increasing trends in the awareness ratio and e-cigarette use prevalence among adolescents exceed those of adults demands immediate attention from the public and policymakers. From the studies of Wang et al.24 and Xiao et al.22, we find that the use of e-cigarettes may increase the likelihood of cigarette-smoking initiation among never cigarette smoking adolescents.Kim et al.The myths of e-cigarettes create a distorted image of e-cigarettes. Therefore, the government should increase the public\u2019s knowledge of e-cigarettes through health promotion activities and regulate e-cigarette advertisements to protect adolescents and young adults from the influence of misleading information and thus from forming wrong perceptions about e-cigarettes.47; in Europe, the European Union Commission revised its regulations on tobacco products on February 2014 to prohibit e-cigarette manufacturers and traders from advertising their products48. However, in China, e-cigarette marketing and advertisement companies play an active role in fueling the popularity of e-cigarettes. The E-commerce platforms are the main channel for e-cigarette sales. So far, there is no regulatory measure of any impact on the circulation of e-cigarette products in the Chinese market29.In China, e-cigarettes are not drugs, health products, medical devices, or tobacco products, in contrast to the United States where the FDA has classified e-cigarettes as tobacco products. The government of the United States limits the supply of e-cigarettes to consumersOur recommendation, for tackling the increasing demand for e-cigarettes, is that communities, schools and media in China should strengthen health promotion education to prevent e-cigarette use and inform the public, both users and non-users, about the health risks associated with e-cigarette use. The government should launch educational campaigns that target the teenagers and non-smokers who tend to use e-cigarettes out of curiosity. Information should also be disseminated to the cigarette smokers who intend to quit smoking because studies show that this group is susceptible to receiving the wrong information about e-cigarette use, i.e. e-cigarette use helps smoking cessation. On the other hand, to regulate the supply side, the Chinese government should strengthen the auditing of e-cigarette advertisements, avoid the exaggeration of the effectiveness and health \u2018benefits\u2019 of electronic cigarettes through advertisements, and avoid the promotion of e-cigarettes to the under-aged population and non-cigarette smokers. Referring to the policies of other countries, the Chinese government should formulate the regulations of e-cigarettes and publish the standards for e-cigarette production as soon as possible.To summarize, this systematic review indicates that e-cigarette awareness, prevalence, and use prevalence have increased and the use of e-cigarettes may pose harm to the public health. Up until 2018, some provinces and cities in China had launched measures of \u2018e-cigarette smoking ban\u2019 independently. For example, Hang Zhou government prohibits e-cigarette use in the smoke-free environment. It is still a long way to go for China\u2019s government to enact effective national policies to address the increasing use of e-cigarettes amid sparse credible evidence from research.We investigated the awareness of e-cigarettes in China and the existing regulation for e-cigarette use and market. Twenty-one studies were identified, and we provided a comprehensive analysis of the e-cigarette awareness ratio in different regions of China and compared the awareness ratios for gender, age group, and smoking status. We also traced e-cigarette use and risk factors, and the relationship between e-cigarette use and smoking cessation. The prevalence of e-cigarette use in China was found to be increasing. The lack of regulation on e-cigarette use and unrestricted e-cigarette marketing practices have fueled consumption of e-cigarettes and misconceptions about the \u2018benefits\u2019 of e-cigarettes, such as being aids to help cigarette smokers to quit or reduce smoking, less harmful than traditional cigarettes, and an enhancement of personal image. Given the rising prevalence of e-cigarette use and the detrimental effects of misleading information by e-cigarette companies, marketers, and advertisers, it is crucial that the government of China prioritize the establishment and implementation of regulations for e-cigarette use and market.Authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none was reported.Click here for additional data file.Click here for additional data file."} +{"text": "E-cigarettes are increasingly popular among both cigarette smokers and non-users. Although smoking cessation yields the most individual and population health benefits, switching to exclusive e-cigarette use offers some individual health benefits for cigarette smokers. However, e-cigarette use is not harmless, and its use among non-cigarette smokers should be prevented. Our study aims to explore the decision-making process about e-cigarettes among an e-cigarette users, cigarette smokers, and non-users. We conducted 12 semi-structured focus group interviews with e-cigarette users, cigarette smokers, and non-users. We performed a thematic analysis of the interview transcripts. First, knowledge reported by e-cigarette users was mainly based on other users\u2019 experiences. Second, cigarette smokers and non-users were more negative towards e-cigarettes than e-cigarette users. Third, e-cigarette users considered switching from cigarette smoking to e-cigarette use by deliberating relevant information, and weighing up the benefits and disadvantages of e-cigarette use versus smoking. Additionally, important factors in the decision-making process were a perception of risks and benefits of e-cigarettes compared to cigarettes, a supportive social environment about e-cigarette use, and trust in information offered about the risks and benefits of e-cigarettes. Our findings provide insight into what we can learn from the conscious decision-making process of e-cigarette users who switched from cigarettes to e-cigarettes. This information can be considered to develop targeted communications strategies to stimulate a conscious decision-making process, these may highlight benefits of switching to e-cigarettes for cigarette smokers, discussing the risks of smoking, and correcting misperceptions about the perceived risks and benefits of e-cigarette use. Electronic cigarettes (e-cigarettes) vaporize a solution of glycerin, propylene glycol, additives, and sometimes nicotine ,2,3,4,5.E-cigarettes have the potential to reduce the harm of cigarette smoking on smokers\u2019 health . Recent Previous research among different types of users and different age groups has described differences in reasons why initiating e-cigarette use is attractive ,24,25. FA conscious decision-making process is defined by deliberating available and relevant information about the options, and by weighing advantages and disadvantages of the choice in the context of the decision-maker\u2019s attitudes ,29,30. PTo explore the decision-making process about e-cigarette use, we conducted focus groups with three homogeneous groups consisting of adult e-cigarette users, adult cigarette smokers, and adolescent non-users who used neither cigarettes nor e-cigarettes. For each of these types of users, four focus groups were conducted. The study was approved by the Medical Ethics Committee of Zuyderland\u2014Zuyd (16-N-84).In June 2016, participants were recruited in the Netherlands, through an advertisement on Facebook\u00a9, Instagram\u00a9, Twitter\u00a9, and scholieren.com (a website targeted at adolescents in the Netherlands). Inclusion criteria were: awareness of e-cigarettes, being a current smoker (18+), e-cigarette user (18+), or non-user (13 to 17 years old), and fluency in Dutch. Adolescent non-users were selected because previous research showed differences in reasons for e-cigarette initiation among adult e-cigarette users, adult cigarette smokers, and adolescent non-users . SupplemThe group discussions were based on a semi-structured protocol with open-ended questions and minimal steering to allow participants to freely discuss e-cigarettes. The topiThe focus groups were conducted between June and July 2016, and lasted approximately 2 hours. Informed consent was obtained before the start of the focus group and after participation, the participants received an incentive of 30 euros. The focus group started with a general introduction. The discussion proceeded in four steps exploring participants\u2019 knowledge regarding e-cigarettes, their attitude towards e-cigarettes, their deliberation regarding e-cigarette use initiation, and their information needs , four focus groups with adult cigarette smokers (n = 17), and four focus groups adolescent non-users (n = 23). All e-cigarette users reported to be former smokers. Overall, the sample was 47% female and 53% male, the average age of adult participants was 29.3 years , and adolescents 15.3 , and the sample was highly educated . We conducted four focus groups with adult e-cigarette users . \u2018You use an e-liquid, there are a lot of flavors, you can choose whether you want with or without nicotine, and there are many different types\u2026\u2019(#3_smoker about e-cigarettes)Compared to cigarette smokers and non-users, e-cigarette users described more detailed information about e-cigarettes. E-cigarette users reported to have acquired much information in order to make a decision about e-cigarette use initiation, for example, about product characteristics, ingredients of e-liquids, and legislation regarding e-cigarettes. Knowledge reported by e-cigarette users was mainly based on user experiences. When asked how informed they felt, cigarette smokers and non-users stated that they did not search for information about e-cigarettes and expressed they did not know much about e-cigarettes. E-cigarette users felt very informed.Participants shared experiences, both positive and negative, with e-cigarettes and cigarettes. Cigarette smokers and non-users, were negative towards e-cigarette use, in general mentioning it was \u2018weird\u2019 (quote 2):\u2018I think it\u2019s [e-cigarettes] really weird and pathetic. Everybody will be laughing at you.\u2019 (#23_non-user)All groups mentioned that using e-cigarettes has the advantage of avoiding smoking restrictions (quote 3). \u2018It [e-cigarettes] is really easy to use [\u2026] and you can even use it inside, great if you\u2019re in the bus [\u2026] Or you can use it for a just bit, instead of smoking an entire cigarette. I think I\u2019d enjoy that\u2026\u2019 (#17_smoker)Compared to e-cigarette users and non-users, many cigarette smokers were positive towards cigarette smoking. Regardless of the negative health effects associated with cigarette smoking, smokers expressed they really enjoy smoking. Several of the e-cigarette users reported a positive attitude about e-cigarettes and negative attitude towards smoking, based on knowledge acquired beforehand. In general, e-cigarette users emphasized the positive aspects of e-cigarette devices and positive experiences, such as the variety of flavors and the adjustability of nicotine levels. Many e-cigarette users consciously deliberated initiation of e-cigarette use versus continuation of cigarette smoking by using knowledge acquired and weighing up the benefits of e-cigarette use and disadvantages of cigarette smoking. For example, the personal health benefits of e-cigarette use, and the negative health issues for their social environment associated with secondhand smoking. They described this decision process of switching from cigarette smoking to e-cigarette use as a deliberation of benefits and disadvantages (quote 4):\u2018Switching from cigarettes to e-cigarettes [\u2026] it was a deliberate choice. I had an e-cigarette laying around and I tried it a few times. I liked the flavor and the satisfaction it gave me [throat hit from nicotine]. In the end I ordered a really nice one because I decided I was going to quit smoking. So yeah, it was a really deliberate choice\u2026\u2019(#13_e-cigarette user)Many cigarette smokers and non-users reported that they had not contemplated using e-cigarettes. Deliberation was often limited to brief passive moments when they were confronted with an e-cigarette in their environment (quote 5).\u2018One of my friends had one [e-cigarette] and I thought: That might be a way to quit smoking.\u2019(#14_smoker)When asked to compare e-cigarette use initiation with cigarette smoking initiation, e-cigarette users and cigarette smokers agreed that cigarette smoking initiation was not a conscious or deliberate choice. Many cigarette smokers expressed they passively discovered themselves to be smokers months or years after they initiated smoking (quote 6).\u2018Everybody smoked, so I started too [\u2026] there wasn\u2019t a point where I thought: OK, from now on I\u2019m a smoker. All of a sudden, you just are one. Smoking is not a deliberate choice you make, something you really think about.\u2019(#1_smoker)In general, non-users did not show an interest in e-cigarettes or cigarettes. Pros and cons were not actively deliberated among non-users, but sometimes curiosity was reason enough to passively experiment with e-cigarettes or cigarettes. When asked about their information need, all three groups stated they had several unanswered questions, such as, \u2018Are e-cigarettes harmful?\u2019, \u2018What are the benefits of e-cigarette use compared to cigarette smoking?\u2019, and \u2018What are the negative long-term health effects of e-cigarette use?\u2019. E-cigarette users, compared to smokers and non-users had more questions about product characteristics, such as \u2018Are there results of quality tests of e-liquids available?\u2019. Cigarette smokers and non-users wanted to know more about the successful quit attempts with e-cigarettes. They wanted to see a risk\u2013benefit analysis of e-cigarette use versus smoking, and they wanted to know what the average duration of use was for e-cigarette use as a smoking cessation tool. All groups agreed that this information should be communicated to the public to facilitate smoking cessation for cigarette smokers. Many participants explained that there is a large body of information available, but that it is difficult to filter useful and correct information from the internet. This section describes additional aspects participants mentioned that played a role in their decision about e-cigarette use.The risks of e-cigarettes versus cigarettes were discussed. E-cigarette users did not perceive any health risks of e-cigarette use (quote 7). \u2018So I thought: \u2018it\u2019s just vape. That can\u2019t be really harmful for you or your lungs. So if I just vape, and I don\u2019t do it every day [\u2026] that won\u2019t be so wrong.\u2019(#4_e-cigarette user)Cigarette smokers stated that although they knew about the common risks of cigarettes, the specifics of how cigarettes causes smoking related diseases were not clear. They also perceived the health risks of cigarettes as a problem for the distant future, something to worry about later, when they were older. E-cigarette users tended to deliberate the risks and severity of cigarette smoking related diseases and acknowledged that the risks applied to them as former long-time cigarette smokers (quote 8):\u2018My grandfather smoked, like me, and you can see his health is getting worse and worse. He suffers from emphysema, I don\u2019t want that. So I wanted to quit.\u2019(E-cigarette user_#26)While non-users were aware of the risks of cigarette smoking, the risks of e-cigarettes were unclear to them. Similar to e-cigarette users, they mentioned that if it is just vape, it did not seem harmful (quote 7). E-cigarette users perceived e-cigarette use compared to cigarette smoking not as a risk, but as a lifestyle (quote 9):\u2018I often say that besides a hobby it\u2019s [e-cigarette use] also a lifestyle.\u2019(#21_e-cigarette user)Similar to e-cigarette users, cigarette smokers perceived cigarette smoking as an addiction, and they described triggers for the desire to smoke, such as alcohol consumption. Non-users perceived both e-cigarette use and cigarette smoking as addictive behaviors.All groups described the importance of their social environment with regard to e-cigarette use and cigarette smoking. In the initiation of e-cigarette use, the social environment was an important factor for many e-cigarette users (quote 10). \u2018My mother had one [e-cigarette], so I tried it too.\u2019(#17_e-cigarette user)Several e-cigarette users further noted that social support for e-cigarette use from their social environment grew after initiation of use, which they described as a sense of belonging to an e-cigarette community (quote 11):\u2018It\u2019s a community [\u2026] Very similar to bikers! You know how they all greet each other on the road? The vaping community is the same.\u2019(#21_e-cigarette user)In general, participants expressed it was more acceptable to smoke than to use e-cigarettes. While cigarette smokers and non-users categorized e-cigarette use as \u201cweird\u201d, cigarette smoking was still considered as acceptable by the several of participants and their social environment. When asked about the role of their social environment in their decision to initiate cigarette smoking compared to switching to e-cigarette use, cigarette smokers expressed that friends who smoked often influenced their decision (quote 12): \u2018One of my friends told me: \u2018Try a cigarette!\u2019 So I did, and I liked it and I continued smoking.\u2019(#15_smoker)Cigarette smokers did not describe the influence of friends as pressure, but they did describe the disappointment their parents expressed in response to finding out about their smoking behavior as embarrassing (quote 13): \u2018I was really afraid to tell my parents that I smoke [\u2026] they were so disappointed.\u2019(#11_smoker)Non-users viewed e-cigarette use and cigarette smoking not as a social activity, but as peer pressure and addiction, not a choice. Non-users felt strongly about parental disappointment related to cigarette smoking and noted this as a specific reason why they would not start smoking cigarettes or start using e-cigarettes.Due to the large body of information available about e-cigarettes, it was difficult for participants to make sure the available information is correct. E-cigarette users and cigarette smokers mistrusted evidence-based information, and relied on anecdotal user experiences (quote 14):\u2018Researchers often don\u2019t know what they are doing (#20_e-cigarette user) [\u2026] yes we need to know about the experiences of e-cigarette users.\u2019(#16_e-cigarette user)Unlike e-cigarette users and cigarette smokers, non-users explained they rather receive scientific information because they trust this information to be true.We explored the decision-making process of e-cigarette use initiation among adult e-cigarette users, adult cigarette smokers, and adolescent non-users. Compared to e-cigarette users, cigarette smokers and non-users did not make a conscious decision to refrain from e-cigarette use. They have acquired different information and have different attitudes. Several of e-cigarette users perceived the risks of cigarette smoking as personally relevant. They acquired information and formed an attitude about e-cigarettes. Finally, their knowledge, attitudes, pros, and cons were deliberated and a conscious decision was made to initiate e-cigarette use. Additionally, lack of information seeking, perception of risk of smoking related diseases, perception of risks and benefits of e-cigarette use compared to cigarette smoking, support to switch to e-cigarettes, a sense of belonging to e-cigarette users, and trust in information offered about e-cigarettes showed to be important factors for a participant in a decision-making process about e-cigarette use. A possible barrier in the decision-making process is the large body of contradicting information available and lack of trust in scientific evidence. Although e-cigarettes users appear knowledgeable, their reported knowledge did not match scientific consensus. We categorized this type of knowledge as beliefs, and these beliefs were primarily based on anecdotal user experiences found online. This finding matches research that shows that experienced e-cigarette users are eager to share their advice and experiences about switching with cigarette smokers . Due to Cigarette smokers did not contemplate to switch to e-cigarettes, or consciously decide to continue smoking. Our results indicate that though cigarette smokers are aware of the health risks of cigarette smoking, they do not perceive the long-term health risks of smoking as personally relevant. Additionally, they perceive e-cigarettes as weird, smoking as a normal, social behavior , and, poSimilar to cigarette smokers, non-users did not actively deliberate e-cigarette use initiation. Adolescent non-users mentioned that if they were to start e-cigarette use, it would be out of curiosity. The risks of e-cigarette use were unclear to non-users, but because it was just vape, e-cigarette use was perceived as harmless. This lack of perceived personal risks of e-cigarette use may diminish the interest to acquire knowledge about the disadvantages of e-cigarette use. Without information seeking and information about advantages and disadvantages of e-cigarette use for non-users, no deliberation can take place . A lack This qualitative study shows that cigarette smokers and non-users did not consciously deliberate information about the risks and benefits of e-cigarettes use to make a decision to refrain from e-cigarette use initiation. The differences and similarities in the decision-making process of e-cigarette use among the different user groups raise a question. Namely, can insights into the conscious decision-making process of e-cigarettes users who switched from cigarette to e-cigarettes be used to stimulate cigarette smokers to consciously deliberate something they consider \u2018weird\u2019? Further research is necessary to investigate how cigarette smokers can be stimulated to consciously deliberate a switch to e-cigarettes. Because of the explorative nature of this study, the results are not meant to convey generalizability beyond the study population, and thus, these findings are limited to a specific geographic context and time. Respondents were asked to participate in a two-hour discussion, which may have attracted individuals more inclined to talk about cigarette smoking and e-cigarette use. Additionally, we asked e-cigarette users, cigarette smokers, and non-users to consider their decisions about initiation in retrospect, knowledge and attitudes may be different when first faced with the decision. Participants\u2019 knowledge, attitudes, and deliberation process might be influenced by their level of education, and living in different areas of the Netherlands. Further research is necessary to explore the impact of education on a decision about e-cigarettes. To conclude, our exploration of the decision-making process of e-cigarette initiation identifies distinct differences between the decision-making process of current cigarette smokers, e-cigarette users, and non-users. Current e-cigarette users (former smokers) in this study made a conscious decision to switch from cigarettes to e-cigarettes, but current cigarette smokers and non-users did not contemplate or deliberate e-cigarette use initiation. E-cigarettes have a potential public and individual health benefit for cigarette smokers, as switching to exclusive e-cigarette use is less harmful than cigarette smoking. E-cigarette use is not harmless, initiation of e-cigarette use and cigarette smoking should thus be prevented among non-users. Several of the e-cigarette users deliberated personally relevant risks of cigarette smoking, which made them interested in switching to a less harmful alternative. These findings underline the importance to explore the possibility to learn from the decision-making process of e-cigarette users, in order to support cigarettes smokers with their decision about switching to e-cigarettes and non-users with their decision to refrain from e-cigarette use."} +{"text": "Similarly, among dual users, nicotine dependence levels were higher when using an e-cigarette (mean 4.7) compared to using traditional cigarettes . Habits and behaviors associated with the use of e-cigarettes did not differ significantly (p > 0.05) between exclusive e-cigarette users and dual users. The findings suggest that e-cigarettes may have a higher addictive potential than smoked cigarettes among young adults.E-cigarettes are often considered less addictive than traditional cigarettes. This study aimed to assess patterns of e-cigarette use and to compare nicotine dependence among cigarette and e-cigarette users in a group of highly educated young adults. From 3002 healthy adults, a representative group of 30 cigarette smokers, 30 exclusive e-cigarette users, and 30 dual users were recruited. A 25-item questionnaire was used to collect information related to the patterns and attitudes towards the use of cigarettes and e-cigarettes. The Fagerstr\u00f6m test for nicotine dependence (FTND) and its adapted version for e-cigarettes were used to analyze nicotine dependence in each of the groups. The nicotine dependence levels measured with FTND were over two times higher among e-cigarette users (mean 3.5) compared to traditional tobacco smokers (mean 1.6; Nicotine is a highly addictive substance and adolescents and young adults may be at high risk of addiction ,2. NicotElectronic cigarettes (e-cigarettes) are a relatively new form of nicotine delivery device and the prevalence of their use is rapidly growing in many countries ,8,9. E-cDue to uncertainty about the dependency potential of e-cigarettes, we decided to carry out a study aimed to: (1) assess the patterns of e-cigarette use by exclusive e-cigarette users and dual users and (2) compare nicotine dependence levels among young adults who use cigarettes or e-cigarettes. We chose to examine the problem in a highly educated group of university students under the assumption that their perception is less biased by commonly held beliefs and stereotypes of e-smoking safety and addiction mechanisms.This study is a continuation of a survey-based and multicentered international project, the YoUng People E-Smoking Study (YUPESS) . From thn = 93) were invited to participate in the second part of the study. Exclusion criteria included occurrence of any chronic diseases, history of lung diseases , presence of any allergic diseases, any medication intake (current or in the last 2 weeks), acute illnesses or infections in the last 2 weeks, influenza vaccination in the last 2 weeks, and current pregnancy or lactation. Among exclusive e-cigarette users, three met the exclusion criteria and six refused. Finally, 30 exclusive e-cigarette users were included. Among dual users, four met the exclusion criteria. To keep an equal number of participants per group, out of 50 eligible dual users, we randomly selected a group of 30 dual users, using randomization procedures available in the Statistica package. An additional group of 30 people was drawn randomly from a group of cigarette smokers (n = 433).A cross-sectional survey was performed between January and March 2018. Three fields of study were randomly selected from a group of 245 different fields of study within Universities in Katowice. The questionnaire was addressed to all the students within each selected field of study. Data were collected from 3002 participants. Out of all the participants, we identified 39 exclusive e-cigarette users and 54 dual users. All subjects who used e-cigarettes . Statistical significance of differences between continuous variables was analyzed using an independent samples t-test or a Mann-Whitney U test where appropriate. Statistical testing to compare categorical variables was completed using an independent samples chi-square test. We assessed strength of association by calculating odds ratios (OR). We used 95% confidence intervals (CI) and defined statistical significance as p = 0.03). Duration of e-cigarette use was comparable between exclusive e-cigarette users and dual users being 29.0 \u00b1 24.1 and 27.7 \u00b1 17.4 months, respectively (p = 0.6). There were no statistically significant differences in sex, age, or the daily number of cigarettes or e-cigarette smoking sessions between groups (p > 0.05).Of the adults included , the mean smoking duration was 50.0 \u00b1 32.0 months among smokers and 67.3 \u00b1 30.5 months among dual users (p > 0.05) between e-cigarette users and dual users. Users of e-cigarettes (n = 60) consumed an average of 4.2 mL of e-liquid per day, with the most frequently chosen e-liquid being that containing 6 mg of nicotine in 1 mL of e-liquid. None of the subjects used nicotine free e-liquids. The study population was dominated by individuals who prepared e-liquids themselves: 66.7% of e-cigarette users and 74.1% of dual users (p = 0.9) had chosen devices that allow technical modifications of the e-cigarette such as voltage, power, and resistance of the heater.Habits and behaviors associated with the use of e-cigarettes, including nicotine content in the e-liquid, e-liquid consumption, type of e-liquid used, and the number of e-cigarettes used, did not differ significantly . The most common reasons stated for starting to use e-cigarettes were it being less harmful to health, price, and desire to quit traditional smoking. The reasons stated for using e-cigarettes are presented in Almost all exclusive e-cigarette users (96%) had ever tried cigarette smoking. Out of the 60 e-cigarette users, 18.5% initiated nicotine use by e-cigarette. E-cigarette, as a first nicotine product, was declared by 16% of exclusive e-cigarette users and 20.7% of dual users indicated that traditional cigarette smoking offers more smoking satisfaction (46.2%) compared to using e-cigarettes (23%). Also, smoking a cigarette in a group of friends, in combination with a cup of coffee, after a meal, or after sex was associated with a greater subjective feeling of satisfaction compared to the feelings associated with the use of an e-cigarette in the same situations. Dual users indicated that the smell (92.9%) and the taste (82.1%) of e-cigarettes as well as the subjective feeling associated with the inhalation and exhalation of vapor generated from e-cigarettes were the primary advantages of e-cigarettes over traditional cigarettes.p = 0.002; p = 0.03) among dual users. E-cigarette users were more likely to use an e-cigarette in the first 30 min after waking and were more likely to find it difficult to refrain from using e-cigarettes in places where it is forbidden (The average FTND score among exclusive e-cigarette users was over twice as high (mean 3.5 versus 1.6) as among traditional cigarette smokers or financial reasons. The participants consumed relatively large amounts of nicotine-containing e-liquid . More than half of the participants chose technically advanced e-cigarette devices.p < 0.001). Similarly, among dual users, nicotine dependence levels were higher when using an e-cigarette (mean 4.7) compared to a traditional cigarette . The high levels of nicotine dependence observed among young adult e-cigarette users compared to smokers in our study suggest this group may be at high risk of addiction.Our key finding was that nicotine dependence levels measured with FTND were over two times higher among exclusive e-cigarette users (mean 3.5) compared to traditional tobacco smokers compared to dual users, although this was not statistically significant.p = 0.03) [Our findings contrast with results obtained by Gonz\u00e1lez et al. , Etter e = 0.03) . Etter e = 0.03) , althoug = 0.03) . When co = 0.03) . Also, p = 0.03) .Several hypotheses should be considered to explain why we found higher levels of addiction among e-cigarettes users compared to smokers in contrast to findings in studies by Gonz\u00e1lez et al. 2017) , Etter e017 , EttIn our study, e-cigarette users consumed an average of 4.2 mL of e-liquid per day. Slightly lower results were obtained by Hajek et al. at 3.3 mL per day and DawkStrengths of this study include the detailed description of patterns of use of young, experienced e-cigarette users and the investigation of the degree of addiction of e-cigarettes and cigarettes using an established measure of addiction among a key population group\u2014young adults. Further separation of this group into groups of exclusive e-cigarette users and dual users allowed for a fuller understanding of the differences in behaviors and addiction presences between these groups.There are several potential limitations of the current study. First, our study was limited to a small sample of young e-cigarette users from one region of Poland. This is a specific group of e-cigarette users which was identified among the 3002 young adults who participated in an across-sectional survey (YUPESS\u2014Katowice branch), with the sample being representative of young adults with higher education (university students) in Poland but not E-cigarettes are promoted as a safer alternative to traditional cigarettes. This view is also shared by e-cigarette users ,36. ExclIn conclusion, in this work the use of e-cigarettes among young adults was shown to result in higher nicotine dependence levels than nicotine dependence related to tobacco cigarette use. In addition, a stronger dependence on the e-cigarette compared to the traditional cigarette identified in dual users indicates that e-cigarettes may be highly addictive. Further research is necessary to provide evidence on the role of e-cigarettes in smoking cessation, especially in young adults. Social normalization of smoking behaviors by allowing e-cigarette use should be monitored."} +{"text": "Drosophila, reduced IIS activity modulates methionine metabolism, through tissue\u2010specific regulation of glycine N\u2010methyltransferase (Gnmt), and that this regulation is required for full IIS\u2010mediated longevity. Furthermore, fat body\u2010specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt\u2010dependent increase in spermidine levels. We also show that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, we found that increased expression of Gnmt occurs in the liver of liver\u2010specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue\u2010specific modulators of IIS\u2010mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. We here show that, in Drosophila. Increased expression of Gnmt in response to reduced IIS activity is conserved in mammals making Gnmt and spermidine possible future therapeutic targets to ameliorate the effects of ageing.Increased tissue\u2010specific expression of Glycine N\u2010methyltransferase (Gnmt), a central enzyme in the methionine cycle, and the polyamine spermidine modulate lifespan through autophagy in response to reduced insulin/IGF signaling activity in These findings indicate that increased Gnmt protein, specifically in the fat body, can extend lifespan.To determine whether increasing the level of Gnmt in the fat body was sufficient to extend lifespan, we over\u2010expressed Gnmt using a Figure Sa,b and e Figure Sa,b and e2.3dilp2\u20103,5 mutants did not reduce lifespan of wild\u2010type flies , with the double mutants living 33% less long than the dilp2\u20103,5 mutants alone , oxidative and xenobiotic detoxification pathway (glutathione metabolism), and the polyamine pathway . We found no change in the survival of flies over\u2010expressing Gnmt using FBGal4, pumplessGal4, or S106GS drivers, suggesting that resistance to oxidative stress is not causal for the longevity of these flies . This is concordant with the finding that oxidative stress resistance may not be causal for the longevity of IIS mutants , suggesting that resistance to xenobiotics is also not causal for lifespan extension in these flies .Ubiquitous, or fat body\u2010specific, over\u2010expression of Gnmt increases SAM catabolism gas . Together, these findings suggest that neither the TSP nor glutathione production branches of Met metabolism underlie the longevity of Gnmt\u2010over\u2010expressing flies.TSP activity leads to the release of hydrogen sulfide and combines with putrescine to form spermidine, or with spermidine to form spermine via spermidine synthase (SpdS) .Met metabolism also branches, through SAM, into the polyamine pathway Pegg, Figure a. SAM isS) Pegg, . In rodeS) Pegg, . FurtherS) Pegg, , 2009. Idilp2\u20103,5 mutant longevity, we quantified key metabolites of the methionine cycle and of polyamine synthesis . Two metabolites of the Met cycle, Met and SAH, were significantly increased in response to reduced IIS, and the level of SAH, but not Met, was dependent on the presence of Gnmt Figure a in wildt Figure b. Interes Figure b, but TSt Figure b. These t Figure b. This sdilp2\u20103,5 mutant flies with spermidine in the presence and absence of Gnmt (1mM) and quantified associated metabolites and dilp2\u20103,5; gnmt double mutant flies or control food. Expression of atg5 and atg8a was induced in the fat bodies of dilp2\u20103,5 mutants, but not in MiGnmt or MiGnmt,dilp2\u20103,5 double mutant flies compared to controls in the fat bodies of control and dilp2\u20103,5 mutants showed a significant increase in autophagy compared to untreated control flies, increasing to the level of untreated 2.7IRS1 KO (Alfp\u2010Cre::IRS1fl/fl) mice control food, and survival was scored every 2\u20133\u00a0days.Flies were maintained in a controlled temperature environment of 25\u00b0C, 65% humidity with a 12\u2010hr day\u2013night light cycle, and fed standard sugar/yeast/agar (SYA) diet . Larval densities were controlled, and adults were once mated. Female flies were sorted under COFood containing RU486 (200\u00a0\u03bcM) and/or spermidine (1\u00a0mM) was prepared every two weeks from a 100\u00a0mM (in ETOH) or a 1\u00a0M (in ddH2O) stock solution, respectively.Dahw) background. The genotypes used were Dahw, dilp2\u20103,5 mutant , and Mi04290Gnmt mutant in a dilp2\u20103,5 background . For over\u2010expression analysis, UAS\u2010Gnmt Spezialdi\u00e4ten GmbH, Soest, Germany] and water. Mice were sacrificed at 3\u20134\u00a0months. Livers were dissected and snap\u2010frozen in liquid nitrogen.Liver\u2010specific knockout of 4.3TM imaging station, and protein bands were quantified using Image Lab software (Bio\u2010Rad).Total protein (30\u00a0\u03bcg) was loaded and separated on precast TGX gels and transferred to nitrocellulose (0.45\u00a0\u03bcm) membranes . Membranes were incubated (1\u00a0hr) in blocking solution (5% nonfat milk in 0.05% TBST) at room temperature and then in primary antibody anti\u2010Gnmt (sc\u201068871) or of fly anti\u2010Gnmt ) (a generous gift from Prof. Miura), anti\u2010p62 (a generous gift from Prof. Juh\u00e1sz), and tubulin overnight at 4\u00b0C. Membranes were washed three times with TBST (0.05%) for 10\u00a0min. HRP\u2010conjugated anti\u2010mouse or anti\u2010rabbit secondary antibodies were incubated for 1\u00a0hr at room temperature. Blots were then washed three times with TBST (0.05%), and detection was performed with ECL Prime reagent (Amersham). Blots were imaged in a ChemiDoc4.4Total RNA was extracted using TRIzol (Invitrogen) to manufacturers\u2019 guidelines and subsequently treated with DNase I (Ambion). cDNA was synthesized using SuperScript\u00ae VILO Master Mix (Invitrogen) cDNA Synthesis Kit, following the manufacturers\u2019 protocol. Quantitative real\u2010time PCR (qRT\u2013PCR) was performed using SYBR Green probes (Applied Biosystems) on a 7900HT Real\u2010Time PCR System (Applied Biosystems).The primers used in the study are listed below:4.52O2 stress assay, 100 female flies were initially fed either SYA food, RU\u2010486 (200\u00a0\u03bcM), or ethanol control food for 7\u00a0days. Flies were then transferred into fresh vials containing 0.03% DDT mixed in SYA food for xenobiotic stress assay and 1% agar food containing 5% v/v H2O2 for oxidative stress assay, respectively. The number of dead flies was counted four times a day and scored in an Excel sheet.For DDT and H4.62S production, 100 female flies were sorted into vials and fed either SYA food, RU\u2010486 (200\u00a0\u03bcM), or ethanol control food. Flies were transferred into fresh vials every second day. H2S measurements were performed as previously described . H2S production was then assayed by densitometry analysis (ImageJ) of lead sulfide darkening and normalized to protein content.For quantification of H4.7TM I\u2010Class System/XevoTM TQ\u2010S (WatersTM) with MassLynx was used. In brief, 25 snap\u2010frozen female flies were homogenized in 71\u00a0\u03bcl of 15\u00a0mg/ml dithiothreitol (DTT) and 429\u00a0\u03bcl 50% methanol and ~35\u00a0mg of frozen mouse tissues were homogenized in 1ml sample buffer (methanol:H2O:chloroform in 7:2:1 ratio). Homogenates were mixed for 5\u00a0min and centrifuged at 9,300\u00a0g at 4\u00b0C for 15\u00a0min. The supernatant was filtered using 0.2\u2010\u03bcm VWR centrifugal filters at maximum speed at 4\u00b0C for 5\u00a0min. The filtrate was then evaporated using SpeedVac for approx. 2\u00a0hr at 30\u00b0C. The pellet was reconstituted into 100\u00a0\u03bcl of running buffer and filtered again. Each metabolite was run in 1:10 and 1:100 dilutions to meet the standard run range. For absolute quantification of metabolites (Sigma) in positive ESI MRM (multireaction monitoring) mode, an Acquity UPLCTM I\u2010Class System/XevoTM TQ\u2010S (WatersTM) with MassLynx and absolute quantification TargetLynxTM (WatersTM) were used. With settings for capillary kv 1.5, desolvation temp. 550\u00b0C, desolvation gas flow 800\u00a0L/Hr, cone 150\u00a0L/Hr, and collision gas flow 0.15\u00a0ml/min, a SupelcoTM DiscoveryTM HS F5\u20103 Column from Sigma 3\u00a0\u00b5m\u00a0\u00d7\u00a02.1\u00a0mm\u00a0\u00d7\u00a0100\u00a0mm was used at 25\u00b0C. Solvent A was 5\u00a0mM ammonium formate (Sigma)\u00a0+\u00a00.15% formic acid (Sigma) and B acetonitrile (VWR). A gradient from 100% A to 0% in 8\u00a0min at a flow rate of 0.35\u00a0ml/min and an equilibration step from 8.3 to 15\u00a0min were used. The following MRM transitions were used as quantifier (M\u00a0+\u00a0H+)+ for putrescine 89.10\u201330.14\u00a0m/z, l\u2010cysteine hydrochloride 121.92\u201375.93\u00a0m/z, ornithine 133.03\u201370.09\u00a0m/z, spermidine 146.14\u201372.16\u00a0m/z, l\u2010methionine 150.03\u201355.99\u00a0m/z, Cys\u2010Gly 178.85\u201375.93\u00a0m/z, l\u2010cystathionine 222.96\u201387.90\u00a0m/z, y\u2010Glu\u2010Cys 250.96\u201383.94\u00a0m/z, l\u2010glutathione reduced 308.02\u201375.92\u00a0m/z, SAH 385.10\u2013136.38\u00a0m/z, and SAM 399.10\u2013250.03\u00a0m/z. Compounds were dissolved in 5\u00a0mM ammonium formate\u00a0+\u00a00.15% formic acid\u00a0+\u00a0100\u00a0\u00b5g/ml DTT. For all compounds, a calibration curve was calculated. Using concentrations from 5 to 75,000\u00a0ng/ml (prepared from stock solutions 100\u00a0\u00b5g/ml), correlation coefficient: r\u00a0<\u00a00.990; response type: external standard, the peak integrations were corrected manually, if necessary. Quality control standards of each standard were used during sample analysis and showed between 0.5% and 40% deviation, respectively. Blanks after the standards, quality control, and sample batch proved to be sufficient. Absolute value of metabolites was normalized to protein content. Relative fold change value was measured to determine the changes in amount of metabolites.For measurement of metabolites (Sigma), an Acquity UPLC4.8For LysoTracker staining, fat bodies from female flies (10\u00a0days) were dissected in PBS and immediately stained with LysoTracker Red DND\u201099 (Invitrogen) dye (1\u00a0\u03bcM in PBS) for 2\u00a0min. Tissues were then washed three times in PBS and mounted in mounting medium (VECTASHIELD H\u20101200) containing DAPI. Images were taken using confocal microscope (Leica TCS SP5X) with 40x 1.25 oil objective. Laser power and optical settings were kept constant between images. Images were then analyzed using Imaris 8.0 software. The number of LysoTracker puncti and DAPI\u2010stained nuclei was quantified according to user manual guidelines and puncti/nucleus calculated. All image analysis and quantification were performed under blinded conditions.4.9Statistical analysis was performed using GraphPad Prism software 5.0f. Individual statistical tests are mentioned in appropriate figure legends. Lifespan assays and stress survival assays were recorded using Excel, and survival was analyzed using log\u2010rank test.LT conceived of the study, participated in its design and coordination, carried out most of the experiments, and drafted the manuscript. CJ participated in the coordination of the study and experimental work. TN generated mouse tissue. JF participated in experimental work. YH participated in experimental work. SG participated in the design and coordination of the study. LP conceived of the study, participated in its design and coordination, and drafted the manuscript.\u00a0Click here for additional data file.\u00a0Click here for additional data file."} +{"text": "Since the discovery of CRISPR-Cas immune systems, astonishing progress has been made on revealing their mechanistic foundations. Due to the immense potential as genome engineering tools, research has mainly focussed on a subset of Cas nucleases that target DNA. In addition, however, distinct types of RNA-targetting CRISPR-Cas systems have been identified. The focus of this review will be on the interference mechanisms of the RNA targetting type III and type VI CRISPR-Cas systems, their biological relevance and their potential for applications. CRISPR-Cas is a prokaryotic adaptive immune system that is present in approximately half of the currently available bacterial and archaeal genomes. It provides sequence-specific defense against mobile genetic elements (MGEs) such as bacteriophages and conjugative plasmids, and plays a role in host gene regulation .cas genes that are generally located in close proximity to the CRISPR array. Together the crRNA and Cas protein(s) form a ribonucleoprotein (RNP) complex that will patrol the cell. In the interference phase, these RNPs use complementarity to the crRNA to detect recurring nucleic acid invaders and utilize the nuclease activity of the Cas proteins to degrade the invading nucleic acids [The mechanism of adaptive immunity by CRISPR-Cas systems can be divided into three different phases: adaptation, expression/processing, and interference. The defense is initiated by a process called \u2018adaptation\u2019 or \u2018spacer acquisition\u2019 during which short, MGE-derived DNA sequences, called spacers, are stored in repetitive loci on the host chromosome. The spacers are separated by repetitive DNA sequences called repeats, collectively forming the CRISPR array. These CRISPRs function as a genetic memory storage that allows the host to recognize future invasions by previously encountered MGEs. In the second phase, a CRISPR array is transcribed into a long RNA transcript that is subsequently processed into multiple mature CRISPR RNAs (crRNAs) . In addiic acids .cas genes. The latest classification divides these systems into two classes [cas genes. Class 1 systems utilize multisubunit RNP complexes, while class 2 systems utilize single protein RNP complexes.A very broad range of CRISPR-Cas systems has been discovered to date, resulting in a classification system and a common nomenclature for the associated classes , each ofbona fide target . Subtypes III-A and III-B (Csm and Cmr) have been described first, while the less frequently occurring variant subtypes III-C and III-D systems have subsequently been discovered. The signature subunit Cas10 typically contains an HD-type nuclease domain and a GGDD-type cyclase domain . The amino acid sequence of the Cas10 subunit of subtype III-C differs substantially from that of the other type III subtypes, probably reflecting an inactive cyclase-like domain . Subtypein trans and have been shown to utilize mature crRNAs derived from other CRISPR arrays [Cas1 and Cas2 proteins are widely conserved amongst CRISPR-Cas systems and were found to be essential for spacer acquisition . SurprisR arrays ,12,24.A well-conserved feature of all CRISPR-Cas systems is that a complex of Cas1 and Cas2, sometimes complemented with additional proteins, is responsible for the selection of protospacers in invading genomes and the subsequent integration as new spacers in the CRISPR array in the host chromosome. A unique additional mechanism for spacer acquisition by some type III systems was recently discovered. In these specific systems, Cas1 was found to be fused to a reverse transcriptase domain (RT) ,26. Sincin vivo experiments with the type III-A Csm\u2013crRNA complex of Staphylococcus epidermidis indicated that immunity relied on the degradation of target DNA [in vitro experiments with the Cmr complex of the type III-B Cmr in Pyrococcus furiosus, which revealed that not DNA but RNA was specifically degraded [Currently, type III systems are the only CRISPR-Cas systems known to utilize three different nuclease activities for invader neutralization. For many years, various targets have been identified for different CRISPR-Cas type III subtypes. Initial rget DNA . This wadegraded . This apdegraded ,29. Intedegraded ,31. In tThe only sequence-specific degradation facilitated by type III systems is that of the target RNA (containing the protospacer sequence complementary to the crRNA guide). Binding of the target RNA to the crRNA of the effector complex positions the target along the Cas7 subunits forming the RNP backbone ,32,33. SInterestingly, the binding and degradation of the target RNA has been reported to be relatively flexible when it comes to mutations within the protospacer sequence ,22,35,38in vitro studies indeed showed that binding of the RNP to a target RNA promotes DNA cleavage [In addition to specific target RNA degradation, type III systems were found to facilitate immunity through DNA cleavage . A majorcleavage ,35,36. Wcleavage ,35,39. Tin vitro work determined that the nuclease activity preferably targets ssDNA, implicating that transcription is required for the production of target RNA as well as for presenting the DNA in a single-stranded form [in vivo data revealing that the non-template strand was cleaved and further degraded while the template strand remained intact [Mutational analysis showed that the HD domain of Cas10 is the metal-dependent DNase responsible for this activity ,35. Addided form . Upon trded form . This wod intact .bona-fide (invader-derived) target RNA. This issue might be resolved by the spatial\u2013temporal restriction of the DNase activity. Since activation of Cas10 requires the RNP to be bound to the RNA target, the complex is locally restricted to the vicinity of invader RNA transcripts. Moreover, it has been shown that upon cleavage of the RNA target, the cleavage products are released, potentially returning Cas10 to a non-active state [Furthermore, it was shown that the three nucleotides complementarity (rPAM) between the crRNA 5\u2032-handle and the 3\u2032 region flanking the RNA protospacer inhibits DNase activity, thereby generating a mechanism to avoid targetting of the host CRISPR array ,39. Howeve state . This hyve state .In addition to the HD domain mentioned before, Cas10 also contains a palm domain (a domain that has been implicated with cyclase activity in other proteins) harboring a conserved GGDD motif. Although the function of the palm domain in Cas10 has long been enigmatic, recent work has shown that it is involved in the conversion of ATP into a range of cyclic oligoadenylates (c(OA)s) in a metal-dependent way ,31. The Streptococcus thermophilus Csm6 (StCsm6) preferentially cleaves at GA or AA. However, the presence of c(OA)s seemed to reduce this apparent preference [CRISPR-Cas type III systems often harbor genes that encode Csm6 or Csx1 family members ,41\u201343. Teference .6 while Thermus thermophilus Csm6 (TtCsm6) is activated by cA4 [in vivo somehow quench this background activity [Cas10 homologs can produce a range of c(OA)s. For example, StCsm6 is activated by cAd by cA4 ,31. Thisd by cA4 . Notablyd by cA4 . Since tactivity ,41.One of the questions that remain to be answered for c(OA)s-induced RNase activity is if, and how, this activity is restrained to invader transcripts. c(OA)s synthesis is regulated through Cas10 activation, and binding of an RNA target induces c(OA)s synthesis while target cleavage by Cas7 terminates c(OA)s synthesis. It is possible that Csm6/Csx1 activity is spatially restricted to the area of c(OA)s production, and that it is temporally restricted by the half-life of c(OA)s-activated Csm6/Csx1 but additional data are required to confirm this hypothesis. Another possibility is that Csm6/Csx1 is not restricted to intruder RNA at all and that it works as a suicide-switch that protects the population, rather than the individual cell, from infection.Type III systems are the only CRISPR-Cas systems that evolved three entirely different nuclease activities. The question remains whether these different nuclease activities are all required to provide efficient protection against invaders. In this section, we will speculate on the origins of type III immunity.CRISPR systems that adopt two different RNase activities and show spacer adaptation from RNA targets might suggest that RNA invaders are the primary target. The RNase activity of Csm6/Csx1 could ensure complete clearance of the invading RNA, while the Cas7 could facilitate temporal control of this activity. In this scenario, Cas7-mediated RNA cleavage is followed by the release of the RNA degradation fragments from the complex, thereby down-regulating the second messenger (c(OA)s) synthesis. The DNase activity could be accounted for by the fact that some RNA phages replicate through a DNA intermediate. The fact that type III systems can indeed convey resistance to RNA phages supports this hypothesis . A countOne aspect of CRISPR-Cas immunity that is often overlooked is that it can target any intruding MGE, regardless of whether the cargo is harmful or beneficial. Temperate phages can proliferate through two different cycles, a lytic cycle that includes avid expression of its genetic information and results in cell death, and a lysogenic cycle in which the viral DNA is incorporated in the host genome and relies on host proliferation for replication. While the lytic cycle is deadly for the host and should be combated, the lysogenic cycle allows for potentially beneficial genes to be incorporated in the host genome ,47. TargType III systems are surprisingly flexible regarding mutations in the target sequence ,35,38. DConsidering that type III systems rely on two sequence-independent nucleases open up the possibility to use it as a \u2018suicide-switch\u2019 or whether it is necessary to cope a high concentration of invader RNA/DNA. It does not seem likely that this is the natural purpose of the system since Cas10 activity is switched off after target RNA degradation and release. Even though it is not currently known how long synthesized c(OA)s will remain present in the cell after Cas10 deactivation, and how long c(OA)s-bound Csm6/Csx1 stays active, thus far, there have been no reports on Csm6/Csx1 causing cell death.Unlike any other CRISPR-Cas system, type VI effector proteins have been demonstrated to exclusively cleave RNA targets . In factLeptotrichia shahii (LshCas13a), Lachnospiraceae bacterium (LbaCas13a), and Leptotrichia buccalis (LbuCas13a) [Currently, there are three published crystal structures of type VI-A Cas13a proteins: Cas13a from uCas13a) . Akin touCas13a) ,59. InstuCas13a) . The NTDuCas13a) ,57.trans-activating crRNA (tracrRNA) or host factors [438 and Lys441 in LshCas13a) abrogated pre-crRNA processing, suggesting that this domain is responsible for the formation and binding of the 5\u2032-handle. In agreement with the lack of conservation of some of these residues, the length of the 5\u2032-handle (determined by the cleavage site) can vary between different Cas13 homologs [In agreement with the absence of a dedicated pre-crRNA processing nuclease (i.e. Cas6 in type I and III systems), the type VI pre-crRNAs are processed by the Cas13 itself . Typical factors . Mutagenhomologs ,60. Aftebona-fide target substrate for type VI CRISPR-Cas systems. Indeed, a heterologously expressed type VI CRISPR-Cas system in Escherichia coli conferred sequence-specific immunity against the lytic phage MS2 (an ssRNA phage that does not have a DNA stage within its life cycle), indicating that Cas13 specifically targets ssRNAs [The presence of two HEPN RNase domains in Cas13 suggested that RNA is the s ssRNAs ,51.cis (provided that the target RNA is long enough to reach the active site), but also conferred promiscuous RNase activity, causing the \u2018collateral damage\u2019 to other non-target RNAs in trans. Although most RNAs appear to be vulnerable to the promiscuous RNAse activity of Cas13, there are subfamily-specific differences in the nucleotide preferences (e.g. targetting mostly adenosine or uridine-rich RNAs) of these ribonucleases [However, it was noted that Cas13-mediated immunity against phage MS2 was accompanied by a growth suppression phenotype. Subsequent biochemical and structural studies unraveled the molecular basis for this phenomenon. It was shown that binding of its cognate target RNA (i.e. a ssRNA complementary to the bound crRNA) converts Cas13 into a sequence non-specific ribonuclease, conferring rapid degradation of neighboring RNAs, an activity often referred to as \u2018collateral damage\u2019. Target RNA binding results in the formation of an A-form dsRNA helix (crRNA\u2013target RNA) which causes substantial conformational changes in Cas13a to accommodate the propagating duplex within a positively charged channel in the NUC lobe. Concomitantly, these rearrangements bring the catalytic residues of the two HEPN domains into close proximity, thereby forming a single, composite catalytic site for RNA cleavage . Strikinucleases ,60,61.csx27 and csx28 appear to have a regulatory role by either repressing or stimulating the collateral activity of Cas13b, respectively [in vitro and in vivo and this result was further supported by crystal structure study of target-bound LbuCas13a [These observations gave rise to several questions regarding outcome on the individual cell undergoing type VI immunity, similar to those arising from the observed collateral damage activity by CRISPR-Cas type III immunity described above. For example: is the collateral damage caused by Cas13 aimed at cleaning up local, high concentrations of foreign RNA after binding its target RNA, or does it contribute to thwarting the phage infection by inducing programed cell death or dormancy ? Regardlectively . Althougectively . MutagenbuCas13a ,56. In abuCas13a ,51,63. IbuCas13a ,56.in vivo RNA editing [These strict requirements dictating the specificity of Cas13 are perhaps counterbalanced by its unprecedented sensitivity to recognize specific target RNAs that meet these criteria within a heterogeneous population of non-target RNAs. It has been reported that Cas13 can detect target RNAs with femtomolar sensitivity , which opened up opportunities to adopt Cas13 for nucleic acid detection applications, for example in disease diagnostics ,65. In a editing . At lastMarinomonas mediterranea [Next to the questions surrounding the biological role and promiscuous ribonuclease activity of Cas13, it is also still open for debate how type VI systems acquire spacers. PSI-BLAST and HHpred analyses suggested that some of the subtype VI-A loci have adaptation modules, indicating that these type VI systems can acquire new spacers from RNA bacteriophages directly . Howeverterranea . NeverthAlthough type III CRISPR-Cas systems were amongst the first to be characterized, only recently are we starting to develop a thorough understanding of how these systems operate. Type III systems are endowed with many different sequence-specific and sequence non-specific (deoxy)ribonuclease activities, which are co-ordinated in a highly organized fashion. The cue for all these activities is the detection and binding of the target RNA, and in this respect, shows conceptual resemblance to the evolutionary distant type VI system . Both systems seem to confer \u2018collateral damage\u2019 toward cellular RNAs, and as outlined in the review, it remains elusive whether this is controlled in a spatial and/or temporal fashion (i.e. to combat phage infection), or whether it kills the infected cell for the sake of the population by inducing programed cell death. The recently discovered involvement of second messenger signaling in conferring this behavior in type III interference was unexpected and intriguing, as it is reminiscent of the usage such molecules in mammalian innate immunity. It also illustrates that there still might be many unexpected aspects in CRISPR-Cas immunity that remain to be discovered."} +{"text": "Nano-clusters are acclaimed to be very efficient absorbers of intense femto second light due to dominant collective mechanisms. Enhanced near 100% absorption due to collective linear plasma resonance compared to a small fraction of absorption by unclustered gas was an important drive in nano-plasma studies. Contrary to such perception, we show that if the pulse duration is (<100\u2009fs), absorption is same irrespective of whether the systems are condensed to large clusters or not. So long as there are same number of similar ionizable systems in the focal volume, absorption is the same and such absorption can be accounted for by single particle response to the field and collisional ionization of atoms. Short pulse absorption by the single particle response can be comparable to the linear plasma resonance absorption for smaller clusters. Of these, the study with matter in the form of nanoclusters4 has its own special place. Having the unique features of solid-like local density confined to dimensions smaller than the laser wavelength and absence of the usual energy dissipation channels of bulk condensed media, allows one to study intense laser plasma physics with much lesser complexity. A combination of these properties result in high absorption5, highest charge densities in the plasma7, MeV ions with only keV electrons9, sub-ps bursts of hard x-rays10 and even acceleration of neutral atoms and negative ions12.The interaction of high intensity(>105. Current understanding of the interaction of intense laser pulse with clusters based on many important studies in this field can be summarised into the following steps: (a) Intense laser pulse ionizes atoms by field ionization (b) Electrons released from atoms are driven by the laser field and contribute to collisional ionization. (c) Increase in electron density leads to formation of a plasma and collective absorption mechanisms like linear plasma resonance begins to enhance absorption of light. (d) Cluster expands/explodes both under hydrodynamic13 or Coulomb15 pressure finally releasing high energy ions and electrons. The first two steps contribute to the absorption of the laser light by single particle response to the external field and is referred to as the \u2018non-collective\u2019 absorption mechanism in this paper. The only major collective absorption mechanism we refer to in this paper is the linear plasma resonance mentioned in step-(c). Here resonant matching of the laser and local plasma frequency lead to boosting the local electric field and an enhanced energy transfer from the electromagnetic field to the plasma fields by increased charge separation and electon-ion collisions. This is referred to as collective because the local field inside the cluster is a function of the dielectric constant, a bulk property.A near 100% absorption when the Xe or Ar gas atoms is condensed to nano-clusters as compared to Ne or He that form very small or no-clusters has driven the nano-plasma science4, still many questions remain for want of answers. Are collective mechanisms important at all for nano-clusters with very short laser pulse interaction? Is absorption by an enhanced local field, a result of collective mechanism of the nanoplasma, the only way of large energy deposition in laser cluster interactions? Answer to many to these questions are often sought by computational models4. Particle-in-cell simulations or analytical computation schemes are used to switch on or switch off the collective mechanisms and the role of the laser energy absorption in the different steps is sought. In the many experiments performed in these systems, it appears that there is no clear strategy to experimentally differentiate absorption due to non-collective processes from collective absorption. We demonstrate in this paper, that a comparative study of laser absorption using Ar and N2 as target gases under the same conditions, provides a clear strategy to probe some of these important questions. We clearly establish that for 800\u2009nm pulses shorter than 100\u2009fs and having pulse energies of ~3.0 mJ focussed to intensities ~1016 W/cm2, absorption is predominantly by single particle response to the external field (steps a and b mentioned above). Absorption for short pulses is the same so long as the same number of atoms/molecules are present in the focal volume irrespective whether they are bound into van der Waals clusters or not. The short pulse absorption is dominated by the single particle response which itself can be as large as that due to linear plasma resonance. Only for longer pulse duration and larger clusters, does the effects of collective mechanisms have any significant contribution in influencing the absorption of energy and ability of Ar to form bigger clusters makes little difference to the absorption. It appears that the absorption is only dependent on the number of atoms of Ar and N2 present in the focal volume. Since, the ionization potentials for the two gases are very similar (one reason why they were chosen in the first place) and the number of the atoms in the focal volume is only dependent on pressure, same absorption for such two gases can then be comprehended if the absorption is predominantly due to the single particle response to the external field with little influence of collective behaviour.Conventional wisdom in the nano-clusters studies covers pulse durations larger than 100\u2009fs. On this timescales one would expect a larger absorption in a cluster aggregates2 has similar first few ionization energies. The first two ionization potentials for Ar are 15.75\u2009eV and 27.69\u2009eV and for N2 they are 15.58\u2009eV and 27.9\u2009eV, respectively16. Higher ionization potentials computed for N2 by the dissociation limits of the atomic ions are slightly higher than that of Ar. Ionization to these states are by barrier suppression or by field driven electron impact ionization. The thresholds for barrier suppression to a 7+ charge state for Ar is about 2.0\u2009\u00d7\u20091016 Wcm\u22122 and it is 2.6\u2009\u00d7\u20091016 Wcm\u22122 for N2. So it would make little difference in ionizability between N2 and Ar, especially for the intensities used here. Computation of the cross sections for electron impact ionization accounted by Lotz formalism17 show that the cross section for ionization of Ar and N2 for 1.2\u2009keV electrons (ponderomotive energy at 2.0\u2009\u00d7\u20091016 Wcm\u22122) differ between 15\u201320%. So if the absorption is due to field driven single particle response , Ar and N2 should be little different, irrespective of the fact that Ar is in the form of clusters while N2 is expected to only have a small fraction of much smaller clusters. Similar absorption with N2 and Ar shown in Fig.\u00a02, unlike that in Fig.\u00a02 reaching a saturation value. It can also be seen that as the pulse width is made larger, the absorption increases even more steeply and saturation is attained with even smaller size clusters or at a lower backing pressure. This and the absence of such a trend in N2 is indicative of collective mechanisms at play in this regime because while Ar forms larger clusters, the poorly aggregated N2 molecules do not allow for such processes and fail to reach the collective resonance leading to increased absorption.Ar and N2 as a continuous function of pulse width. This is presented in Fig.\u00a02 clusters there is a systematic reduction in absorption with increase in pulse width. However, for Ar a different feature appears. For very short pulses (23\u2013100\u2009fs) the absorption is similar to that of N2. Absorption decreases till about 100\u2009fs but after that there is a reversal in the trend and absorption increases again with increase in pulse width. Depending on the cluster size, the absorption increases to a peak value and further increase in pulse width decreases the absorption. The raise in absorption, peaking to large value depending on the cluster size and decrease further, is a well known feature of linear plasma resonance in clusters. This has been reported before with laser pulses of longer duration5 and is comprehended in many computation models18. It is attributed to the occurrence of a resonant coupling of the laser light with the collective oscillatory motion of the electrons having a characteristic natural frequency, the local plasma frequency. The field inside the cluster nanoplasmas that are much smaller in size as compared to the laser wavelength can be described as5\u03b5 and v are the plasma dielectric constant and the electron-ion collision frequency respectively and E0 is the laser electric field. With the internal electric field defined as above, the rate of energy deposition in the clusters, can according to the Poynting\u2019s theorem, be written in terms of the rate of change of the energy density U as,\u03b5 and the plasma frequency \u03c9p,3 gives a good exposition of this plasma resonance scheme. Owing to the much slower motion of the ions and the trapping of the quasi-free electrons in the potential of these background ions, the initial ionization of the cluster medium generates an almost solid density plasma with an ne close to 1023 cm\u22123 and thus a plasma frequency in the ultra-violet. But as the hydrodynamic forces expand the clusters over a few hundred fs(s) and the ne decreases to 3nc, where nc is the critical density for 800\u2009nm, both the field inside the cluster nano-plasma as well as the rate of energy deposition reaches a maximum. This can be easily verified by plotting Eq.\u00a0e/nc. This resonance clearly is a collective phenomena as indicated by the inclusion of collective properties like the dielectric constant in the equations above. The energy deposited due to these processes should also likewise be dependent on the size of the collective systems. Thus linear plasma resonance heating mainly due to the inverse Bremsstrahlung processes4 gives the maximum absorption.It should be kept in mind that the maxima of absorption due the linear plasma resonance and the resonance widths are strong functions of the laser intensity, wavelength and the cluster size. The physics issues brought out in this papers are limited to smaller clusters (<1/10 of the laser wavelength) and in the non-relativistic intensity regime (1015\u201317 Wcm\u22122). However if the cluster expansion is too slow, it is possible that the critical density is not reached before the end of the laser pulse and there is no resonance absorption. For smaller clusters, formed especially with N2, this resonance mechanism does not appear to affect the absorption significantly as it does for the larger Ar clusters at longer pulse duration. The expansion velocity in coulomb explosion depends on both the charge (average charge/atoms times the number of atoms/cluster) and mass of the atomic systems. Smaller cluster with smaller total Coulomb charge will expand slowly, even if the mean charge per atom is the same. For the same backing pressure, the energy of N is several times smaller than that of Ar.19. As a result, N2 clusters do not exhibit collective behaviour for the range of pulse duration studied here. This is clearly seen in the data presented in Fig.\u00a02 while for Ar in Fig.\u00a0In order to present the complete picture of the transition from one regime to another, a scan of the pulse durations was conducted to measure absorption in Ar and N20 could also be safely disregarded because of the similitude of the absorption values between the smaller N2 and larger Ar clusters as observed in this regime.A reversal and increase in absorption as the pulse duration is made shorter than 100\u2009fs, not withstanding the changes in the intensity, is contrary to the expectations of linear plasma resonance absorption in clusters and has not been reported in contemporary literature. Short pulse absorption clearly depend only on the number of atoms present in the focal volume and can be attributed to the field dependent ionization of atoms, without the influence and modifications due to collective properties. Possibilities of anharmonic resonance due to increase in intensities10 Wcm\u22122, ionization is possible by tunnel ionization. The probability of ionization of an atom at a given position is computed using ADK tunnel ionization which is given to be to be21\u03c9 is the atomic frequency, E is the laser electric field, and Ip is the ionization energy. At higher intensities, beyond the over barrier ionization threshold22 we use the direct ionization rate. The corresponding energy from the field is depleted so that in the next spatial slice of the laser pulse propagation is at lower laser intensity. The electrons generated at that point is assumed to be ponderomotively accelerated by the field and energy dispersed in collision ionization is calculated using the lotz formula17. The energy transferred to the atom by laser drive electron-impact ionization is again depleted. In the next spatial slice the laser pulse propagates with the corrected reduced intensity and similar computation is repeated. At the end of the propagation, the energy remaining in the field gives the unabsorbed light (It) transmitting out of the focal area. Absorbance is computed as (I0\u2009\u2212\u2009It)/(I0). Thus the total absorption of light is computed by integrating the fractional absorption through the different intensity regions and also accounting for loss in propagation through each cross-section.A computation of the absorption by field ionization is presented in Fig.\u00a02 has small clusters is also incorporated by taking the appropriate electron density the clusters expand in time. To account for cluster expansion the hydrodynamic expansion rate has been considered with the average electron temperature taken to be about the ponderomotive energy.The experimental measurements for N2 at a pulse valve backing pressure of 40\u2009bar is normalised with the computed absorption for the shortest pulse as shown in Fig.\u00a02 can be effectively modelled as systems where the primary mode of light absorption is only due to the single particle - field responses: multi-photon ionization, over the barrier ionization and field driven electron impact ionization. We note that the computed absorption for pulse duration around 100\u2009fs is smaller than that from experiments. The model that assumes only atomic ionisation processes does not include collective contributions like for example, there could be longitudinal drift of electrons as the laser propagates. For the experimental conditions used, there will be small N2 clusters and at larger pulse duration there will be some contribution to the absorption by collective mechanisms. The model that does not account for these effects is expectedly shows smaller absorptions than the experimental measurements. This difference is evidently much smaller in the very short and very long pulse durations where collective effects should not make much difference. Till about 100\u2009fs where the absorption becomes minimal absorption at 25\u2009fs is same as it is at 250\u2009fs. Larger the cluster, stronger is the absorption due to collective mechanisms. At 30\u2009bar or Ar\u2009\u2243\u2009122, 500 atoms, absorption at 25\u2009fs is 60% as compared to 75% at 700\u2009fs. The shaded region in the Fig.\u00a02 and Ar respectively.Also as is seen, for smaller clusters absorption due to collective mechanisms is comparable to the absorption due to single particle response. Figure\u00a013. So models with non-uniform density are proposed such that the resonance condition is met for longer duration as different radial expansion zones come into resonance. It may be anticipated such a non-uniform expansion feature should also work for shorter pulse duration and there should have been signatures of resonance absorption even with shorter pulses. The experiments shown here does not support this possibility. For pulses shorter than the 100\u2009fs and in the parameter space explored here, absorption is characteristically different and seem understandable well by single particle response to the external field. We note that the short pulses here in fact have higher intensity so the ability to produce stronger ionization is only larger. For clusters upto about 5\u2009nm, absorption due to single particle response is comparable to that from the collective mechanisms in a direct experimental setting. We also note that recently theoretical simulations in the 5\u2009fs pulse regime20 have demonstrated the possibility of frequency shifts in anharmonic plasma resonance absorption. The present experiments used longer pulse durations So, although the linear plasma response phenomenon is understood there are difficulties in comprehending the magnitude of absorption. An expanding nano-plasma passes the resonance point for a very short duration and power transferred to the cluster ensemble is not large enough to account for the x-ray/electron emission5. N2 with very similar ionizability as Ar works well as a system to compare the absorption due to collective and non-collective mechanisms.To summarize, intense ultrashort pulses impinging on matter are absorbed to start with by single particle response to field like multi-photon ionization, tunnel ionization or over the barrier ionization. Collisional ionization by the field driven electron further contribute to absorption by single particle response. Once the electron density increases and if the plasma frequency matches with the laser frequency, linear plasma resonance enhances the laser absorption. Our experiments show that in nano-clusters upto a size of about 14\u2009nm, for pulse width shorter that 100\u2009fs, absorption is predominantly due to the single particle response and there is a very little contribution to absorption by collective mechanism. Larger pulse duration with Ar clusters do show linear plasma resonance effect very similar and in concurrence to the previous experiments16 Wcm\u22122 intensity. The cluster jet is produced using a pulsed solenoid valve fitted with 500\u2009\u03bcm conical nozzle. The mean size of the clusters in the beam is systematically varied by varying the backing pressure of the pulsed solenoid valve. The experiments are carried out in the regime where there is near 100% clusterisation with Ar and clusterisation is about 86% in case of N223. The cluster systems are characterised by Rayleigh scattering experiments carried out on this supersonic jet system earlier24.The mean cluster radius \u2329r\u232a and number of atoms per cluster \u2329N\u232a for the gases used Ar and N2 was estimated using the Hagena scaling parameter \u0393*25. The pulsed valve is operated with a repetition rate of about 3\u2009Hz and is synced to operate with the laser so that every gas pulse would interact with one laser pulse. We compare the signal level of the laser pulse that interacts with the cluster jet and those that does not, using a photo-diode placed after the region of interaction. The scattering losses are less than 5% as measured by a photodiode calibrated to a power meter. The average reduction in transmission as measured over The experiment is performed with an 800\u2009nm 3.5 mJ laser having a 1\u2009kHz pulse repetition rate and generates shortest pulses of 23\u2009fs that are focused onto a cluster jet using an off-axis parabolic mirror to achieve 5\u2009\u00d7\u2009102 have very similar ionization potentials. Ar with larger polarizability forms large clusters even at low pressure in the backing chamber while on the other hand N2 has very low polarizability and does not efficiently form clusters. At any given backing pressure and identical operating conditions , the number of the atoms in the focal volume would be same except that in the case of Ar there would be nano-clusters and in the case of N2 it would be nascent molecules along with a smaller number of smaller clusters. Absorption of light in these two systems is the measure that we use to probe the role of collective mechanisms or the lack of it.The key strategy in this experiment is the choice of the gases made for comparison. Both Ar and N"} +{"text": "TP63 gene, which encodes the p63 protein, is involved in multiple biological processes, including embryonic development and tumorigenesis. \u0394Np63\u03b1, the predominant isoform of p63 in epithelial cells, acts as an oncogene in early\u2010stage tumors, but paradoxically acts as a potent antimetastatic factor in advanced cancers. Here, we report that \u0394Np63\u03b1 is a direct target of hsa\u2010miR\u2010522 (miR\u2010522). Induced expression of miR\u2010522 reduced the levels of \u0394Np63\u03b1, predisposing breast epithelial cells to a loss of epithelial and acquisition of mesenchymal morphology, resulting in accelerated collective and single\u2010cell migration. Restoration of \u0394Np63\u03b1 repressed miR\u2010522\u2010induced migration. Interestingly, overexpression of miR\u2010522 did not affect breast epithelial cell proliferation, suggesting that miR\u2010522 acts specifically through \u0394Np63\u03b1 in this context. Furthermore, expression of miR\u2010522\u20103p and p63 was negatively correlated in human cancer samples. Thus, miR\u2010522 might be a causative factor for breast tumorigenesis and cancer metastasis. In summary, our results reveal a novel miR\u2010522/p63 axis in cell migration and thus suggest a potential strategy for therapeutic treatment of cancer metastasis.The p63 is a p53 family member with pivotal roles in tumorigenesis. Here, we report that hsa\u2010miR\u2010522 (miR\u2010522) is a direct regulator of p63. miR\u2010522 could promote scattering and migration of mammary epithelial cells through repressing \u0394Np63\u03b1. The expression of miR\u2010522\u20103p and p63 was negatively correlated in human cancers. The miR\u2010522/p63 axis might be a potential therapeutic target for cancer metastasis. EMTepithelial\u2013mesenchymal transitionFforwardmiR\u2010522hsa\u2010miR\u2010522RreverseSCCsquamous cell carcinomaTCGAThe Cancer Genome AtlasTGF\u2010\u03b2transforminggrowth factor\u2010\u03b2TNBCtriple\u2010negative breast cancerWTwild typeTP63 gene, which encodes the p63 protein, is a member of the tumor suppressor TP53 family with fundamental roles in multiple biological processes, including embryonic development, cell proliferation and tumorigenesis [TP63 gene generates distinct protein isoforms, whereas \u0394Np63\u03b1, which lacks the N\u2010terminal transactivation domain, is the predominant one in epithelial cells and epithelial\u2010originated tumors [The igenesis . Alternad tumors .\u0394Np63\u03b1 may act as either a tumor promoter or a suppressor based on precise cellular scenarios. High expression of \u0394Np63\u03b1 is frequently observed in early\u2010stage cancers and function to stimulate tumorigenesis. It may enhance proliferation and survival of squamous cell carcinoma (SCC) through antagonizing p53/p73 signaling , 5. HoweIn contrast, growing modulators have been identified that can regulate \u0394Np63a to control the cell motility and cancer metastasis. Oncogenic PI3K, HRAS and ERBB2 have been reported to be able to suppress \u0394Np63\u03b1 expression to promote EMT and metastasis of lung and breast cancers . TGF\u2010\u03b2 aAmong these factors, only a few microRNAs have been identified to be able to modulate the expression of p63 to regulate cellular migration. miR\u2010301a is able to directly inhibit p63 expression and induce EMT and invasion of prostate cancer cells . In conthsa\u2010miR\u2010522 (miR\u2010522) is a member of the primate\u2010specific chromosome 19 microRNA cluster , 26, 27.in\u00a0vitro and in\u00a0vivo. The system includes Tet\u2010on and Tet\u2010off types, based on whether gene expression is allowed in the presence or absence of Tet or its derivative doxycycline [The Tet\u2010responsive expression system has been widely applied in biological studies to control the timing of gene expression ycycline . The Tetycycline , 39.Here, we report that miR\u2010522 is a regulator of \u0394Np63\u03b1. The expression of \u0394Np63\u03b1 was directly repressed by miR\u2010522 in MCF10A, MCF7 and MDA\u2010MB\u2010231 breast epithelial cells. To examine the role of miR\u2010522 in breast tumorigenesis, we used a Tet\u2010on system to drive the inducible expression of miR\u2010522. The results showed that overexpression of miR\u2010522 led to morphological scattering and increased migration of breast epithelial cells, which could be interrupted by reintroduction of \u0394Np63\u03b1. miR\u2010522 did not change the proliferation of these cells. Moreover, the expression of miR\u2010522\u20103p was found to be inversely correlated with p63 RNA and protein levels in human cancers.\u22121 epidermal growth factor , 100\u00a0ng\u00b7mL\u22121 cholera toxin , 10\u00a0\u00b5g\u00b7mL\u22121 insulin , 500\u00a0ng\u00b7mL\u22121 hydrocortisone and 5% of horse serum (Invitrogen). MCF7 breast cancer cells were cultured in minimum essential medium supplemented with 10% FBS and 10\u00a0\u00b5g\u00b7mL\u22121 insulin. MDA\u2010MB\u2010231 breast cancer cells, HEK293 cells and HeLa cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium with 10% FBS .Human mammary epithelial cells MCF10A were cultured in Dulbecco\u2019s modified Eagle\u2019s medium/F\u201012 (1\u00a0:\u00a01) with 20\u00a0ng\u00b7mLMicroRNA screen vectors were generated by PCR amplification and subcloned into pLenti\u2010m3\u2010blasticidin vector. TP63\u20103\u2032 UTR sequence was amplified by genomic PCR using primer pair TP63\u20103\u2032 UTR\u2010forward (F) and TP63\u20103\u2032 UTR\u2010reverse (R), and then cloned into pmirGLO Dual\u2010Luciferase reporter to construct TP63\u20103\u2032 UTR\u2010wild type (WT) vector. Vectors with deletion of the predicted binding site of miR\u2010522\u20103p in TP63\u20103\u2032 UTR, namely, TP63\u20103\u2032 UTR\u2010\u22061\u20104, were constructed using primer pairs: 522DL1\u2010F and 522DL1\u2010R, 522DL2\u2010F and 522DL2\u2010R, 522DL3\u2010F and 522DL3\u2010R, and 522DL4\u2010F and 522DL4\u2010R, respectively. To construct the vector with inducible expression of miR\u2010522, we amplified genomic DNA using primer 522TRE\u2010F and 522TRE\u2010R and cloned into pLVX\u2010TRE3G vector of Lenti\u2010X\u2122 Tet\u2010On 3G Inducible Expression System . All primers are listed in Table\u00a0HEK293 or HeLa cells were cotransfected with 400\u00a0ng microRNAs and 100\u00a0ng TP63\u20103\u2032 UTR\u2010WT, TP63\u20103\u2032 UTR\u2010\u22061\u20104 or pmirGLO vector alone. Cells were harvested 24\u00a0h later and lysed in Passive Lysis Buffer. Lysates were analyzed for firefly and renilla luciferase activities using the dual\u2010luciferase reagent assay system according to the manufacturer's instructions. Relative luciferase activity was calculated as the ratio of firefly/renilla activities and normalized to control.\u22121 neomycin to get stably infected cells. These cells were then infected with pLVX\u2010TRE3G\u2010microRNA viral particles and selected with 2\u00a0\u00b5g\u00b7mL\u22121 puromycin and 1\u00a0mg\u00b7mL\u22121 neomycin to establish the inducible cell lines.For viral harvest, 293T cells were transfected with pLVX\u2010Tet3G or pLVX\u2010TRE3G\u2010microRNA constructs along with psPAX2/pMD2.G packaging vectors by Lipofectamine 2000 . Forty\u2010eight\u00a0hours later, the media were collected and filtered to remove debris. Viral particles were then concentrated by ultracentrifugation . MCF10A, MCF7 or MDA\u2010MB\u2010231 cells were infected with pLVX\u2010Tet3G viral particles and selected with 2\u00a0mg\u00b7mLWestern protocols were described previously . Antibod6 (MCF10A and MDA\u2010MB\u2010231) or 1.5\u00a0\u00d7\u00a0106 (MCF7) cells per well. Twelve hours later, the monolayer of cells was wounded by performing a scratch with a sterile 200\u2010\u03bcL micropipette tip. The cells were washed with PBS and further cultured in media with 2% horse serum (MCF10A) or without serum (MCF7 and MDA\u2010MB\u2010231). Wound areas were monitored for 24\u00a0h (MCF10A and MDA\u2010MB\u2010231) or 72\u00a0h (MCF7).Cells were seeded into six\u2010well plates at a density of 1\u00a0\u00d7\u00a0104 for MCF10A, 2\u00a0\u00d7\u00a0105 for MCF7 and 2.5\u00a0\u00d7\u00a0104 for MDA\u2010MB\u2010231) were plated into the upper chamber with 200\u00a0\u00b5L serum\u2010free medium; 500\u00a0\u00b5L complete medium was added to the lower chamber as a chemoattractant. After incubation for 24\u00a0h (MCF10A and MDA\u2010MB\u2010231 cells) or 6\u00a0days (MCF7 cells), cells were fixed with methanol and stained with 0.1% crystal violet. Migrated cells in the lower chamber were imaged, and cell numbers (MCF10A and MDA\u2010MB\u2010231 cells) were counted by image j and normalized to control. Clones formed by migrated MCF7 cells were counted.Twenty\u2010four\u2010well transwell chambers were used according to the manufacturer's protocol. Cells \u20105\u2010(3\u2010carboxymethoxyphenyl)\u20102\u2010(4\u2010sulfophenyl)\u20102H\u2010tetrazolium, inner salt) was performed using the CellTiter 96 kit according to the manufacturer's instructions.For growth analysis of MCF7 and MDA\u2010MB\u2010231 cells, 1\u00a0\u00d7\u00a010https://xenabrowser.net/). Genomic Data Commons Data Portal The Cancer Genome Atlas (TCGA) Breast Cancer datasets were analyzed for RNA level of p63 or miR\u2010522. Datasets of lymph\u2010node\u2010negative breast cancer [All data were downloaded from the University of California Santa Cruz Xena portal was used for data processing, calculation and graphing. Pearson's correlation coefficient was used to rank the correlation. Median expression value of miR\u2010522 or p63 was used as a cutoff to classify samples into high\u2010 and low\u2010expression groups. Student's t test was used for group comparisons.P\u00a0<\u00a00.001), suggesting a putative inhibition of p63 expression by miR\u2010522. miR\u2010203 decreased the reporter activity to a similar extent, whereas miR\u2010944 showed no significant effects Fig.\u00a0. Westernes) Fig.\u00a0. Furtheres) Fig.\u00a0. These rP\u00a0<\u00a00.001), indicating that these three sites were critical for miR\u2010522\u20103p to bind to TP63 3\u2032 UTR (Fig.\u00a0A bioinformatics algorithm found four potential miR\u2010522\u20103p binding sites at the 3\u2032UTR of TP63 Fig.\u00a0. To checUTR Fig.\u00a0. These rCultured MCF10A epithelial cells associated tightly with cell\u2013cell junctions. When miR\u2010522 was induced to be expressed in them, the cells became dissociated and shifted toward mesenchymal\u2010like morphology Fig.\u00a0. MeanwhiWe then investigated the function of p63 in 522\u2010induced scattering phenotype. The results indicated that ectopic expression of \u0394Np63\u03b1 in MCF10A and MCF7 cells could repress the scattering response and make cells associated tightly again Fig.\u00a0. Thus, \u0394EMT is the pivotal step in cancer metastasis; our results suggested that the miR\u2010522/p63 axis might play important roles in epithelial cell scattering and thus contribute to cell migration.To further investigate the function of miR\u2010522 in cell migration, we performed wound\u2010healing assays in MCF10A, MCF7 and MDA\u2010MB\u2010231cells cultured with reduced serum concentration. We confirmed that treatment with doxycycline did not significantly affect the wound closure speed in control MCF10A cells Fig.\u00a0. HoweverWe then conducted transwell assays to check whether miR\u2010522 can modulate the single\u2010cell migration. When miR\u2010522 was induced to be expressed by doxycycline treatment, more MCF10A cells migrated through the transwell and showed 52.0% higher migration rate. Infection of \u0394Np63\u03b1 into these cells led to a dramatic reduction of the migration rate, indicating that \u0394Np63\u03b1 could abolish miR\u2010522's migration\u2010promoting effect Fig.\u00a0.In transwell assays, MCF7 cells associated so tightly that they could not pass the membrane. Thus, we cultured the cells for 6\u00a0days and counted the cell clones formed by migrated cells. The results showed that induced expression of miR\u2010522 empowered MCF7 cells with the ability to pass through the membrane, and \u0394Np63\u03b1 could reverse this effect Fig.\u00a0. SimilarFurther experiments demonstrated that doxycycline treatment did not significantly change the growth rate of cultured MCF10A, MCF7 and MDA\u2010MB\u2010231 cells harboring inducible miR\u2010522 vector Fig.\u00a0. Taken tTo further define the function of miR\u2010522 on p63, we explored bioinformatics databases for the clinical relevance of miR\u2010522 and p63 expression. Significant up\u2010regulation of miR\u2010522 was found in TCGA breast invasive carcinoma when compared with normal breast tissue Fig.\u00a0. On the In TCGA breast invasive carcinoma samples, Pearson\u2019s analyses revealed a clear negative correlation between miR\u2010522\u20103p mature RNA and p63 mRNA levels Fig.\u00a0. We thenHere, we identified miR\u2010522 as a direct upstream regulator of p63. By targeting \u0394Np63a, miR\u2010522 could promote migration of mammary epithelial cells while not affecting their proliferation. Furthermore, the expression of miR\u2010522\u20103p demonstrated an inverse correlation with p63 level in human cancers.In Tan et al.'s work, puSeveral experimentally validated targets of miR\u2010522 in human cancers have been documented, including ABCB5, BLM, MAOB, PHLPP1, DKK1, SFRP2, DENND2D, SOCS5 and PPM1A , 31, 45.miR\u2010522 has been reported as a migration\u2010promoting factor for cancer cells. Overexpression of miR\u2010522 led to enhanced expression of EMT marker genes and invasive ability in MDA\u2010MB\u2010468 breast cancer cells . In keepmiR\u2010522 is frequently considered as an oncogene, yet distinct results have been observed regarding its role in cell proliferation. Overexpression of miR\u2010522 could promote proliferation of hepatocellular carcinoma through repressing DKK1 and SFRP2 . By targIn our experiments, we did not detect a significant change of cell proliferation on miR\u2010522 overexpression in MCF10A, MCF7 and MDA\u2010MB\u2010231 cells. This discrepancy might reflect the unique profile of the miR\u2010522/p63 axis in mammary epithelial cells. \u0394Np63\u03b1 may act either as a tumor promoter or a suppressor based on precise cellular scenarios. It may initiate tumorigenesis via promoting cell proliferation but inhibit tumor metastasis and progress in advanced cancers , 10, 11.Taken together, this study is the first to show that miR\u2010522 plays an important role in cell migration through repressing the expression of \u0394Np63\u03b1. Our finding provides clues for the functions of the miR\u2010522/\u0394Np63\u03b1 axis in tumor metastasis. Molecular drugs targeting miR\u2010522 could be evaluated for cancer treatment, especially for metastatic tumors with reduced \u0394Np63\u03b1 level.The authors declare no conflict of interest.YT conceived and designed the project. YD, JL and XL performed experiments and data analysis. YD and YT wrote the manuscript. GX, Z\u2010XJX and YT contributed to the discussion and manuscript revision."} +{"text": "Distant metastasis is the leading cause of treatment failure in patients with hepatocellular carcinoma (HCC). However, the underlying mechanisms have not been fully elucidated. Here, we report that Leucine zipper tumor suppressor 2 (LZTS2) is downregulated and correlated with poor prognosis in HCC. Furthermore, we provide evidence that LZTS2 associates with p85 to inhibit the activation of PI3K/AKT signaling and impairs HCC tumorigenesis and metastasis in vitro and in vivo. Moreover, we identify LZTS2 as a bona fide substrate of the E3 ligase \u03b2-Trcp and protein kinase CK1\u03b4, which are responsible for the ubiquitination and degradation of LZTS2. Importantly, we show that the \u03b2-Trcp and CK1\u03b4-mediated degradation of LZTS2 promotes HCC progression and metastasis by activating PI3K/AKT signaling. Collectively, our study not only illustrates the roles of LZTS2 in regulating HCC tumorigenesis and metastasis but also reveals a novel posttranslational modification of LZTS2 by \u03b2-Trcp and CK1\u03b4, indicating that the \u03b2-Trcp/CK1\u03b4/LZTS2/PI3K axis may be a novel oncogenic driver involved in HCC progression and metastasis. Hepatocellular carcinoma (HCC), the most common histological type of primary liver cancer, is the third leading cause of cancer-related death worldwide . At presThe ubiquitin-proteasome system (UPS) is the principal protein degradation machine that facilitates the proteasomal destruction of key regulatory proteins , 5. ThisLeucine zipper tumor suppressor 2 (LZTS2), also known as LAPSER1, is located on the 10q24.3 chromosomal region that is frequently deleted in various cancers . EmerginIn this study, we present evidence that LZTS2 is downregulated and inhibits HCC progression and metastasis in vitro and in vivo. We further uncover that the E3 ubiquitin ligase \u03b2-Trcp and the protein kinase CK1\u03b4-mediated destruction of LZTS2 contribute to tumorigenesis and metastasis by activating the PI3K/AKT signaling pathway in HCC. Our study reveals a previously unknown antimetastatic role and a novel posttranslational modification of LZTS2, indicating that LZTS2 may be an attractive therapeutic target for HCC.P\u2009<\u20090.001). Kaplan\u2013Meier survival curve analysis demonstrated that HCC patients with low LZTS2 expression levels had a shorter overall survival than patients with high LZTS2 expression levels (P\u2009=\u20090.0043) . As shown in Fig. 43) Fig. , suggest43) Fig. . Next, wPrevious studies showed that LZTS2 binds to and controls the subcellular localization of \u03b2-catenin , 26, indWe previously demonstrated that LZTS2 binds to the PI3K subunit p85 to inhibit the activation of the PI3K/AKT signaling pathway and overcomes radioresistance in nasopharyngeal carcinoma . Given tWe previously conducted an unbiased tandem affinity purification (TAP) and mass spectrometry (MS) approach to identify LZTS2-binding proteins in HEK293T cells . In addiIt has been well documented that the proper phosphorylation of substrates by one or a combination of specific kinase(s) is required for the recognition and binding by the E3 ligase \u03b2-Trcp . To inveIt has been demonstrated that most \u03b2-Trcp substrates contain a conserved DSGXXS (X represents any amino acid) degron sequence that is critical for the recognition of \u03b2-Trcp , includiWe have reported that LZTS2 participates in HCC progression and metastasis in a p85-dependent manner. Accordingly, we next explored the significance of LZTS2 ubiquitination modification in these biological processes in HCC. To this end, we reconstituted LZTS2-deficient cells with vectors expressing LZTS2-WT or the LZTS2-M3 mutant in the HCC cell line SMMC-7721. As shown in Fig. In this study, for the first time, we reveal the roles of LZTS2 in impairing tumorigenesis and metastasis in HCC in a p85-dependent manner. Moreover, we provided evidence that \u03b2-Trcp ubiquitin ligase and CK1\u03b4 protein kinase are responsible for the binding and subsequent ubiquitination of LZTS2. Clinically, LZTS2 is significantly downregulated, and a low level of LZTS2 is associated with poor clinical outcomes in HCC, implying that LZTS2 may act as a tumor suppressor and serve as a potential prognostic biomarker in HCC.It has been demonstrated that LZTS2 could be regulated at the transcriptional level in different ways , 30. ForLZTS2, a candidate tumor suppressor protein, has been proposed to play vital roles in tumorigenesis and radioresistance , 24. HowIn conclusion, our findings demonstrated that the ubiquitin ligase \u03b2-Trcp and protein kinase CK1\u03b4 are crucial upstream modulators that control the stability of LZTS2 and reveal that the PI3K/ATK signaling pathway is the main downstream effector involved in LZTS2-mediated biological processes, as proposed in Fig. 2 at 37\u2009\u00b0C.Five hepatocellular carcinoma (HCC) cell lines , LO2, HeLa and HEK293T were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco\u2019s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS) and 100\u2009\u03bcg/mL penicillin and streptomycin with 5% COThe pDOR-LZTS2 vector was purchased from DF/HCC DNA Resource Core. The Myc-\u03b2-Trcp plasmid was kindly provided by Dr. Wenyi Wei . The pDOR-LZTS2 vector was transferred to the Gateway compatible destination vector with the indicated SFB, Myc and HA tags using Gateway Technology (Invitrogen). Mutations were conducted using the KOD Hot Start Kit (Novagen), followed by DNA sequencing validation. Plasmids were transfected using polyethylenimine reagent or Lipofectamine 2000 (Invitrogen).Human anti-LZTS2 antibodies were obtained from Proteintech (15677-1-AP) (for western blotting) and Bethyl (A304-938A-T) (for IP). Human anti-\u03b2-Trcp (#4394), anti-HA (#3724), anti-phospho-AKT (#4060), anti-AKT (#4685), anti-p85 (#4257), anti-p110 (#3011), anti-CK1\u03b5 (#12448), anti-Snail (#3879), anti-Slug (#9585), anti-N-Cadherin (#13116), anti-E-Cadherin (#3195), anti-ZO-1 (#8193), anti-\u03b2-Catenin (#8480), anti-Vimentin (#5741), anti-TCF8 (#3396), and anti-Claudin-1 (#13255) antibodies were obtained from Cell Signaling Technology. Human anti-S6K1 (sc-8418), anti-GAPDH (sc-47724) and anti-Myc (sc-40) antibodies were purchased from Santa Cruz Biotechnology. Anti-CK1\u03b4 (14388-1-AP), anti-CK2\u03b1 (10992-1-AP), anti-CDK1 (19532-1-AP), anti-GRK2 (13990-1-AP), anti-VRK2 (12946-1-AP), anti-RSK1 (16463-1-AP), anti-RSK2 (23762-1-AP) and anti-PKC\u03b1 (21991-1-AP) antibodies were obtained from Proteintech. Anti-Flag (F7425) antibody was purchased from Sigma-Aldrich. Anti-CK1\u03b1 (ab82426) antibody was purchased from Abcam. Anti-ATM (A19650), anti-\u03b1-tubulin (AC012) and anti-Lamin B1 (A11495) antibody were obtained from ABclonal Technology. Anti-phospho-p85 antibody (AF5905) was purchased from Beyotime Biotechnology. Cycloheximide and MG132 (474790) were purchased from Sigma-Aldrich and Millipore, respectively.siRNA transfection was carried out using RNAiMAX transfection reagent (Invitrogen) according to the manufacturer\u2019s instructions. Transfected cells were cultured for 48\u2009h before use. To construct the LZTS2 siRNA-resistant plasmid, seven nucleotide substitutions were introduced into the LZTS2 siRNAs target sequence. The following target siRNA sequences used in this study were as follows:\u03b2-Trcp siRNA: 5\u2019-AAGUGGAAUUUGUGGAACAUC-3\u2019LZTS2 siRNA-1: 5\u2019-GGAGGAGAUCACUGCUACU-3\u2019LZTS2 siRNA-2: 5\u2019-GUGUGAGCAGCCUUAUCUC-3\u2019CK1\u03b4 siRNA-1: 5\u2019-UGAUCAGUCGCAUCGAAUA-3\u2019CK1\u03b4 siRNA-2: 5\u2019-GGUCUAGGAUCGAAAUGUU-3\u2019.HEK293T cells were transfected with the indicated shRNAs and the packaging plasmids pSPAX2 and pMD2G. Forty-eight hours after transfection, lentiviral supernatants were collected with a 0.45\u2009mm filter and then used to infect MHCC-97H cells in the presence of 10\u2009mg/mL Polybrene . Stable cell lines were selected with media containing 2\u2009mg/mL puromycin and confirmed by western blotting. The shRNA sequences were as follows:Control shRNA: 5\u2019-TCTCGCTTGGGCGAGAGTAAG-3\u2019LZTS2 shRNA-1: 5\u2019-TGATGTAGGTGAAGCTGGT-3\u2019LZTS2 shRNA-2: 5\u2019-TGAGCGGTCTGATCTTGCT-3\u2019.Cell lysates were prepared using NETN buffer (20\u2009mM Tris HCl (pH 8.0), 100\u2009mM NaCl, 1\u2009mM EDTA, and 0.5% Nonidet P-40) on ice for 30\u2009min, separated by SDS-PAGE and then transferred to PVDF membranes for western blotting using ECL detection reagents. For exogenous immunoprecipitation, the samples were incubated with S-protein beads (Millipore) or anti-Myc agarose (Santa Cruz Biotechnology) overnight at 4\u2009\u00b0C. For endogenous binding, the supernatants were incubated with anti-LZTS2 for 2\u2009h and protein A/G-agarose overnight. The precipitates were washed five times using NETN buffer and analyzed by western blotting.Cells were co-transfected with the indicated siRNAs and plasmids and lysed using NETN buffer solution with the addition of MG132 (10\u2009\u03bcM) 4\u2009h prior to collection. The lysates were incubated with S protein beads overnight at 4\u2009\u00b0C. After being washed five times with NETN buffer, the beads were boiled and the pull-down proteins were resolved by SDS-PAGE.The cells were transfected with the indicated siRNAs and plasmids. Alternatively, the cells were directly transfected with HA-tagged LZTS2-WT (wild-type) or LZTS2-M3 (mutant) plasmids. After transfection, 20\u2009\u03bcg/mL CHX was added to the medium at the indicated times. The protein levels were detected by western blotting.4 cells/well, and cell numbers were counted every other day. For the colony formation assay, transfected cells were seeded in a six-well plate at a density of 500 cells/well and then cultured for 2 weeks. After fixation and staining, the number of colonies (more than 50 cells) was calculated.After transfection with the indicated siRNAs or plasmids, the cells were seeded into six-well plates at 1\u2009\u00d7\u2009105 cells/well in 96-well plates and incubated with 50\u2009\u03bcM EdU for 2\u2009h at 37\u2009\u00b0C. After fixation with 4% formaldehyde and permeabilization with 0.5% Triton X-100, the cells were treated with 100\u2009\u03bcL of 1\u00d7Apollo\u00ae reaction cocktail for 30\u2009min. Subsequently, the nuclei of the cells were stained with 100\u2009\u03bcL of DAPI for 30\u2009min. The cells were visualized and photographed under a fluorescence microscope.Cell proliferation was measured by the 5-ethynyl-20-deoxyuridine (EdU) Assay Kit . The cells were plated at a density of 3\u2009\u00d7\u200910SMMC-7721 or MHCC-97H cells were transfected with the indicated siRNAs and seeded on coverslips. After being fixed, the cells were blocked with 5% bovine serum albumin and then incubated with anti-Snail and anti-Slug antibodies overnight. The samples were incubated with the secondary antibody for 1\u2009h and then counterstained with DAPI for 10\u2009min and visualized using a fluorescence microscope.4 cells in 200\u2009\u03bcL of serum-free Dulbecco\u2019s modified essential medium (DMEM) were added to the upper chamber. For the invasion assay, 1\u2009\u00d7\u2009105 cells in 200\u2009\u03bcL of serum-free DMEM were plated in the upper chambers of the transwell plates with Matrigel-coated membranes. The cells were allowed to migrate or invade for 24\u2009h toward the lower cambers with 10% fetal bovine serum (FBS) media, and then they were fixed and stained with crystal violet. The stained cells were photographed and counted.These assays were performed as described previously , 35. In 6 MHCC-97H stable cells (sh-Control and sh-LZTS2). Tumor growth was measured by a blinded reader with callipers every four days, and tumor volumes were calculated according to the formula V\u2009=\u2009(L\u2009\u00d7\u2009W2)/2, where V\u2009=\u2009volume (mm3), L\u2009=\u2009length (mm), and W\u2009=\u2009width (mm). For the tail vein lung metastasis model, the mice were intravenously injected with 3\u2009\u00d7\u2009107 MHCC-97H stable cells (sh-Control and sh-LZTS2) in 150\u2009\u03bcL of phosphate-buffered saline (PBS) through the tail vein. After 8 weeks, the mice were sacrificed, and all lung tissues were collected, fixed and sectioned, followed by haematoxylin and eosin staining. Macroscopic metastases were quantified by counting lesions in all five lobes of the lung per mouse.All animal experiments were approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. BALB/c nude mice (5-6 weeks old) were randomly divided into three groups consisting of five mice each and subcutaneously injected with 5\u2009\u00d7\u200910An HCC tissue microarray, which contained 86 carcinoma tissues and paired para-carcinoma tissues, was obtained from Shanghai Outdo Biotech . All patients were pathologically diagnosed with HCC. IHC analysis was performed as previously described \u201337. In bt test . Kaplan\u2013Meier survival analysis was performed to illustrate the prognostic value of LZTS2 in HCC patients. A P value\u2009<\u20090.05 was considered statistically significant.Individual experiments were repeated independently three or more times. The data were analyzed using GraphPad Prism V6.0 and are presented as the mean\u2009\u00b1\u2009SD unless otherwise stated. Statistical significance was analyzed using Student\u2019s Supplemental MaterialSupplementary Fig S1Supplementary Fig S2Supplementary Fig S3"} +{"text": "Diabetes is one of the leading causes of death in developing countries. Existing mobile health app design guidelines lack a description of the support of continuous self-monitoring of health status, behavior change to improve and adopt a healthy lifestyle, and communication with health educators and health care professionals in case of any need.This paper presents the development of a specialized set of heuristics called heuristic evaluation for mHealth apps (HE4EH) as an all-in-one tool and its applicability by performing a heuristic evaluation of an mHealth app.An extensive review of heuristics and checklists was used to develop the HE4EH. The HE4EH was evaluated by domain experts for heuristics, checklist items, severity ratings, and overall satisfaction. The OneTouch app, which helps individuals with diabetes manage their blood glucose levels, was evaluated using HE4EH to identify usability problems that need to be fixed in the app.The expert evaluation of HE4EH revealed that the heuristics were important, relevant, and clear. The checklist items across the heuristics were clear, relevant, and acceptably grouped. In terms of evaluating the OneTouch app using the HE4EH, the most frequently violated heuristics included Content, Visibility, Match, and Self-monitoring. Most of the usability problems found were minor. The system usability scale score indicated that the OneTouch app is marginally acceptable.This heuristic evaluation using the OneTouch app shows that the HE4EH can play a vital role for designers, researchers, and practitioners to use HE4EH heuristics and checklist items as a tool to design a new or evaluate and improve an existing mHealth app. The leading reason for morbidity and mortality, especially in developing countries, is chronic disease . About 8Designers typically use an existing set of guidelines or heuristics as a base to design a new app; they also evaluate and improve the usability aspects of a developed app in light of an existing set of guidelines or heuristics. A search on guidelines and heuristics did not reveal an all-in-one package that supports self-monitoring, behavior change, and communication with health care professionals, as already mentioned, but it did reveal related guidelines and heuristics. These include mHealth apps , privacyOnce an app is developed, a potential end user of the app or a domain expert can be recruited to evaluate the app. End users are typically exposed to the app towards the last stage of app development; therefore, the expertise of domain experts is utilized to quickly evaluate the app on behalf of end users. In this research, domain experts evaluated the OneTouch app. Methods that can be used for an expert evaluation include heuristic evaluation ; cognitiThis paper presents the development of a specialized set of heuristics based on existing heuristics, called the heuristic evaluation for mHealth apps (HE4EH), that can be used as a tool to design or evaluate an mHealth app. This paper is structured as follows. The Methods section presents the mHealth app design framework. The Results section presents the evaluation of the HE4EH by domain experts and its applicability to evaluate the OneTouch app. Last, the article is concluded in the Discussion section.To compile the heuristics, we searched for existing sets of mobile heuristics, mHealth heuristics, behavioral change, and self-monitoring of blood glucose in electronic databases, including Scopus, Web of Science, PubMed, and Google Scholar. The research revealed 8 sets, by Shneiderman , NielsenThere are similar heuristics across the sets; for instance, consistency and standards, strive for consistency, and consistency mean the same thing. Similarly, flexibility and efficiency of use, efficient interaction, customizability, and efficiency also mean the same thing. There are also other examples. Therefore, all such heuristics were grouped together using a concept similar to an affinity diagram.Once all the related heuristics were grouped, it became obvious that there would be multiple related checklist items. The next step was to identify all those related checklist items and group, merge, and expand the checklist items wherever needed. The checklist items were mostly in the form of statements. Each statement was converted into a question, so the checklist items could be used to identify which items satisfied, did not satisfy, and were not applicable for the app to support the researchers and practitioners in evaluating the mHealth app.This subsection presents the overall study design of the expert evaluation study 1, while the results are presented in the Results section.Experts are widely used in research studies to mainly gather their opinion and improve the instrument based on their feedback. Experts were also used in this research to improve the instrument . All the participants were recruited through convenience sampling and snowball sampling.The HE4EH is a proposed set of heuristics that contains 25 heuristics see and a toThe prestudy questionnaire included questions related to the demographic information of the experts. The demographic questions included gender; occupation; industry; experience (in years); familiarity with types of diabetes; level of human-computer interaction (HCI), usability, user interface, or user experience design experience; experience working with patients with diabetes; and involvement in a number of projects for patients with diabetes.The expert review questionnaire consisted of 4 parts, namely heuristics, checklist items, severity ratings, and satisfaction. A brief description of each follows.The heuristics part of the expert review questionnaire presents all 25 heuristics provided to the experts; for each heuristic, the expert had to answer one question each for the importance of the heuristic, the relevancy of the heuristic, and clarity of the heuristic in the set. Each question was rated using a 5-point Likert scale. The items corresponding to an odd-numbered value for importance were \u201cNot Important,\u201d \u201cModerately Important,\u201d and \u201cVery Important\u201d; similarly, the items corresponding to an odd-numbered value for relevancy included \u201cNot Relevant,\u201d \u201cModerately Relevant,\u201d and \u201cVery Relevant.\u201d Last, the items corresponding with odd-numbered values for clarity were \u201cVery Poor,\u201d \u201cBarely Acceptable,\u201d and \u201cVery Good.\u201dA total of 436 checklist items were presented to the experts in the expert review questionnaire. Similar to the heuristics, for each checklist item, the expert had to answer one question each for the clarity, grouping, and relevance of the checklist item. Each question for clarity, grouping, and relevance was rating using a 5-point Likert scale. The items associated with odd-numbered values for clarity included \u201cVery Poor,\u201d \u201cBarely Acceptable,\u201d and \u201cVery Good\u201d; similarly, the items associated with odd-numbered values for grouping were \u201cStrongly Disagree,\u201d \u201cUndecided,\u201d and \u201cStrongly Agree.\u201d Last, the items associated with odd-numbered values for relevance were \u201cNot Relevant,\u201d \u201cModerately Relevant,\u201d and \u201cVery Relevant.\u201dResearchers in this space typically use a 5-point severity rating scale where 0 means \u201cNo problem,\u201d 1 means \u201cCosmetic,\u201d 2 means \u201cMinor,\u201d 3 means \u201cMajor,\u201d and 4 means \u201cCatastrophe.\u201d We decided to investigate 3 alternate severity rating scales. For the individual items in the 3 alternative scales, experts were asked to choose a suitable option on a 5-point Likert scale from \u201cVery Difficult\u201d to \u201cVery Easy.\u201d Then, we used descriptive statistics to identify the most suitable of the 3 scales.The first alternative scale was scored as follows: 0, \u201cNo Violation\u201d; 1, \u201cLow\u201d; 2, \u201cModerate\u201d; 3, \u201cHigh, Severe.\u201d The second alternative scale was scored as follows: 0, \u201cNot Applicable\u201d; 1, \u201cNo Violation\u201d; 2, \u201cMinor\u201d; 3, \u201cMajor\u201d 4, \u201cCatastrophe.\u201d The third alternative scale was scored as follows: 0, \u201cNo Violation\u201d; 1, \u201cLow\u201d; 2, \u201cMedium\u201d; 3, \u201cHigh.\u201dThe last part of the expert review evaluation asked the expert about their level of satisfaction in terms of using HE4EH for the evaluation of an mHealth app. They were asked to answer 13 questions see using a Each expert received a separate email to provide consent for the evaluation of a set of heuristics and the checklist items. In the email, they were briefly informed about the background of the study and its objectives and were introduced to the set of heuristics and its checklist items. The experts who agreed to participate in the study were sent another email with 3 instruments: (1) prestudy questionnaire, (2) heuristics and checklist items, and (3) expert review questionnaire. They were asked to complete the prestudy questionnaire first and then review all the heuristics and associated checklist items. Last, they were asked to complete a comprehensive expert review questionnaire and return the completed questionnaires via email.Microsoft Excel software was used to analyze the data collected in this study.For each heuristic, checklist item, severity rating, and satisfaction rating, the expert was asked to answer one or more questions using a 5-point or 4-point Likert scale. The frequency was calculated as a sum of responses for each Likert-scale score for each question.This subsection presents the overall study design of expert evaluation study 2, while the results are presented in the Results section.In studies evaluating heuristics, at least 3 participants are recommended, as they can contribute to identifying \u226575% of the usability problems in the app or the system used for the evaluation . In thisWebsites with rankings of apps to manage diabetes were searched. The selection criteria used to choose a webpage and the app were as follows: (1) webpage provided the names and descriptions of apps to manage diabetes, (2) app was available for both Android and iOS devices, (3) app was free to download, and (4) app did not have in-app purchases.One of the pages entitled \u201cThe best diabetes apps of 2019\u201d was alsoThe proposed set of heuristic HE4ED and its checklist were used as the instruments in this research. The details of heuristics and checklists are discussed in detail in the earlier sections.The OneTouch app helps manage blood glucose levels; this app complements the OneTouch Verio Flex meter. Once the blood glucose readings are added, the app automatically searches for and highlights trends and provides push notifications so the user can take the necessary actions. Physicians have access to their patients\u2019 data; physicians can change their patients\u2019 diet plans based on their history. The app also provides the opportunity to share data about progress with family members and friends. Furthermore, users can log in to their accounts online from their desktop or laptop. This app was carefully chosen because it may be useful for an individual with diabetes and obesity in the Middle East and North Africa region.The prestudy questionnaire contained questions related to demographic information. The questions included gender, age, highest degree or level of education, designation (if working), HCI-related courses taken, previously evaluated apps, and set of heuristics or guidelines used.The usability problem reporting form allowed participants to report identified usability problems with the OneTouch app. For each identified problem, the participant needed to report the broken heuristic and checklist, description of the problem, possible solution from their perspective, and severity rating. The severity rating ranged from 0 to 4 inclusive: \u201cnot a problem,\u201d \u201ccosmetic problem only,\u201d \u201cminor usability problem,\u201d \u201cmajor usability problem,\u201d and \u201cusability catastrophe.\u201d The typical severity rating scale was chosen for 2 reasons. First, the experts\u2019 opinions on the severity rating scales were almost the same. Second, the new ratings may not have given a better picture as each expert only received about 25% of the checklist items. Researchers conducting heuristic evaluation studies typically use the same severity scale mentioned in this subsection rather than the one proposed in this study.The system usability scale (SUS) by Brooke was usedWe sent an email to the participants to brief them about the set of heuristics developed and the evaluation of the OneTouch app. They were informed that they would be divided into 5 groups, with 1 participant in each group, and groups would be randomly formed. They were asked to give consent by replying to the email. The participants consenting to participant were sent another email with the following:Set of instructions to download and install the OneTouch app on their Android or iOS device and to register an account for themselves. They were informed that they would evaluate the app on their own.Set of heuristics and either 107 or 108 checklist items . We provided about one-fourth of the checklist items to limit workload and, at the same time, define a minimal set of checklist items that could be useful to evaluate the app. These checklist items were equally distributed in a way that each heuristic has one or more checklist item. There were some similarities in the checklists between groups.Pre-study questionnaire, usability problems reporting form for the OneTouch app, and poststudy questionnaire. They were informed to complete these in the following order: prestudy questionnaire, the evaluation of the OneTouch app and reporting of usability problems in the reporting form, and poststudy questionnaire. We requested that they return all completed files via email.They were also informed that, in case of any confusion or query, they could always communicate via email, and a Skype call could be initiated for discussion, if needed. They were also asked to nominate a potential list of participants who would be interested in evaluating the app.Microsoft Excel software was also used to analyze the data collected during expert evaluation study 2.For each heuristic, the usability problems represent the sum of usability problems found for all the severity ratings.For each heuristic, the average severity rating represents the average of usability problems found for all the severity ratings.The standard calculation method for the SUS score was used in this research.The numeric values in the 3 columns (from left to right) for the importance of a heuristic represent the number of responses for \u201cVery Important,\u201d \u201cImportant,\u201d and \u201cModerately Important,\u201d respectively. Similarly, the numeric values in the next 3 columns (from left to right) for the relevance of a heuristic represent the number of responses for \u201cVery Relevant,\u201d \u201cRelevant,\u201d and \u201cModerately Relevant.\u201d The numeric values in the last 3 columns (from left to right) for the clarity of a heuristic represent the number of responses for \u201cVery Good,\u201d \u201cGood,\u201d and \u201cBarely Acceptable.\u201d There are only 3 negative responses for the \u201cLittle Importance\u201d item of the Likert scale for a question related to the importance of the heuristic; the 3 heuristics marked with an asterisk \u201c*\u201d had 1 negative response each. Therefore, the 2 columns for the negative items on the Likert scale are not shown in the table. Similarly, 1 expert did not answer a question related to the importance and relevance of the heuristic for 3 heuristics; these heuristics are marked with a hash \u201c#.\u201dOf the responses, 90.4% (113/125) showed that the heuristics are important, 4.8% (6/125) showed that they are moderately important, and 2.4% (3/125) showed that heuristics are of little importance, while the remaining 2.4% (3/125) were unanswered.Of the responses, 93.6% (117/125) showed that the heuristics are relevant, and 4.0% (5/125) of the responses showed that they are moderately relevant, while the remaining 2.4% (3/125) of the responses were unanswered.Of the responses, 74.4% (93/125) showed that the heuristics are clear, while the remaining 25.6% (93/125) showed that they are barely acceptable.The results show that the experts had a neutral opinion about the clarity of 2 of the 436 (0.5%) checklist items . The experts agreed for 173 of the 436 (39.6%) checklist items, while they strongly agreed for the remaining 262 of the 436 (60.0%) checklist items.In terms of the grouping of checklists for each heuristic, the results show that the experts agreed for 149 of the 436 (34.1%) checklist items, while the experts strongly agreed for the remaining 288 of the 436 (66.0%) checklist items.The results show that the experts had a neutral opinion about the relevance of 2 of the 436 (0.5%) checklist items . The experts agreed for 141 of the 436 (32.3%) checklist items, while they strongly agreed for the relevance of the remaining 294 of the 436 (67.4%) checklist items.According to the experts, sufficient or enough material was used to develop te HE4EH; it is not complex to use and provides useful information to design or evaluate an mHealth app. The terminologies used in the HE4EH are clear and easy to understand, learn, and use; the length of the HE4EH is suitable for the evaluation of mHealth apps that aim to improve the health of patients, especially those with diabetes in the context of this research, and allow them to self-monitor and change their behaviors. They were satisfied with the number of checklist items and their categorization, and they could use them to evaluate an app.The demographic information of the recruited participants is presented in The results of the heuristic evaluation study are shown in The cumulative number of usability problems found by all the groups was 137: severity rating 4: 15/137, 11.0%; severity rating 3: 49/137, 35.8%; severity rating 2: 54/137, 39.4%; severity rating 1: 19/137, 13.9%.The results show that one or more usability problems were found for most of the heuristics . The most frequently broken heuristics included content (30/137), visibility (12/137), and match and self-monitoring (9/137 each).During the process of dividing the checklists into the groups, it was expected that some groups might find a usability problem in the app but that they may not find suitable checklist items to which they could map the problem. This was expected because each group received about 25% of the entire checklist. During the analysis of the usability problems, the groups found 14 usability problems for which they could not find a matching checklist item. In The average severity rating of all usability problems found was 2.4; this shows that most of the usability problems found were minor.There are different ways to interpret the SUS scores ; these iAccording to the grading scale interpretation of SUS scores by Bangor and colleagues , the OneThis research presents a modified set of heuristics called the HE4EH and its applicability by evaluating the OneTouch app. The HE4EH consists of Nilsen\u2019s heuristic, mobile heuristics, health heuristics, and other heuristics . The HE4EH is a tool to assist HCI experts and mobile app developers when designing or evaluating an mHealth app for individuals with diabetes, by measuring the usability problems that can influence the user experience with the app. First, an expert evaluation study was conducted to improve heuristics and associated checklist items. Then, another expert evaluation study was conducted to evaluate a popular app called OneTouch intended to help with blood glucose management. The findings of the usability evaluation are as follows:Although the participants had a partial list of checklist items, they were able to identify the usability problems and link them to the given checklist items.The participants were also able to identify usability problems that could not be mapped to the partial list of given checklist items. This shows that, if given a complete list of checklist items, more usability problems could be identified.The top 3 frequently violated heuristics included Content, followed by Visibility, Match, and Self-monitoring.Although minimal, the participants were able to identify usability problems in the OneTouch app associated with the heuristics incorporated explicitly for behavior change and self-monitoring of blood glucose. The lack of incorporation of these and related heuristics and the associated checklist items means the identified usability problems had remained undetected.The average severity rating of all the usability problems found was minor.The mean SUS score showed that the OneTouch app is marginally acceptable and needs enhancements to improve the overall usability of the app.As with all studies, this research has a few limitations. First, we used only selected bibliographic databases and search terms to identify a set of related heuristics. Second, a limited set of checklist items was given to each expert in the study. Last, the severity rating scale typically used in heuristic evaluation studies was also used in this research due to a subtle difference between the severity rating scales proposed in this research.The current research can be extended in different ways. First, we intend to evaluate the mHealth apps by giving a complete set of checklist items to the HCI experts and investigate the impact on identifying usability problems in the apps, especially in terms of the specialized heuristics added to the HE4EH. They can consider using one of the severity rating scales discussed in this research. Second, future research can evaluate the effectiveness of different severity ratings proposed in this research. Third, the accessibility heuristic contains one generic checklist item; future research can, therefore, enrich it with a specialized set of guidelines like the Web Content Accessibility Guidelines and then"} +{"text": "Mycobacterium abscessus among patients with cystic fibrosis.Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is thought to be caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of M. avium-PD, a couple with M. intracellulare-PD, and a second couple, one of whom was infected with M. intracellulare and the other of whom was infected with M. abscessus. Whole genome sequencing was performed using patients\u2019 NTM isolates as well as environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs). By comparison with the genetic distances among 78 publicly available NTM genomes, NTM isolates derived from the two pairs of patients infected with the same NTM species were not closely related to each other. In phylogenetic analysis, the NTM isolates from patients with M. avium-PD clustered with isolates from different environmental sources.We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting patients with NTM-PD who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15\u2009years were enrolled: a mother and a daughter with In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal. The prevalence of nontuberculous mycobacterial pulmonary disease (NTM-PD) is increasing in developed countries \u20134. SeverVibrio cholera [Staphylococcus aureus [Pseudomonas aeruginosa [The development of next-generation sequencing (NGS) technology enables the identification of massive numbers of single nucleotide polymorphisms (SNPs) by whole genome sequencing (WGS). Using SNPs as genetic fingerprints and comparing them among multiple samples, phylogenetic analysis has been able to identify the source of infection for pathogens such as cholera , Staphyls aureus , Pseudomruginosa , and NTMruginosa \u201311.Mycobacterium abscessus isolates from patients with cystic fibrosis, performed WGS, and analysed phylogenetic relationships among these isolates. Surprisingly, they found strong evidence supporting human-to-human transmission of M. abscessus among patients with cystic fibrosis, and identified some M. abscessus isolates that were widespread globally [Since NTM are ubiquitous microorganism, it is generally assumed that patients with NTM-PD acquire NTM from their environment, not from other infected individuals. However, recent NGS studies showed that this might not be the case, at least for patients with cystic fibrosis , 9. Bryaglobally , NGS stuRecently, we diagnosed three pairs of NTM-PD patients with no immunodeficiency who had been co-habiting for at least 15\u2009years. We investigated the genomes of NTM isolates derived from the patients and environmental samples in their houses to understand the source of infection using WGS.M. avium PD had lived in an apartment in an urban area (HOME-1) for 15\u2009years. A couple (Patients C and D) with M. intracellulare PD had lived in a house in a rural area (HOME-2) for 30\u2009years. A second couple (Patients E and F) with M. intracellulare PD and M. abscessus subsp. massiliense PD had lived in an apartment in an urban area (HOME-3) for 30\u2009years. No patients were suspected of any immunodeficiency disorders. They were HIV-negative, were not taking any immunosuppressants and had no history of recurrent infection of any organs.Three pairs of patients with NTM-PD who had been co-habiting for at least 15\u2009years were enrolled . The core region of M. avium subsp. homonissuis consisted of 4.30 Mbp of the 5.15 Mbp genome (83%), and that of M. intracellulare consisted of 4.23 Mbp of the 5.40 Mbp genome (78%). On average, 38,377 high confidence SNPs were detected in M. avium subsp. hominissuis and 16,464 high confidence SNPs were detected in M. intracellulare within these regions (Supplementary Table\u00a0The mean sequencing depth of identified isolates was 313\u00d7 210\u2013487\u00d7). To reduce the potential impact of recombination and mobile genetic elements on our results, we defined core regions for each species as common sequences observed across the 17\u200910\u2013487\u00d7. M. avium subsp. hominissuis and M intracellulare genome, respectively, and used for further analysis. Pairwise SNP distances between every pair of isolates at those positions were calculated and clusters on the histogram of SNP distances were observed , and the SNP distance was not calculated.In HOME-2, the SNP distance between the M. abscessus among cystic fibrosis patients [M. abscessus-PD. However, these guidelines were only designed for patients with underlying risk factors and contain no statements for patients without immunodeficiencies. In this context, it is clinically important to understand if there is any evidence of patient-to-patient transmission among patients without predisposing factors.Recent NGS studies suggest the patient-to-patient transmission of patients , 9. In rpatients , US Cystpatients recommenM. avium-PD isolates that were genetically distinct but similar to separate isolates from environmental specimens in their homes. Although we could not identify environmental sources for the remaining two pairs of patients, one patient pair was also infected with genetically distinct M. intracellulare isolates (SNP distance >\u2009104), and the last pair of patients was infected by different species of NTM (M. intracellulare and M. abscessus subsp. massiliense). Therefore, patient-to-patient transmission is less likely in our study.Among the three pairs of co-habiting NTM-PD patients without overt immunodeficiency examined here, no patient pairs had near-identical NTM isolates Fig. . A pair M. avium. One report showed that samples from 17 (46%) of 37 households yielded the same species of NTM found in the patient. In seven of those households, the patient isolate and a plumbing isolate exhibited the same repetitive sequence-based PCR DNA fingerprint. Another study reported that seven of 20 (35%) patients with NTM-PD had NTM isolates with identical genotypes to isolates from household water or shower aerosols [M. avium-PD had genetically matched isolates to household isolates [M. avium-PD patients living together (HOME-1). Patient A seemed to have acquired NTM from the kitchen, while Patient B acquired NTM from the bathroom or kitchen.Household water sources are considered a major reservoir for NTM, especially for aerosols . Recentlisolates . In our M. intracellulare was isolated from soil samples, especially in high soil environments [M. intracellulare for these patients could be soil instead of water.Although we thoroughly collected environmental specimens from water and biofilms on the faucets and showerhead, we could not culture any NTMs from HOME-2, and identified only one NTM from HOME-3. A previous study reported that NTM species could be isolated from 22 of 37 (59%) households of NTM-PD patients. In addition, a positive correlation was observed between the number of samples collected per house and the number of NTM-positive samples . We collronments , 16, 17,k-mer based taxonomic classification, these isolates were not NTM. The sequencing reads of NTM isolates from Patients E and F aligned to NTM reference genomes, but those from environmental specimen isolates did not. Thus, these environmental isolates were not closely related to NTM in HOME-3. A previous study showed that, among 19 patients with M. avium-PD or M. intracellulare-PD living in low soil environments, no patients had genetically identical isolates compared with soil sample isolates from their houses [M. intracellulare-PD (Patient E) in HOME-3 (low soil) may have been exposed to the NTM outside the house. The lag time between the acquisition of NTM from the environment and diagnosis of NTM-PD also makes it more difficult to identify the source of NTM [In HOME-3, three morphologically distinct colonies were cultured from one environmental specimen and sequenced separately. However, using r houses . Our rese of NTM .M. avium in patients in one pair was identified: one acquired infection from the kitchen and the other from the bathroom or kitchen. The likelihood of patient-to-patient transmission appeared minimal in these three pairs of NTM-PD patients.NTM isolates derived from three pairs of co-habiting NTM-PD patients were determined to be different strains based on WGS data. The sources of ClinicalTrial.gov (NCT03532438).Pairs of adult patients living together and diagnosed with NTM-PD at Seoul National University Hospital satisfying the following inclusion criteria were enrolled: age\u2009>\u200918\u2009years; typical respiratory symptoms such as chronic cough, sputum, or haemoptysis; findings suggestive of NTM-PD on computed tomography; identification of NTM in \u22652 sputum cultures or in \u22651 bronchoscopic specimen; living in the same household prior to diagnosis with NTM-PD; and consented to collection of environmental samples in their home. This study was approved by an institutional review board (IRB Number: 1804\u2013064-936) and registered at The most recently-isolated NTM from participant sputum or bronchial wash specimens from participants was collected. In addition, we visited patients\u2019 homes to collect environmental specimens. One litre of water was aseptically collected into sterile containers directly from all faucets and showers in bathrooms and kitchens. Swabs were taken from the inside of faucets and showerheads in bathrooms and kitchens. As previously described , 18, eacrpoB gene [All biomass from Middlebrook 7H11 plates or Ogawa media plates taken from sweeps of colonies was mixed with 425\u2013600\u2009\u03bcm glass beads, vortexed for 5\u2009min, incubated at 80\u2009\u00b0C for 10\u2009min and then centrifuged. DNA was extracted from the supernatant using a QIAamp DNA mini kit as previously described and rpoBpoB gene , 21 sequk-mer matches [M. avium subsp. homonissius, reads were mapped to M. avium subsp. homonissius TH15 using BWA [M. intracellulare, reads were mapped to M. intracellulare ATCC 13950. Isolates assigned by Kraken2 as non-NTM species were excluded from further analyses. Variants were called using SAMtools and bcftools [M. avium subsp. homonissius and 14\u2009M. intracellulare genomes available in three publicly available datasets deposited in the short read archive were downloaded and processed using the same procedures [Kraken2, a taxonomic classification system using matches , was usesing BWA . For isobcftools with theocedures , 25, 26.Additional file 1: Table\u00a0S1. Characteristics on environmental specimens. The numbers of collected specimens, culture-positive, morphologically distinct isolates from each habitat. Table\u00a0S2. NTM species Identified and Sequencing Results. Description on the source of each sample and its sequencing quality , species, clonality, and morphologic feature.Additional file 2."} +{"text": "The purpose of this study was to examine the role of using a gym as a mechanism for adherence to regular physical exercise among individuals with aged 55 and over. This was a grounded theory research design. Data were collected from face-to-face interviews and observations among 15 people who attended the gym regularly and then analysed via content analysis. Three categories emerged from the present study regarding the adherence to regular physical exercise: (1) meaningful starting points, (2) reinforcement by motivators, and (3) adherence through positive changes. Results of this study can help health policymakers, managers of gyms/fitness centres, and gym instructors to design and implement programs Adherence to regular physical exercise is greatly beneficial for one\u2019s health. It is also a very challenging health behaviour. According to the World Health Organization WHO, , insuffiSeveral studies were conducted to identify barriers and facilitators of adherence to regular physical exercise. Some common barriers were observed, including symptoms of swollen, stiff, painful joints and swollen ankles and legs aged 40\u00a0years and older, (2) with a monthly or annual gym membership for 12\u00a0months or longer, (3) ability to speak Mandarin or Taiwanese, and (4) agreement to participate in a tape-recorded interview.The face-to-face, in-depth, and individual interviews were conducted by the primary author (Ming-Hsin Chen) in a private, quite, and comfortable room with the goal of uncovering the views of each participant. Interviews began with an open-ended question: \u201cWhat makes you adhere to regular physical exercise?\u201d Each interview lasted from 30 to 60\u00a0minutes. All interviews were conducted in Mandarin or Taiwanese and tape-recorded. The interview data were transcribed verbatim prior to coding.Translations of transcribed interviews were performed after data collection. According to Mariano , data coThe content analysis was used to analyse the data with original language. We analysed interview data into units, then summarized into categories. Next, they both discussed developing categories and coding scheme. After agreeing on the general categories and coding schema, the principle investigator coded each interview. All disagreement was discussed until a consistency of coding employed. After most of data were analysed and the categories were clearly draw, identifying the relationship of uncover patterns coding was used to group categories into a smaller number of themes. The coding was performing with original language, then translated by two authors (the second author and PI).The rigour of this study included three criteria: creditability, transferability, and dependability . To ensure credibility, the PI and study subjects read and discussed the meanings derived from the analysis. To establish transferability, the PI made observation notes during the interviews and recorded these. These observation notes were used to compare or clarify a particular subject\u2019s transcript. To achieve dependability, all interviews were conducted by the PI and tape-recorded. Hence, the interview data were transcribed verbatim by a research assistant, and the accuracy of the transcription was re-examined by the PI.The study was approved by the Human Subjects Protection Programme at National Cheng Kung University, Tainan, Taiwan. To recruit the subjects, a sponsor or administrator of the gym first introduced the primary investigator (PI) to members when they attended the gym for physical exercise activities or classes. Next, the PI explained the purpose, rationale, and background of the study to the subjects. Gym members were encouraged to ask questions and participate in the study. If a member of the gym agreed to participate, she or he signed a consent form.Fifteen subjects participated in this study. Most were female , and married . The average number of children were 1.8 . The average age was 56.93\u00a0years , the average length of time of attending the gym was 15.24\u00a0years , and the average number of hours spent doing physical exercise weekly was 5.67 . Three categories emerged from the interviews regarding the adherence to regular physical exercise: (1) a meaningful starting point, (2) reinforcement by motivators, and (3) adherence through positive changes. The category of \u201ca meaningful starting point\u201d was defined as what motivated the subject to join a gym and attend exercise classes regularly. This category was divided into four subcategories: (1) making the body perfect, (2) making friends, (3) having more free time, and (4) experiencing a comforting environment. One participant (participant 5) stated that \u201cI am looking for a place where I don\u2019t need to worry about air pollution, or it is a raining day, when I am doing exercise \u2026 . A place makes me feel comfortable, then I will go and do exercise regularly \u2026 . I can make body sharp perfect if I do exercise regularly. That is why I am here [a gym].\u201d Participant 2 stated that \u201cI retired, I have more free time \u2026 . And, a friend of mine asked me to come here [a gym] and to do exercise together \u2026 . That is why I am here [in gym].\u201dThe category of \u201creinforcement by motivators\u201d was defined as including factors or experiences that had an effect on the participants\u2019 continuing to exercise regularly. Four subcategories were defined: (1) experienced positive outcomes, (2) experienced social support, (3) developed an exercise habit, and (4) received understanding through an instructor. One participant (participant 8) indicated that \u201cexercise makes me happy and healthy. When I feel muscle sore, I will do stretch out. That makes me feel more relaxed and happy due to pain release.\u201d Another participant (participant 15) stated that \u201cI improve health due to regular exercise here [gym] \u2026 . Exercise makes me happy, heathy and relax. I also make a lot of friends \u2026 . My husband also has been very supportive \u2026 . Sometimes, he takes care of children, so I am able to attend the aerobics class \u2026 . Because my husband and I love to attend exercise classes. Due to our influence, my children love to exercise too \u2026 . Doing exercise regularly not only become my habit, but also a habit of my whole family.\u201d Participant 14 indicated that \u201cexercise with friends makes me happy \u2026 . I usually like to attend a class, if the instructor can offer multiple class-time options, and if he/she has a well-organized teaching strategy. The instructors in this gym are also charming enough to attract students without absence. That is why I am here and do exercise regularly for many years.\u201dThe category of \u201cadherence through positive changes\u201d was defined as the changes that the participants experienced that led them to attend physical exercise classes regularly. The positive changes included two subcategories: (1) improvement in health status and (2) lack of pain. Participant 3 indicated that \u201cdoing exercise regularly improves my body health \u2026 . For example, I reduced body soreness. My immunity is improving [less cold and sickness]. I also feel physically comfortable.\u201d Another participant (participant 13) indicated that \u201cdoing exercise regularly makes my vitality gradually better. Many outcomes of my physical examination are encouraging and some reach the standard, which enable me to have higher motivation to maintain exercise regularly.\u201d\u201cConstant daily exercise keeps the doctor away\u201d was the major theme of the current study. The average period of attending a gym were 15.23\u00a0years among study participants. The majority of them were female and receiving support from their family members. They adhered to physical exercise to maintain or improve health. These are facilitators to motivate them to adherence to an exercise program. This principal result is consistent with several previous studies. For example, Stodle et al. conducteSimilarly, Marcus-Varwijk et al. conducteRichardson et al. investigThis study has four limitations. First, although study subjects have many years\u2019 experiences of using a gym, they are not expert panellist that we can accept their comments as the scientific truths. Second, subjects used a gym might be related to physical health issues. In this study, the assertion of the participants could have been verified (triangulated) by clinical data like the blood pressure for the participants that confirmed he/she is hypertensive. Third, a non-random recall bias might exist due to opinions of the participants which will be subjective in favour of gym usage. Thus, data of method triangulation may be used in further investigation. Fourth, the participants of the study were Taiwanese individuals. The regional culture might influence individual perceptions of using gym and affect outcomes of the study.The gym is a space where individuals can improve their health to reach a status of controlled comfort. It also offers a safe environment and access to qualified instructors. This study showed that the participants reinforced their popular knowledge by their perceptions of \u201ca person\u2019s health condition can be improved via adherence to regular physical exercise.\u201d These findings provide instructors with some guiding principles to design physical exercise classes of higher exercise effectiveness. The principles include providing a comforting environment, providing a supportive instructor via assessing participants\u2019 ability thoroughly, using a well-organized teaching strategy to design a class. These findings can also be used to design and implement further research aimed at increasing adherence to physical exercise among middle-aged and older individuals. To promote health-enhancing behaviours, the results of this study can be applied by health policymakers or managers of gyms/fitness centres to design interventions for community-dwelling people."} +{"text": "Background: Older people with diabetes have an increased risk for disability and cognitive dysfunction, which may impede self-care capacity. These are not evaluated routinely in current health systems. In the Center for Successful Aging with Diabetes, patients over the age of 60 undergo multi-disciplinary evaluation days and are provided with an integrated treatment plan. Among individuals with below-normal cognitive function, self-adherence to these recommendations poses a challenge. Thus, the aim of this study was to test the feasibility of a multidisciplinary intervention amongst older people with diabetes with below-normal cognitive function and sub-optimal glucose control.Methods: Patients with a MoCA score under 26 and A1C >= 7.5% participated in a two-arm intervention: (A) a medical intervention: monthly meetings with a diabetes nurse-educator, supervised by a diabetes specialist and study psychologist during which changes in their pharmacological regimen of glucose, blood pressure, and lipid control were made and (B) a cognitive/physical rehabilitation intervention. This arm consisted of (1) an intensive phase-group meetings which included computerized cognitive training, aerobic, balance, and strength exercise, and group discussions and (2) a monthly consolidation phase. Outcomes included change in A1C, change in strength, balance, and aerobic exercise capacity as well as change in quality of life.Results: After 12 months there was a 0.7% reduction in A1C. After 3 months there was a statistically significant improvement in physical indices, including aerobic capacity , balance (FSST) and indices assessing the risk of fall . There was no additional improvement in physical indices between the 3 and 12 month visits. For some of the physical measures, the improvement observed after 3 months persisted partially to the 12-month visit.Conclusions: This feasibility study provides preliminary data that support the efficacy of the complex interventions described. The findings suggest that this older population would require an ongoing \u201cintensive phase\u201d intervention. Larger prospective randomized trials are needed. The prevalence of diabetes increases with age. In the US it has been reported that ~25\u201330% of the population over 65 have diabetes . In IsraType 2 diabetes is a disease that requires complex self-care management capacities, including a variety of health-related behaviors such as diet, physical activity, adherence to medication, medical surveillance, and self- inspection. Self-care management in diabetes has been shown to be important in all age groups, with positive effects on glucose control and in the prevention of the long-term negative consequences of diabetes . There iMany diabetes clinics provide patients with routine surveillance and multi-disciplinary treatment for the well-known complications of diabetes (for example eye and kidney disease) as well as glucose, blood pressure, and lipid control as standard of care in treatment of people with diabetes. However, these clinics cater to all age groups and disregard the special needs of older patients, including mobility, frailty, and cognitive and communication difficulties, which impact the potential utilization of health care services. At the center for successful aging with diabetes we aim to overcome these barriers by offering a novel comprehensive evaluation that incorporates cognitive, physical and emotional assessments that together enable the tailoring of a personalized treatment plan . Among iThis was a feasibility study. From the cohort of individuals who had undergone evaluation at the center for successful aging with diabetes people wIndividuals participated in a 2-arm intervention .(A) A medical intervention involving monthly meeting with a diabetes nurse-educator supervised by a diabetes specialist and consulted by a study psychologist, in which changes in pharmacological regimen of glucose, blood pressure, and lipid control were made.(B) A multi-disciplinary group intervention consisting of:(1) An intensive phase. Weekly 4-h group meeting which included individually tailored computerized cognitive training sessions, aerobic, balance, strength exercise, and group discussion which were dedicated to cognitive rehabilitation strategies development and implementation with emphasis on disease management and physical activity as well as psycho-education on various disease management aspects . Computerized cognitive training was conducted using the Brain HQ cognitive training program. The program has 29 online exercises that work on many cognitive domains including attention, speed, memory, people skills, navigation, and intelligence. The participants got free access to BrainHQ and were asked to exercise at least three times a week. In the weekly meeting sessions, participants were provided with a tablet and each session was dedicated to a different cognitive domain. The importance of each cognitive domain in diabetes self-care performance was explained. Each participant was required to perform cognitive tasks in increasing level of complexity according his/her abilities. Neuropsychologists led the sessions.Each week they received physical and cognitive tasks to conduct on a daily basis at home, and the performance of these tests was monitored during the weekly meeting.(2) A consolidation phase monthly 2-h group discussions on challenges of implementation and coping strategies.Both phases were conducted by a neuropsychologist and physiotherapist. Psycho-education on medical and nutritional aspects were conducted by a diabetes nurse educator, diabetes specialist and dietitian.Outcomes included change in A1C (primary), change in aerobic, strength, and balance indices measures the distance an individual is able to walk over a total of 6 min on a hard, flat surface. The goal is for the individual to walk as far as possible in 6 min. The individual is allowed to self-pace and rest as needed as they traverse back and forth along a marked walkway. The six minute walk distance in healthy adults has been reported to range from 400 to 700 m. People with lower vs. higher scores on the 6-min walk are at higher risk for falls, disability, frailty, hospitalization, and death.The test examines the pace and number of steps it takes a person to pass 10 meters. A route of 10 meters is marked by two lines and a chair is placed two meters past the runway end line. The subject starts the test two meters before the runway and goes 14 meters (two meters for acceleration at the beginning and two meters for deceleration at the end). The score achieved is determined by the time lapsed by the participant during walking along the middle 10 meters. Subject performs the test four times, the first two times are for practice: measurement occurs only during the third and fourths time. In addition to measuring the speed, the number of steps required to cross the short distance are also counted. Studies have a shown that better gait speed is associated with a lower risk for functional decline, hospitalization and mortality.The test evaluates dynamic balance in a high functional level and features walk forward backwards left and right above 2, 90 and 2.5 cm high long sticks that divide the floor into four squares. The participant is stands in square 1 facing no. 2 square. The goal is to walk as quickly as possible in all the squares in the following order: from 1 to- 2,3,4,1,4,3,2, and 1 without touching the sticks. The score is the time required to complete the entire route. A score of above 15 s has a high positive predictive value for a high risk for falls.The maximum grip strength test is measured using the Jammer dynamometer. The score is the average in kilograms. This score is compared to the general population according to age and sex . StudiesThis test examines the strength of the lower extremities. The participant is instructed to stand up for a full session as many times as he can, without the help or push of the hands (his hands crossed on his chest) for 30 s. The score is determined by the number of times the subject is able to achieve full compliance. The score on this test has been shown to have good discriminatory ability with respect to the risk for falls.Medical variables were collected through interview, physical examination and collection of information from medical records. Neuropathy was defined as either bilateral reported neuropathy or bilateral reduced pain, touch vibration sensation on physical examination of lower limbs. Retinopathy was defined as evidence of diabetic retinopathy on eye examination. Diabetic Kidney Disease (DKD) was defined as either an elevated creatinine level or a microalbumin/creatinine ratio of above 30 mg/gram. Severe hypoglycemia was defined as a reported episode of hypoglycemia requiring the aid of another.The study was approved by the Sheba medical center IRB committee and all participants signed an informed consent form.p-values < 0.05 were considered significant.Baseline demographic, medical, and psycho-social characteristics collected during the evaluation processes were presented using mean (SD), N(%), medians, and Intra quartile Range (IQR). The difference in median scores between baseline measurement and after 3 months of the interventions were calculated, and statistical significance was tested using the Kruskal-Wallis/Mann-Whitney U test. The statistical significance of the trend between measurements and baseline parameters was calculated using a mixed model with repeated measures analysis with the Tukey-Kramer correction for multiple comparisons using Proc Mixed in SAS Version 9.1.3 . All Twenty-one individuals with type 2 diabetes with a MoCA score of below 26 and also an A1C of >=7.5 were included in the study. The demographic and medical characteristics of study participants is depicted in Average overall satisfaction with the program was 9.5, and score for the question \u201cwill the program have an impact on your long-term health\u201d was 9.5. p < 0.05). There was no change in quality of life. There was a statistically significant reduction in grip strength by the end of the study. For the FSST and 30-s chair (lower limb strength) there was an improvement after 3 months and some of the improvement experienced during this interval persisted to end of the study. For the 6-min walk (aerobic capacity) stand there was an improvement after 3 months and at the end of the study. For the TUG , there was a statistically significant improvement after 3 months that did not persist to the end of study assessment.A complex intervention conducted in older people with diabetes with sub-optimal glucose control and below-normal cognitive function was feasible and after 12 months demonstrated an improvement in A1C. After 3 months there was an improvement in physical indices related to aerobic capacity, strength and balance and a reduction in the risk for falls, possibly mediated through an improvement in self-care capacity. These improvements were generally diminished or did not persist to the end of study period.Previous studies in people with and without diabetes have demonstrated the efficacy of a MDT intervention. Thus, the FINGER trial a 2 year multi-domain group intervention of diet, exercise, cognitive training and vascular risk monitoring conducted in people at high risk for cognitive decline, demonstrated that individuals randomized to the intervention arm experienced less cognitive decline then those randomized to the standard care arm and included ~162 (13%) older people with diabetes . The shoThis study has several limitations including its small size, the lack of a control group and the relatively short follow-up, However, it does provide data regarding the feasibility and the possible efficacy of such an intervention including its effects on glucose control and physical indices cardinal to the aging person with diabetes such as aerobic capacity, strength and balance measures. To the best of our knowledge, this study is the first to include cognitive training and rehabilitation as part of a multi-disciplinary approach aimed at improving adherence to self-care management in an older diabetic cohort. This approach enables the older individual to learn compensatory cognitive strategies and adjust the self-care management plan to the cognitive challenges they are facing, while simultaneously utilizing in an optimal way existing cognitive assets.To conclude this small feasibility study provides preliminary data that supports the efficacy of the complex intervention described. In light of the change in physical indices that did not persist into the consolidation phase, it seems that this population would need an on-going \u201cintensive phase\u201d intervention. On a public health level, given the high prevalence of diabetes among older adults and the fact that this population is at a high risk for disability, even a small improvement in physical indices will have high impact as it may reduce the rates of disability in this high-risk population. Further studies are needed in order to assess how this type of intervention may be applied to a larger population.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Sheba Medical Center IRB. The patients/participants provided their written informed consent to participate in this study.An abstract of the current work was presented as a poster at the annual meeting of the Israel Endocrine Society, 2019.MA, OG, TC-Y, RN, HM, NG, SR, and TY contributed to acquisition and interpretation of data. TC-Y and RN contributed to acquisition, interpretation, analysis of data, and drafting this manuscript. OK contributed to the drafting, revising, and formatting of the manuscript. TC-Y also made substantial contribution to conception and design of the research described. MA, NG, OG, TY, RN, OK, SR, and TC-Y have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Malformations of cortical development (MCD) comprise a broad spectrum of structural brain lesions frequently associated with epilepsy. Disease definition and diagnosis remain challenging and are often prone to arbitrary judgment. Molecular classification of histopathological entities may help rationalize the diagnostic process. We present a retrospective, multi-center analysis of genome-wide DNA methylation from human brain specimens obtained from epilepsy surgery using EPIC 850\u00a0K BeadChip arrays. A total of 308 samples were included in the study. In the reference cohort, 239 formalin-fixed and paraffin-embedded (FFPE) tissue samples were histopathologically classified as MCD, including 12 major subtype pathologies. They were compared to 15 FFPE samples from surgical non-MCD cortices and 11 FFPE samples from post-mortem non-epilepsy controls. We applied three different statistical approaches to decipher the DNA methylation pattern of histopathological MCD entities, i.e., pairwise comparison, machine learning, and deep learning algorithms. Our deep learning model, which represented a shallow neuronal network, achieved the highest level of accuracy. A test cohort of 43 independent surgical samples from different epilepsy centers was used to test the precision of our DNA methylation-based MCD classifier. All samples from the test cohort were accurately assigned to their disease classes by the algorithm. These data demonstrate DNA methylation-based MCD classification suitability across major histopathological entities amenable to epilepsy surgery and age groups and will help establish an integrated diagnostic classification scheme for epilepsy-associated MCD.The online version contains supplementary material available at 10.1007/s00401-021-02386-0. Human brain malformations present with a broad spectrum of anatomo-pathological lesions , geneticHerein, we address DNA methylation as an objective molecular diagnostic biomarker that can be reliably detected and analyzed from archival human brain FFPE tissue , 60, 66.n\u2009=\u200912), FCD type 2A (n\u2009=\u200929), FCD type 2B (n\u2009=\u200929), FCD type 3A (n\u2009=\u200914), FCD type 3B , FCD type 3C , FCD type 3D , hemimegalencephaly , mild malformation of cortical development , mMCD with oligodendroglial hyperplasia in epilepsy , polymicrogyria , cortical tuber of tuberous sclerosis complex , or temporal lobe epilepsy . Based on MRI and histology, all 15 TLE patients were diagnosed with hippocampal sclerosis, but we used only histologically normal temporal neocortex. All cases included into this study have been extensively studied at the microscopic level with Hematoyxlin\u2013Eosin and Cresyl Violet \u2013 Luxol Fast Blue stainings available from all FFPE surgical tissue blocks. An immunohistochemistry panel of antibodies recommended for the neuropathology work-up of epilepsy surgery specimens [n\u2009=\u200911; Table We reviewed clinical, and MRI data of individuals who underwent surgery for the treatment of their focal pharmaco-resistant epilepsy and were diagnosed with FCD type 1A was identified on H&E slides as described above and macro-dissection performed by punch biopsy or by hand Fig.\u00a0. DNA washttps://github.com/FAU-DLM/Methylr2py. Additionally, we stratified quantile normalized data using the \u2018minfi\u2019 \u2018preprocessQuantile\u2019 function [M values (data not shown). Then unsupervised dimensionality reduction for cluster analysis on the data was performed. Uniform Manifold Approximation and Projection (UMAP) for general non-linear dimensionality reduction was used for visualization [M values using python-based seaborns, clustermap plotting method [As described previously, samples were analyzed using Illumina Infinium MethylationEPIC 850\u00a0K BeadChip arrays , 38. Brifunction . After tfunction . Consequfunction . All paifunction . We alsolization . The follization . The folg method . The fol\u2018Scikit-learn\u2019, \u2018fastai\u2019, and \u2018Pytorch\u2019 were used as python-based packages to leverage machine and deep learning , 55, 56.Using \u2018Scikit-learn\u2019, various types of, classic machine learning algorithms were spot-checked on their performance on the dataset via stratified fivefold cross-validation. We separately trained an, extra trees classifier, a, nearest neighbor classifier, a, support vector classifier\u2019 and a, \u2018random forest classifier. We then modeled via stratified fivefold cross-validation an ensemble stacking model using, vecstack . StackedUtilizing, fastais tabular-learner, we modeled a deep linear neuronal network consisting of three subsequent layers. The first layer contained 500, the second layer 250, and the last layer only one neuron. In a stratified fivefold cross-validation manner, we then trained neuronal networks with a batch size of 32 by cycling the learning rate between 0.0001 and 0.08 for a total of four epochs which was identified by early stopping. Conventionally, the learning rate is decreased as the learning starts converging with time. It is helpful to oscillate the learning rate toward a higher learning rate as it may help get out of saddle points. This method was found to be most efficient in training neuronal networks . To be cThe performance of the resulting classifier predictions generated by the cross-validation for machine and deep learning models was evaluated by the balanced accuracy, precision, recall, F1 score, and the multiclass area under the receiver operating characteristic (ROC) curve (AUC). Results were plotted into a normalized confusion matrix. The balanced accuracy takes class imbalances into account. Precision and recall were chosen as additional metrics to measure how good samples were classified concerning the fraction of correctly and incorrectly classified samples. Precision is also known as the positive predictive value, and recall is also known as sensitivity. To easier assess these metrics during the training process, we additionally captured the harmonic mean between these scores: the F1 score.To establish a comprehensive MCD reference cohort, we generated genome-wide DNA methylation profiles using Infinium HumanMethylation850K BeadChip arrays from 239 surgical cases representing the majority of MCD disease entities . We also included the intact temporal neocortex of 15 non-MCD TLE patients. Furthermore, we selected 11 samples from 4 autopsy cases representing non-neoplastic, non-MCD, non-epilepsy controls , a so-called ensemble method that combines the predictions of several \u2018weak\u2019 classifiers to improve prediction accuracy, and compared it to a three-layer shallow neuronal network . Both ML and DL classifiers raw predictions were UMAP reduced to two dimensions, plotted, and showed excellent methylation class separation Fig.\u00a0a, b. WheTaken together, our findings provide evidence that methylation profiles are distinct for different epilepsy-associated disease entities and can be discriminated by machine and deep learning methods, which may help to rationalize disease classification and patient stratification.n\u2009=\u200943), including 18 samples obtained from the most recent ILAE FCD agreement trial [Next, we tested both models against an independent test cohort to good (k\u2009=\u20090.68) depending on the additional amount of information being available for the neuropathologist, i.e., immunohistochemistry or gene panel analysis [Histopathology diagnosis of epilepsy surgery brain tissue poses a particular challenge to everyday clinical practice, especially in FCD . This haanalysis . In factanalysis , 54. Difanalysis . While ianalysis , it was analysis . Hence, analysis .SLC35A2 [DNA methylation analysis already fosters detection and molecular characterization of more specific and new disease entities in the broad group of brain tumors, particularly those characterized by specific pathogenic variants or treatable by targeted therapies , 64, 68.SLC35A2 , 27, 61.SLC35A2 .Further, we recently identified polymicrogyria (PMG) with mosaic trisomy of the long arm of chromosome 1 as a molecularly defined MCD subgroup . Its speYet another ongoing interest and research area has been low-grade developmental brain tumors associated with early-onset epilepsy, with many new categories implemented in each novel WHO classification scheme . DNA metIn contrast, the genomic DNA methylation in bulk epileptic brain tissue has been highly specific to the seizure phenotype across species and model systems irrespective of cellular composition and appeared further specific for etiology and histopathology , 38\u201340. Supplementary file1 (PDF 1328 KB)Below is the link to the electronic supplementary material."} +{"text": "We investigate the propagation of waves in dense static granular packings made of soft and stiff particles subjected to hydrostatic stress. Physical experiments in a triaxial cell equipped with broadband piezoelectric wave transducers have been performed at ultrasound frequencies. The time of flight is measured in order to study the combined effect of applied stress and rubber content on the elastic properties of the mixtures. The bulk stiffness deduced from the wave speed is nonlinear and non-monotonic with the increasing percentage of rubber with a more prominent effect at higher pressures. Moreover, in the frequency domain, a spectral analysis gives insights on the transition from a glass- to a rubber-dominated regime and the influence of rubber particles on the energy dissipation. Mixtures with rubber content below 30% show enhanced damping properties, associated with slightly higher stiffness and lighter weight. In addition, the importance of wave propagation into a granular media comes up in application such as oil exploration, earthquake and roads construction.In geotechnical engineering, it is common to incorporate recycled materials into earth constructions for conventional designs and soil improvement projects \u20133. SimilExploring the effect of granular composition on the effective physical properties of mixtures can help optimizing industrial processes, and engineering structures ,6\u20138. TheIn early experimental works \u201313, inveAlthough several methods are commercially available to determine the stiffness of geomaterials, both in the laboratory and in the field, (ultrasound) wave propagation techniques are widely accepted for their rapid, non-destructive, and low-cost evaluation methods. The use of piezoelectric transducers to estimate small-strain stiffness of soils from wave velocity has been well established nowadays \u201322.Mechanical waves are perturbations moving through space and time in a medium, where the small deformations lead to elastic restoring forces. This causes a transfer of momentum and energy through particle contacts, with little mass transport. The propagation of the mechanical wave through the medium provides valuable effective information about the medium itself. By applying this method to samples, one can deduce their mechanical bulk response ,24. For Along with the characterization of the material bulk stiffness and dispersion, wave analysis provides information about the attenuation properties of the medium. When a mechanical wave propagates through a medium, a gradual decay of wave amplitude can be observed. In certain materials, wave amplitude is only reduced by the spreading of the wave, so-called scattering. \u2018Scattering\u2019 is the reflection of the sound waves in directions other than propagation due to energy distribution on an expanding wavefront. Another reason for amplitude decay is the energy absorption due to viscous momentum interaction, as related to material properties. The combined effect of scattering and absorption is called attenuation. Intrinsic attenuation caused by viscous case can be experimentally obtained via the spectral ratio method.Q defined as the ratio between the energy stored and energy loss per frequency cycle due to viscous momentum interaction. It has long been believed that attenuation is an important quantity for the characterization of particulate systems like sands, rocks and pore fluid properties, e.g. saturation, porosity, permeability and viscosity because the attenuation is more sensitive than the velocity [In physics, the energy loss (intrinsic attenuation) is usually characterized by the quality factor velocity ,27\u201330. Avelocity . Its detvelocity .Q\u22121 in a certain frequency range, along with high stiffness and low density. This allows for a novel design methodology for calm, smooth, and smart materials that can be better in various aspects than their separate components.In this study, we apply ultrasound through transmission techniques in order to study stiffness and attenuation in granular materials made of mixtures of glass and rubber beads. The focus is on the effect of the volumetrical composition of the mixture and the stress level, where these factors can be exploited to optimize the material stiffness and density . In addiThis paper is organized as follows: In \u00a7. 2(a)dr\u2009=\u2009dg\u2009=\u20094\u2005mm) are used to prepare cylindrical specimens with different volume fractions of glass and rubber beads. Using similar size particles will isolate results due to the difference in particle properties with regards to their elastic or viscoelastic behaviour. The material characteristics for both glass and rubber beads are reported in The experimental set-up and the applied methodology are described in this section. Glass and rubber particles with similar size (\u03bd\u2009=\u2009dvr/dv be the volume fraction of the rubber particles (fraction of space possessed by the rubber particles), with the total volume of rubber particles given by dvr and the total volume of the mixture, dvr\u2009+\u2009dvg, given by dv. Glass-rubber samples were prepared with variable rubber content, \u03bd\u2009=\u20090, 0.1, 0.2, \u2026, 0.9, 1.0, where a mixture with \u03bd\u2009=\u20090 is composed of glass particles only and \u03bd\u2009=\u20091.0 of rubber particles only. In general, differences in size, density, stiffness and shape of particles could lead to segregation in granular mixtures. Thus, care is taken to prepare homogeneous mixtures and avoid segregation by minimizing vibration during specimen preparation. Each sample was prepared by first mixing particles to ensure that the glass and the rubber beads were evenly mixed. After that, specimens were prepared by allowing particles to fall freely from a funnel into a flexible latex membrane (with 0.304\u2009mm thickness and fit tightly around prepared specimens) stretched by a mold for isotropic tests. Densification is attained by tapping. All granular specimens are tested without any pore liquid, i.e. they are dry. In order to quantify intrinsic attenuation of the sample by the spectral ratio method, two sample heights (100 and 70\u2009mm) are considered in this study.Let P-wave broadband piezoelectric transducers (Olympus-Panametrics Videoscan V1011), an arbritrary waveform generator (Tektronix AFG 3101), a broadband power amplifier (E&I 1040L), a pre-amplifier/low-pass filter (Olympus-Panametrics 5077PR) and a digital oscilloscope (PicoScope 5444B). Figure 1b shows a schematic drawing of the set-up and peripheral electronics.The ultrasound instrumentation of the system consists of a pair of 100\u2009kHz pc and a mechanically applied axial load is applied to the sample having a diameter of 100\u2009mm (a). In subsequent stress increments, the granular samples are compressed in axial direction via the piston of the mechanical testing device. We are aiming for hydrostatic stress states. By hydrostatic stress state, we refer to a configuration in which normal stresses are equal and shear stresses are zero, i.e. the loading and radial directions are principal directions of stress. Such a stress state is also referred to as uniform or isotropic. Thus, at each load step, the confining pressure pc is adopted to the axial stress state. Since the samples are tested in isotropic state, we express the hydrostatic stress state with the pressure p, i.e. pc\u2009\u2261\u2009p, in this work. Water is used as a confining fluid for the samples enclosed by a rubber membrane. In total, 10 hydrostatic stress states are analysed, between p\u2009=\u200950 and 500\u2005kPa. At each stress level, a high voltage burst signal is excited to measure the time of flight. The top plate and the bottom plate of the cell are instrumented with the piezoelectric transducers. The piezoelectric transducers have a diameter of 38\u2009mm (around one-third of the sample diameter) and are in direct contact with a 10-mm thick polymethylmethacrylate contact plate (delay block) with a diameter of 100\u2009mm adjusted to the sample size. We use two identical vertically aligned piezoelectric transducers. The top transducer is acting as ultrasound source while the transducers at the bottom of the triaxial cell acts as receiver. The acoustic sound pressure is small enough to not destruct the granular samples, i.e. rearrangements of the micro-structure (fabric) are prevented. In this configuration, all through-transmission ultrasound investigations have been performed. The transmitted signal is a \u00b1400\u2009V square wave pulse. The signals transmitted and received are pre-amplified, filtered and recorded with a digital oscilloscope (LeCroy WaveSurfer 1\u2009GHz). The signal-to-noise ratio is improved by repetitive averaging of 100 detected signals using the digital oscilloscope and then sent to a computer for further processing. Thanks to a new generation of oscilloscopes, averaging of signals, which are sent into the samples repeatedly, is done automatically. Therefore, a clean (not noisy) signal is generated as an output. Sending electronic signals and averaging them is quite fast (less than 10\u22126\u2005s), which means there is no time dependence in the collected signals.A confining pressure f 100\u2009mm a. In subAll experiments performed for a certain rubber content and a stress state are repeated five times in order to avoid configuration-dependent results. We would like to emphasize that repeating the experiment means setting up a new granular packing in the triaxial cell. For each experiment, particles are mixed and poured into the cell again. Later on, when the results are shown, an error bar indicates the standard deviation of the five experimental results.(b)\u03bd, the mass density of the sample is given by\u03c1g and \u03c1r are the bulk densities of glass and rubber beads that are calculated at fraction \u03bd\u2009=\u20090 and \u03bd\u2009=\u20091, respectively. The numbers of particles used in each experiment, Ng (number of glass particles) and Nr (number of rubber particles), are estimated by knowing the weight of particles poured into the chamber and the weight of each particle.For each rubber content Vbox\u2009=\u2009(\u03c0/4)D2H, with D\u2009=\u2009100\u2005mm (diameter) of the sample and H\u2009=\u2009100\u2005mm (height) of the sample, before and after deformation, and volume of every single particle, Vbead\u2009=\u2009(2\u03c0/3)d3, one can determine the porosity \u03d5 of the sampleKnowing the cylindrical geometry (volume) of granular sample In . 3\u03bd\u2009=\u20090) at pressure p\u2009=\u2009300\u2005kPa. In highly attenuated granular media, there is always a significant uncertainty and difficulty associated with the determination of the wave travel time (see appendix B). Suggested criteria and recommendations vary depending on the installation, application and input signal. Here, we adopt a consistent peak-to-peak approach [In this section, the time of flight of the wave through the samples, for each rubber content and hydrostatic stress state, is measured, which eventually is converted to the bulk stiffness. A typical output is presented in approach .Figure P-wave signals recorded during hydrostatic loading and unloading for several rubber volume fractions. Regarding the acquired signals, it is obvious that the waveforms are sensitive to changes in material composition and applied hydrostatic stress state. For samples prepared with a low rubber content , the waveforms show a clear peak followed by a deep valley, which moves to the left on the time axis with increasing hydrostatic stress. For \u03bd\u2009=\u20090.3, additional features start to appear associated with high-frequency transmission, especially for high-pressure levels. At \u03bd\u2009=\u20090.5, the behaviour resembles that of lower rubber contents only at high pressure, while at low pressure a new peak appears later in time, and high-frequency ripples are visible in both the peak and post-peak parts. For \u03bd\u2009\u2265\u20090.7, the pressure-dependent peak disappears and high-frequency features dominate. Comparison between \u03bd\u2009=\u20090.3, 0.5 and 0.7 clearly shows a pronounced change in the waveforms associated with a transition from stiff to soft dominated regimes. Adding soft particles to the sample not only leads to a delay in the events but also to additional features in the post-peak part of the signal. Furthermore, waveforms of \u03bd\u2009=\u20090.7 and 1 reveal that soft dominated samples are insensitive to pressure.P-wave (tp) and the tip-to-tip distance between transmitting and receiving transducers (L), the P-wave velocity in the specimen (v) is obtained as [By measuring the travel time of the ained as 3.1v=Ltpa,b shows the wave velocity versus rubber content and pressure. The wave velocity remains fairly constant up to \u03bd\u2009=\u20090.3. By increasing the volume of rubber, there is a considerable drop in the wave velocity for all pressure levels, which can be associated with the end of the glass-dominated regime. The wave velocity is again relatively stable for \u03bd\u2009=\u20090.7 to 1, where the medium is, eventually, controlled by rubber particles.Figure 5P-wave modulus, M, is related to the velocity, v, by\u03c1B is the mixture\u2019s bulk density of the sample as given by equation , uppb, the P-wave modulus M is plotted against the hydrostatic stress state expressed in p. While the sample with \u03bd\u2009=\u20090 shows a different behaviour, the relative increase of the modulus with pressure is similar for mixtures with \u03bd\u2009\u2264\u20090.4. The behaviour dramatically changes when rubber content moves from \u03bd\u2009=\u20090.4 to 0.5 and 0.6. Further on, at higher rubber contents, the modulus becomes almost independent of pressure. We associated the change in the material behaviour with the transition from a glass-dominated regime, where waves travel through glass-glass contacts to a rubber-dominated case [In figure 6ted case , where tted case ,45.. 4Looking at a mechanical wave in time-series profile, we can see that the wave signals can be decomposed into two parts, the initial coherent wavefront followed by an incoherent multiply scattered signal also known as \u2018coda\u2019. The initial wavefront is of low frequency in nature, opposite to the coda, which contains high frequencies . The codP-wave velocity (and the effective stiffness of the sample) and discard the contribution of the incoherent coda wave. It is worth mentioning that a filtering function has not been applied so as to not lose any information.In order to understand the transition in the material behaviour as observed in figures A for a given frequency f and thus the Fourier power spectrum |A(f)|2. In figure 7a, the spectrum is plotted for samples with rubber contents of \u03bd\u2009=\u20090, 0.1, 0.2 and 0.3 at two different hydrostatic stress states of p\u2009=\u2009200 and p\u2009=\u2009500\u2005kPa. For these low rubber volume fractions, the main frequency remains unchanged despite the increase in the effective stiffness and a bimodal behaviour is appearing at the higher stress state at a frequency about f\u2009=\u20090.055\u2005MHz. For these intermediate rubber volume fractions, the first arrival of the P-wave (d) seems to be related with the glass network, while the energy is mainly concentrated at higher (rubber-related) frequencies.Furthermore, we focus on the frequency response in the transition regime. Figure 7e P-wave d seems tf\u2009=\u20090.055\u2005MHz is associated with a transitional stage of the system from stiff- to soft-dominated regime. For intermediate rubber contents, \u03bd\u2009=\u20090.4, 0.5 and 0.6, the system is controlled by disorganized stiff and soft clusters. The peak at f\u2009=\u20090.055\u2005MHz is possibly associated with one (or more) of these clusters, while other, smaller/bigger clusters become dominant when the rubber content changes from \u03bd\u2009=\u20090.5 to \u03bd\u2009=\u20090.6. Similar transient features, at these intermediate rubber contents, are also related to changes in pressure. In fact, the peak appears when moving from p\u2009=\u2009200 to p\u2009=\u2009500\u2005kPa, where it is expected new/more clusters will form. For higher rubber content, larger and organized clusters of rubber particles form and finally behave as a single cluster, i.e. the system is dominated by the soft phase. This is associated with a persistent peak at f\u2009=\u20090.055\u2005MHz, which becomes more pronounced with rubber content and pressure.The peak observed at \u03bd\u2009=\u20090.7 and 1 are depicted in figure 7c. Unlike the earlier plots, this figure does not show any significant frequency at 0.01\u2009MHz that was associated with glass particles, but the energy is concentrated at 0.05\u2009MHz, that is, the peak frequency of the pure rubber media. Therefore, it can be concluded that glass particles, and therefore the discrete granular packing, do not play an important role in samples with high(er) rubber volume fractions.Finally, rubber contents y-axis is different between figure 7a\u2013c, meaning that the amplitude in the peak changes by three orders of magnitude from \u03bd\u2009=\u20090 to \u03bd\u2009=\u20091. Looking at these plots, observations related to energy per frequency lead to an interesting conclusion on the nature of the medium in the different stages. The energy associated with low frequencies is related with the \u2018discrete nature\u2019 of the glass beads phase. The low-pass filter associated with particle width, d\u2009=\u20094\u2005mm, survives the filtering of the granular medium, which instead traps high frequencies.It is important to point out that the scale of the This has been observed earlier in . Increas\u221229\u2005J, which is very small and negligible in comparison with the pressure at which experiments were carried out. As expected, the energy is very small in comparison with the output signals, which ensures that samples have not been burst by the input, i.e. no network rearrangement or particle breakage. In this respect, wave propagation is often referred to as a constant-fabric experiment.It is worth mentioning that applying the FFT analysis on the input signal gives the dominant frequency of pressure wave at around 100\u2009kHz, which is normal for granular media. The amount of energy exerted by transducers at 100\u2009kHz is approximately 6.6\u2009\u00d7\u200910. 5\u03bd\u2009\u2264\u20090.3. The attenuation of seismic waves is an important property of the Earth, which is of great interest to material scientists, geo-mechanical engineers and physicists. During acoustic wave propagation, some of the elastic energy is lost (e.g. transforms into heat) per cycle in a propagation waveform through the media. Therefore, we introduce a quantity commonly used in materials science, geomechanics and (geo)physics. The ability of a material to attenuate acoustic waves is measured by the dimensionless quantity Q called the quality factor (or sometimes loss factor):A(f)|2 is the energy of the wave as introduced in \u00a7A(f)|2 is the change in energy in a single cycle.The purpose of this section is to study intrinsic attenuation of the sample, i.e. the viscous loss of energy during wave propagation with a focus on samples with a low rubber volume fraction of Q [t1 and t2 , varies as a function of frequency during the propagation of the acoustic wave [H1 and H2 yields to:A1(f) and A2(f) are the amplitude spectra at different lengths, f is the frequency, t1 and t2 are the travel times from the source to the receiver at distance H1 and H2, and c is a constant which contains all frequency-independent terms like transmissivity, geometrical spreading, source and receiver response, etc. A major strength of the spectral ratio method is that any frequency-independent scaling factor, as the geometrical spreading, falls into the intercept term of the linear regression, c, thus, it does not affect the quality factor.Several methods have been developed to compute experimentally the quality factor Q ,50,51. Htic wave \u201354. CompQ factor can be estimated by fitting a straight line to the logarithmic spectral ratio over a finite frequency range. Q is directly related with the slope, m, of the best-fit straight line asThen the quality factor Q can significantly deviate from the true value because the selection of the first peak from the received noisy signal is hard.The above derivation is the basic idea of the classic spectral ratio method, which is originally derived for the application to vertical seismic profile data . It musta shows raw signals recorded in the time domain for two samples with the same rubber fraction at the same hydrostatic stress state but having different sample heights, namely H1\u2009=\u2009100 and H2\u2009=\u200970\u2005mm. It is not surprising to see the shift of the signal to the left side of the time axis for the case of shorter samples, H2, i.e. signal arrives earlier. From the time domain plot (a), the difference between the time of flight (\u03b4t\u2009=\u2009t2\u2009\u2212\u2009t1) of the two samples is obtained, i.e. the first peak difference, where the first peak is considered since Q is measured for a single cycle consistently. To measure Q, the signals must be taken from the time domain to the frequency domain by the aid of FFT. After that, the quality factor is measured by fitting a line .Figure 8ain plot a, the dig a line to the le energy b.FigureQ\u22121 and is plotted against rubber volume fractions (\u03bd\u2009\u2264\u20090.3) in figure 9a for different pressure levels. When the amount of rubber increases, the quality factor parameter Q\u22121 increases in a linear fashion, i.e. the mixture behaves more dissipatively when the amount of soft inclusions is higher. On the contrary, increasing pressure leads to a decrease of damping as shown in figure 9b, where Q\u22121 is plotted versus the confining pressure.Intrinsic attenuation is expressed by the inverse quality factor \u03bd considered here. Combining the results in figures . 6P-wave modulus (stiffness) and intrinsic attenuation of the biphasic mixture. The experimental data indicate that the \u2018stiff\u2019 skeleton composed out of glass beads controls the effective mechanical response at small rubber fractions, \u03bd\u2009\u2264\u20090.3, while the \u2018soft\u2019 rubber skeleton prevails at larger rubber volume fractions of \u03bd\u2009\u2265\u20090.7. There is a considerable drop in the P-wave modulus M, only at intermediate mixtures (0.3\u2009<\u2009\u03bd\u2009<\u20090.6), where the transition from a stiff to soft regime occurs. Interestingly, we found that waves propagate faster in a range of \u03bd\u2009\u2248\u20090.2 compared with a mixture composed only of glass beads, \u03bd\u2009=\u20090. Such an effective stiff behaviour, which could not be explained by classical mixture rules or effective medium theories, could be observed at all hydrostatic stress levels, but is enhanced by high pressure. The relevant feature can be explained in many industrial applications where processes and bulk material properties have to be optimized.In the present experimental investigation, acoustic wave propagation experiments at ultrasound frequencies have been performed to examine the behaviour of biphasic granular mixtures consisting of glass (stiff) and rubber (soft) beads. Acoustic signals inferred from transmission tests using broadband piezoelectric transducers were acquired and interpreted to infer both effective \u03bd\u2009=\u20090.4 to \u03bd\u2009=\u20090.5, with a new sharp peak appearing. Comparing the peak frequencies, the transition from stiff (discrete mixtures) to soft (continuum-like) media was clearly observed.Furthermore, the frequency spectra, obtained from FFT, show that the dominant frequency for fixed rubber volume fractions is independent of the applied hydrostatic stress, while the majority of the elastic energy moves from low to high frequencies when the rubber fraction is increasing in a small range from Finally, intrinsic attenuation in samples composed of low rubber volume fractions was determined. As expected, a systematical increase of viscous energy loss with increasing rubber content was attained, while an increasing hydrostatic stress state reduces attenuation.P-wave modulus M and the inverse quality factor 1/Q for samples with low rubber volume fractions reveals interesting phenomena: although the stiffness remains almost unchanged with increasing rubber volume (or even increases slightly), the damping increases continuously. Especially, an optimal mixture was attained for a rubber volume fraction close to \u03bd\u2009=\u20090.2 at high hydrostatic stress states, showing the highest P-wave modulus and significantly high attenuation.The relationship between the S-waves in glass-rubber mixtures and numerical simulations (using discrete element methods and/or finite-element methods) to reproduce the behaviour of glass-rubber mixtures tested experimentally and gain more micro-mechanical insights.Future work includes the experimental investigations of"} +{"text": "Mobile health and web-based technological advances allow for new approaches to deliver behavioral interventions for chronic diseases such as obesity and diabetes. African American and Hispanic adults experience a disproportionate burden of major chronic diseases.This paper reviews the evidence for mHealth and web-based interventions for diabetes and obesity in African American and Hispanic adults.Literature searches of PubMed/Medline, The Cochrane Library, EMBASE, CINAHL Plus, Global Health, Scopus, and Library & Information Science Source were conducted for relevant English-language articles. Articles identified through searches were reviewed by 2 investigators and, if they met the inclusion criteria, were extracted and assessed for risk of bias. Findings were summarized in tabular and narrative format. The overall strength of the evidence was assessed as high, moderate, low, or insufficient on the basis of risk of bias, consistency of findings, directness, precision, and other limitations.Searches yielded 2358 electronic publications, 196 reports were found to be eligible for inclusion, and 7 studies met the eligibility criteria. All 7 included studies were randomized control trials. Five studies evaluated the effectiveness of an mHealth intervention for weight loss, including one that evaluated the effectiveness for diabetes and two studies focused on diabetes. Of all the studies that focused on weight loss, 3 reported significant differences in weight loss in participants in the intervention group compared with those in the usual care group. Although all studies on diabetes control showed greater improvement in glycemic control for the intervention group compared to that in the control group, only one study showed a significant difference between the 2 groups.This analysis indicates that there are few published studies that assessed mHealth interventions among minority populations and focused on weight or diabetes. Although the overall strength of evidence was low for diabetes control, it was moderate for weight loss, and our findings suggest that mHealth and web-based interventions may provide a promising approach for interventions among African American and Hispanic adults who have obesity or diabetes. Mobile health and web-based technological advances allow for new approaches to delivering behavioral interventions for chronic diseases such as obesity and diabetes ,2. TheseAfrican American and Hispanic adults experience a disproportionate burden of major chronic diseases, have a high prevalence of diabetes and obesity, and end up with worse health outcomes compared to their White counterparts . For insmHealth and web-based technology can mitigate some of these access issues and treatment challenges by providing pertinent information for patients with regard to their condition and offering elements such as monitoring of blood glucose levels and insulin dose adjustment, carbohydrate and calorie counting, and physical activities, which can be incorporated into their routine to improve outcomes . SeveralFurthermore, minority populations such as African American adults are known to be underrepresented in mHealth research despite existing health disparities . mHealthWhile a growing body of literature supports the ease and effectiveness of mHealth and web-based interventions for diabetes and obesity, to our knowledge, no systematic review has evaluated the effectiveness of such interventions for improving health outcomes of African American and Hispanic patients. The objective of this study is to provide a review of the evidence for mHealth and web-based interventions for diabetes and obesity in African American and Hispanic patients.Our literature search was conducted in accordance with the PRISMA guidelines . We searA research librarian performed searches for English-language articles on human adults in scientific journals (aged \u226518 years), which combined terms for (1) minority population terms, (2) mHealth intervention terms, and (3) chronic condition terms for type 2 diabetes mellitus and obesity. The last search was conducted on September 23, 2020. Searches were limited by study design to controlled trials. 1c, which is a standard measure of glycemic control in patients with diabetes.Studies with any of the aforementioned interventions in combination with other types of activities such as keeping a journal of meals or extra group or one-on-one coaching were also included. Studies that focused on patients with diabetes or overweight with other health conditions as a group such as stroke, pregnancy, or depression were excluded from this study. There was no limit on the publication time, duration of the intervention, or participants\u2019 age. For this review, primary outcomes of interest were objective measures related to obesity and diabetes, including BMI, weight change, waist circumference, or hemoglobin ATwo reviewers (CE and MH) extracted the data, and each reviewer independently extracting data from all studies. Extracted elements included the following: publication date, authors, study aims and objectives, study design, number of participants in the intervention and control arms, components and duration of the intervention, and results of the study. Outcome measures were extracted at all timepoints for studies that included multiple assessments. Reviewers checked each other\u2019s extractions for accuracy and completeness. Discrepancies were discussed until arrival at an agreement and when necessary, a third reviewer was involved.Each included study was assessed for risk of bias, using the Cochrane tool for risk of bias in randomized trials . The folWe summarized our findings for each question in tabular and narrative format. We did not conduct a meta-analysis owing to the limited number of studies that met the inclusion criteria. The overall strength of the evidence (SOE) was assessed as high, moderate, low, or insufficient on the basis of the overall risk of bias of included studies, the consistency of findings across studies, directness, precision, and other limitations . The combined search strategies yielded 2358 electronic citations, which were screened to assess for eligibility . In totaThe primary aim of 5 studies was to evaluate the effectiveness of an mHealth intervention for weight loss or diabetes, while 2 studies included an evaluation of the feasibility and acceptability of the mHealth intervention as their primary aim. All included studies were randomized control trials ,28-31. I1c values for the diabetes studies ranged from 9.02% to 10.2%.The total number of participants in all studies was 942, with sample sizes ranging from 18 to 371. Three studies had all African American or Hispanic participants, and the other 3 studies had greater than 30% of participants who identified as African American or Hispanic minorities. Study durations ranged from 3 months to 12 months. Mean participant ages across studies ranged from 24 to 53 years. BMI of the participants ranged from 31.5 to 38.0. Mean hemoglobin AAll studies had mobile phones and SMS text messaging as the main mHealth device and medium of communication . SMS texmHealth technology was also used to check on adherence to or progress on set physical activity and dietary goals.In addition to SMS text messages, 3 studies that focused on obesity had additional activities as part of the intervention. One study included all elements of the Women, Infants, and Children (WIC) program , another1c, weight, and BMI of participants at 3 months and 6 months.Obesity trials reported mean weight loss and weight change from baseline, percentage weight loss, and change in BMI. One study reported the proportion of participants who achieved a \u22655% weight loss and a \u226510% weight loss from baseline. The diabetes trial reported hemoglobin AWith regard to the risk of bias of studies included, a randomization sequence generation process was reported by all studies. Bias for allocation concealment and selective outcome reporting were determined to be low for all studies. Only two studies provided information on blinding of personnel and outcome assessors ,30. One Overall, 3 studies reported significant differences in weight loss between participants in the intervention group and those in the usual care group ,29,30 T. ImproveFor the studies that focused on diabetes, all reported an improvement in glycemic control in participants in intervention groups compared to those in control groups ,12,28, bOverall, some evidence supports the efficacy of mHealth and web-based interventions for weight loss among obese African American and Hispanic adults (moderate SOE; 1c among those who received mHealth interventions compared to their control group counterparts, suggesting that the evidence may be unclear for the role of mHealth interventions in diabetes control among patients in ethnic minorities. This systematic review shows that mHealth and web-based interventions for 6 months to a year can be effective for weight loss in African American and Hispanic adults.We identified 7 trials that reported the benefit of mHealth interventions directed toward obesity and diabetes among African American and Hispanic adults. We found an association between receiving the mHealth intervention and weight loss compared to those in control groups, which suggests that mHealth and web-based interventions are effective for minority patients who are overweight. Of the three studies that addressed diabetes, only one reported an improvement in hemoglobin AOur findings in support of mHealth interventions for weight loss among patients in ethnic minorities are similar to those of other studies -34. A sy1c with mHealth interventions compared to those with usual care. Our findings are similar to those of other studies that have shown improved glycemic control with mHealth interventions in the general population [1c [We also observed an improvement in hemoglobin Apulation ,14,36, epulation . One majpulation ,39. Bettpulation . The lacpulation ,41. Furtpulation . The destion [1c .Another important finding from this study is the limited number of studies with African American and Hispanic adults in mHealth interventions for obesity and diabetes. The underrepresentation of participants from minority groups in research studies has been well-documented -46 and iThe limited number of studies with primarily African American and Hispanic participants is a limitation of this study, which has also been observed in several other studies . AnotherAlthough the overall strength of the evidence was moderate for weight loss and low for diabetes, our findings suggest that mHealth and web-based interventions may provide a promising approach. mHealth technology can be used to meet different health needs such as the delivery of motivational messages, health education, prompts or reminders, and personal coaching . Further"} +{"text": "This paper analyses the most ethnically diverse spaces in England. We define multi-ethnic neighbourhoods as spaces where no one group is in a majority and at least five ethnic groups have representation. Around 4% of all English neighbourhoods (Lower Layer Super Output Areas) met these criteria in 2011. Often mislabelled as \u201csegregated\u201d spaces, the growth of ethnically diverse neighbourhoods helps benchmark increased inter-ethnic contact, yet we know very little about their spatial extent and the dynamics of their expansion. We use Census data for 1991, 2001, and 2011 to consider how neighbourhood-level diversity has changed during a period of substantial increase in ethnic diversity at the national scale. To what extent did these highly diverse areas grow, and what is the geography of that growth? Which types of areas did these neighbourhoods transition from? For example, were multi-ethnic neighbourhoods formerly low or moderately diverse, and which groups dominated these locales? We also consider if multi-ethnic neighbourhoods are here to stay, or if they are compositionally unstable. We reveal a surprising aspect in England\u2019s neighbourhood transitions: multi-ethnic neighbourhoods are highly stable, and increasingly so. Some 88% of neighbourhoods that were multi-ethnic in 1991 retained their high-diversity status in 2001, while over 95% of 2001 multi-ethnic neighbourhoods remained highly diverse by 2011. This is a different story to that of the USA, where high-diversity neighbourhoods have received more scholarly attention, and where these neighbourhoods have high attrition rates, functioning as stepping stones to another type of space. We explore the demographic and housing dynamics associated with this stability. They arWe have a complementary interest. This paper reports on the results of a large-scale quantitative analysis of the dynamics of multi-ethnic neighbourhoods in England, what we take to be highly diverse places in which at least five ethnic groups have meaningful representation. We paint a picture of England\u2019s ethnic landscape and explore how this has altered between 1991 and 2011 \u2013 a period of major population, policy, and political shifts. Against this backdrop, we ask: To what extent did the number of multi-ethnic residential neighbourhoods increase, and what is the geography of that growth? Who is exposed to high diversity, and how has that changed over time? Are multi-ethnic neighbourhoods transitory spaces, and what demographic dynamics are associated with their (in)stability?Several factors spur our curiosity about the transitions associated with multi-ethnic neighbourhoods. High diversity cannot be understood as a fixed state \u2013 it must emerge, and it may also dissipate. Social science research on neighbourhood dynamics has a long history. Most recent studies have moved from binary thinking about Black\u2013White transitions to accounting for multiple groups in contexts of both segregation and diversity e.g., . DespiteThe paper begins with a brief review of the literature concerning neighbourhood transition theory and recent changes in the ethnic and racial make-up of UK neighbourhoods. We then explain our neighbourhood classification scheme, and show its application to neighbourhood transitions in England between 1991 and 2011. English neighbourhoods remain predominantly White, but we show how this is changing and that one of the main pathways is towards multi-ethnicity. Accordingly, we devote much of the analysis to offering insights into why multi-ethnic spaces came about, which groups are in them, and their stability. The conclusions broaden the discussion to contemplate the implications of our findings for inter-ethnic mixing.2 \u2223Many approaches to neighbourhood (ethnic) transition trace to the USA and the mass-migration period in the early 20th century. Just as they do today, the majority of newly arrived immigrants settled in major metropolitan areas. One of these, Chicago, became the place where influential scholars studied the profound changes at work in urban space e.g., . Among tThe accent at the time was on neighbourhood turnover, not on ethnic and racial mix. Increased immigration in the last few decades has produced demographic change across the Global North. In the USA, as most new immigrants are Latinx or Asian, different racial and ethnic spatial dynamics are in play, requiring a reassessment of earlier methods and theory e.g., . OverallThe UK has a different history of immigration, demographic change, colonialism, and racial and ethnic relations. Nevertheless, the foreign-born proportion of the UK population was, in 2019, similar to that of the USA (~14%). Also, as in the USA, Black, Asian and Minority Ethnic (BAME) populations have increased their overall share, and there has been a discernible reduction in levels of residential segregation . So how Shifting scale to local authorities, London is demonstrably special in this context. London\u2019s \u201cdiversity of diversity,\u201d including and beyond ethnic affiliations , means that it is often described as \u201cthe world in one city\u201d . The capNone of the aforementioned studies focus on highly diverse, multi-ethnic neighbourhoods. Accordingly, we connect the growing interest in high-diversity neighbourhoods in the USA e.g., to multi3 \u2223The literature concerned with the measurement of multi-ethnic spaces is almost as diverse as the mix it seeks to capture e.g., . To undeEj is the entropy of neighbourhood j and pi is the proportion of group i in a given area, and there are k groups in the area (k = 8 in our case). A scaling constant s equal to 1/ln(k) is included so that Ej ranges from 0 to 1. Like low-diversity neighbourhoods have a scaled entropy value of less than or equal to 0.3436, or when one ethnic group makes up equal to or greater than 80% of the population of that neighbourhood. High-diversity,xs or multi-ethnic, neighbourhoods have a scaled entropy of equal to or higher than 0.6766, and no group constitutes higher than 45% of the neighbourhood\u2019s population. These criteria ensure people from at least five groups must be present in these neighbourhoods. They also require that ethnic groups other than the largest two constitute, in combination, at least 20% of that neighbourhood\u2019 s population, with this minimum percentage inversely dependent on the number of groups . The remaining neighbourhoods are classified as moderate-diversity. For low- and moderate-diversity neighbourhoods, we also identify which ethnic group is numerically dominant. Based on the neighbourhood\u2019s largest group, often with well over a majority share of the population, the resultant classification labels include, as examples, \u201cmoderate-diversity Indian\u201d and \u201clow-diversity White.\u201d This step makes less sense for neighbourhoods classified as highly diverse (multi-ethnic), since no group can exceed 45% of a neighbourhood\u2019 s population for the area to qualify for this category.To adapt this taxonomy for the English context, we must account for the differences in census classifications of the English and US cases. To do this, we adjust the six-group entropy-based schema of diversity as segregation, interpreting diverse places that lack a White majority as evidence of separation . Consequently, we spend some time unpacking this particular grouping in our analysis.We recognise that the ethnic group categories we use obscure their internal diversities: ethnic groupings such \u201cIndian,\u201d \u201cBlack African,\u201d and \u201cPakistani\u201d each encompass large variation in culture. The same applies to \u201cWhite,\u201d by far the largest ethnic group we include. With recent immigration, \u201cWhiteness\u201d in the UK has arguably never been more complex e.g., , 2019b oEthnic group categories are inconsistent between the 1991, 2001, and 2011 Censuses. Among other alterations, the broad White ethnic category was disaggregated into three sub-groups in 2001, and four in 2011. Since 2001, given an increasingly mixed population, four mixed ethnicity categories were included. These changes yielded the categorisation of 16 ethnic groups in 2001, and 18 in 2011, increasing from 10 when the ethnic group question was first introduced in the 1991 Census. Ethnic groups must therefore be aggregated for analyses that cut across time periods. 2 Data processing and analyses were undertaken using the R software environment . Introduced in 2001, these units were designed so that each zone had approximately the same size of population. This provides an advantage over Census wards, which are much more variable. In 2011, England had 32,844 LSOAs, with a mean size of 1,614 people. They are the best unit for this study because they offer an appropriate balance of spatial granularity compared to smaller (Output Area) or larger (Middle Layer Super Output Area) zones. The former, with a mean population of 300 people, would be too small to allow us to identify any meaningful diversity, while the latter zones are too large to be considered a \u201cneighbourhood.\u201d We harmonised the ethnic group information into 2011 boundaries for all three census years.ironment .4 \u2223Moderately diverse Pakistani majority neighbourhoods were the most common moderately diverse category for BAME groups since 2001, and their count increased between 1991 and 2011. Moderate-diversity, rather than low-diversity, was by far, and consistently, the most common diversity type for any neighbourhood that had a majority BAME group. As with White spaces, increased diversity, rather than segregation, was the dominant trend for majority minority neighbourhoods.five ethnic groups must be present in these neighbourhoods. Thus, by 2011, 1,417 of England\u2019s neighbourhoods were profoundly ethnically diverse.While moderately diverse neighbourhoods dominated by an ethnic minority group are rare between 1991 and 2011, the steady growth of the neighbourhood type that is the main interest here is striking. High-diversity \u2013 multi-ethnic \u2013 neighbourhoods grew from just over 0.5% of the total in 1991, to over 1.5% in 2001, to over 4% in 2011. While on first consideration this may seem small, it represents the most common neighbourhood type that is not White majority. Our classification scheme\u2019s entropy threshold and the maximum share constraints for the largest two ethnic groups necessitates that at least 3 that, in our case, rescale the LSOAs by the square root of their original area , the Midlands , and in east London.Low diversity is the almost exclusive domain of people claiming White ethnicity in England. Yet the geography of ethnic 4 Yet while multi-ethnic neighbourhoods were concentrated in the capital, and to a lesser degree, Birmingham, a steady growth of high-diversity neighbourhoods has occurred in other places. The number of multi-ethnic neighbourhoods grew almost threefold between 2001 and 2011, with London\u2019s proportion remaining stable over this decade. Therefore, by 2011, multi-ethnic neighbourhoods existed in sizeable numbers outside of London, in parts of Birmingham, Greater Manchester, Bradford, Leeds, Leicester, and other metropolitan places in mid- and northern England. As we shall see, most of these transitions to high diversity came from moderately diverse White neighbourhoods. But we turn next to the question of the population composition of multi-ethnic neighbourhoods.The growth of multi-ethnic neighbourhoods is a key feature of England\u2019s evolving ethnic landscape, especially in London. London was home to over 70% of England\u2019s multi-ethnic neighbourhoods in 2011, up from just over 50% in 1991.5 \u2223exposed to high diversity in their residential environment. Of course, every multi-ethnic neighbourhood will tell a different story, but there is much to be learned from these summary data, and we concentrate on a few particularly interesting points. Most fundamentally, the total population living in multi-ethnic neighbourhoods in 2011 was over 2.5 million, equating to about 5% of the total population of England . The nexmix of ethnic identities. This flies in the face of common perceptions of Muslim self-segregation and isolation to just under 3% higher (Indian).away from high diversity are relatively few and produced moderate-diversity Pakistani (17) neighbourhoods between 1991 and 2001, and moderate-diversity Pakistani (10) and Indian (9) neighbourhoods in the following decade. In stark contrast to the USA, multi-ethnic neighbourhoods in England are remarkably stable. Of the 170 multi-ethnic neighbourhoods identified in 1991, 149 (88%) had retained that status by 2001. The stability of multi-ethnic neighbourhoods increased in the subsequent 10 years. Some 504 multi-ethnic neighbourhoods, or over 95% of multi-ethnic neighbourhoods in 2001, were highly diverse in 2001 and 2011. Most English multi-ethnic neighbourhoods are not avenues to another type of space.The main transitions less transitory.Just as London dominated the story of multi-ethnic neighbourhood formation, the capital takes the lead in neighbourhood stability. Of all neighbourhoods that retained their high-diversity state between 2001 and 2011, some 73% were in the capital. This was a significant increase on the period 1991 to 2001, where just 56% of that stability occurred in London\u2019s multi-ethnic neighbourhoods. In other words, as London\u2019s diversity has grown, at the neighbourhood scale, it has become 7 \u2223multi-ethnic neighbourhoods? To better understand the mechanisms behind the stability of English multi-ethnic neighbourhoods, we next examine the age of their residents and their housing profiles. This choice is influenced by fundamental demographic concepts. We would expect that stable neighbourhoods would be older on average and have relatively fewer renters. But what about stable overall Chinese population in this age range . Also of note are the relatively large shares of people across several younger age groups, from young children to those in their early 40s, living in multi-ethnic neighbourhoods.The age profiles of multi-ethnic neighbourhoods are, on average, slightly younger than other neighbourhood types, with a higher proportion of their population in their 20s and 30s. Students, however, are not dominating this story. For example, while a large share of the Chinese population living in multi-ethnic neighbourhoods are aged 20\u201324, this equates to a small fraction of the replacement, likely through dual processes of natural change and in-migration. Rather than the growth of a single group, or groups, representing a pathway towards new forms of non-White segregation or gentrification by in-migrating White people \u2013 as in the USA \u2013 in the English case, the population profiles of multi-ethnic neighbourhoods suggest stable mixing among White and minority groups.Disaggregation of the White category sheds light on whether White youthfulness in multi-ethnic neighbourhoods is attributable to the younger Other White population, with origins in recent immigration. A considerable proportion of this group can indeed be found in multi-ethnic neighbourhoods (as for other neighbourhoods), yet multi-ethnic neighbourhoods are also home to a relatively large share of young adult White Britons. Thus while ethnic minority groups typically have younger age structures than the White British (and White Irish) populations , the WhiOur consideration of housing relates to levels of home ownership in multi-ethnic neighbourhoods, a tenure traditionally associated with less mobility and greater commitment to neighbourhood than (particularly private) renting. Home-ownership rates were particularly low for multi-ethnic neighbourhoods in 2011, compared to other neighbourhood types. Just 42% of the population in multi-ethnic neighbourhoods in England owned their property , compared to nearly 67% of residents of other neighbourhood types. In contrast, both social and private renting rates were very high in multi-ethnic neighbourhoods. This is a persistent observation from 1991 (not shown given space constraints).and multi-ethnicity, and the younger age profiles of these neighbourhoods . Are we observing layers of ethnic diversity as populations move in and out of these neighbourhoods, according to their housing, employment, and education needs? Future research making use of longitudinal data to explore length of stay in these places would shed further light on these processes. The evidence presented here suggests a different evolution \u2013 and retention \u2013 of high diversity in England compared to the USA, and therefore different processes at work in each context.and might well retain that status. Likewise, based on the observed trajectories, there is no evidence of an emergence of low-diversity non-White spaces in the future. Indeed, some moderate-diversity White-dominated neighbourhoods were transitioning into other forms of diversity (most commonly into high-diversity). It therefore might be reasonable to expect that these shifts will be associated with new forms of diversity.Our story also challenges dominant UK policy-political narratives of increasing diversity as a pathway to increasing polarisation . Increasall \u2013 White British and ethnic minority \u2013 groups, despite differing population profiles at the national level , in nurturing inter-ethnic tolerance. Additionally, the uncertainties posed by the impact of Brexit loom large. The implications of leaving the European Union (EU) on future immigration/emigration scenarios are difficult to predict, and every possible scenario will have at least some impact on the UK\u2019s diverse future . Reduced"} +{"text": "Recurrent Pregnancy Loss (RPL) affects 2\u20134% of couples, and with increasing numbers of pregnancy losses the risk of miscarrying a euploid pregnancy is increased, suggesting RPL is a pathology distinct from sporadic miscarriage that is due largely to lethal embryonic aneuploidy. There are a number of conditions associated with RPL including unspecified \u201cimmune\u201d pathologies; one of the strongest candidates for dysregulation remains T regulatory cells as depletion in the very early stages of pregnancy in mice leads to pregnancy loss. Human endometrial Treg and conventional CD4T cells were isolated during the peri-implantation period of the menstrual cycle in normal women. We identified an endometrial Treg transcriptomic signature and validated an enhanced regulatory phenotype compared to peripheral blood Treg. Parous women had an altered endometrial Treg transcriptome compared to nulliparity, indicating acquired immune memory of pregnancy within the Treg population, by comparison endometrial conventional CD4T cells were not altered. We compared primary and secondary RPL to nulliparous or parous controls respectively. Both RPL subgroups displayed differentially expressed Treg gene transcriptomes compared to controls. We found increased cell surface S1PR1 and decreased TIGIT protein expression by Treg in primary RPL, confirming the presence of altered Treg in the peri-implantation RPL endometrium. Recurrent Pregnancy Loss (RPL) is the consecutive loss of two or more pregnancies before 24 weeks\u2019 gestation,3 and can affect nulliparous (primary RPL) or parous women (secondary RPL). RPL is partly associated with genetic variation,4 uterine anomalies, endocrine dysfunction, parental balanced chromosomal translocation and specific maternal autoantibodies,5 however these associated clinical factors are identified in fewer than 50% of cases. The maternal immune system is implicated in some cases of recurrent pregnancy loss.6The female reproductive tract (FRT) is a mucosal barrier tissue and like other mucosa, immune cells infiltrate the stromal layers adjacent to the epithelial surface. Throughout the hormonally controlled menstrual cycle host immunity provides protection from pathogens that may enter the FRT, however when an embryo implants into the endometrium, the immune system is temporarily modified to permit attachment and invasion of trophoblast cells from the developing conceptus. At the time of embryonic implantation NK, T, B and macrophage cells are present in the human endometrium.5 and CD8T cells are also known to be phenotypically altered.7 The CD4T cell compartment has not been extensively studied. Of specific interest are the Treg cells, which in mice have been show to prime tolerance, as depletion during early stages of pregnancy during embryonic implantation leads to pregnancy loss.8 Murine Treg cells are also pivotal to tolerance of paternal alloantigen.9 The transcription factor FOXP3 confers regulatory lineage commitment to Tregs, multiple mechanisms of action are known such as high CD25 expression which consumes IL-2 preventing effector T cell functions, CTLA4 driven inhibition of antigen presenting cells, and inhibitory cytokine production .10 Decreased numbers of FOXP3+ cells are found in endometrium of patients with RPL compared to fertile controls,11 however in humans FOXP3 can also be expressed by activated T cells and other cells12 and the phenotype and functional nature of these cells has not been clearly elucidated.NK cell densities are associated with RPL,In this study human endometrium was collected during the peri-implantation period of the menstrual cycle, from normal fertile women and from women with RPL, with the aim of identifying if Treg are defective in tolerance mechanisms in women with RPL, leading to pregnancy loss. The methods applied in this study are detailed in Fig.\u00a0Endometrial biopsies were taken during the mid luteal phase of the menstrual cycle, when the tissue is receptive to embryo implantation. Single cell suspensions were obtained and flow cytometry performed to identify the total CD4T cell population (CD45/CD3/CD4), and the Treg subpopulation (CD25hi/CD127lo/FoxP3+), a representative example is shown in Fig.\u00a0n\u2009=\u20097) or parous controls (n\u2009=\u20093)). In addition we isolated cells from women with primary (n\u2009=\u20098) or secondary (n\u2009=\u20097) recurrent pregnancy loss (PRPL or SRPL respectively), and Treg were isolated from 5 peripheral blood samples from RPL patients. RNA-sequencing was performed and principal component analysis revealed that, as expected, endometrial conv-CD4T cells and endometrial Treg cells have distinct signatures, Fig.\u00a0We isolated Treg cells or Tconv from endometrial tissue biopsy digests, from fertile women (nulliparous (IL2RA (CD25) was the most significantly differentially expressed gene, along with other well described Treg genes such as FOXP3, TIGIT and IKZF2 (HELIOS). One of the highest differentially expressed genes in endometrial Tconv compared to Treg counterparts was EOMES, encoding a transcription factor involved with CD4T cell differentiation. The most abundantly expressed, significantly differentially expressed genes from endometrial Treg cells and Tconv derived from nulliparous control women are shown in Fig.\u00a0CTLA4, TIGIT, TNFSRF9 and the chemokine CCR8. Along with the classical master Treg transcription factor FOXP3 gene, the endometrial Treg express greater levels of the transcription factor genes BATF, IKZF2 (IKAROS) and IKZF4 (HELIOS). We confirmed endometrial Treg express significantly higher protein levels of the transcription factors FOXP3 and HELIOS, regulatory molecules CTLA4, TIGIT and CD39 and also the chemokine CCR8, than CD4-Tconv, Fig.\u00a0Differential gene expression between endometrial Tconv and Treg was investigated, depicted by Volcano plot, Fig.\u00a013 therefore we explored if parous women also have an altered transcriptomic endometrial Treg or Tconv profile. Endometrial Treg or Tconv from controls were segregated into groups of nulliparous (n\u2009=\u20096) or parous women (n\u2009=\u20093), Treg from parous women had a large number of uniquely expressed genes compared to those from nulliparous women and SRPL (n\u2009=\u20092) groups, . Next we compared peripheral blood Treg cells (n\u2009=\u20095) and endometrial Treg cells (n\u2009=\u20095) from RPL patients to determine the endometrial tissue specific Treg signature, a volcano plot of differentially expressed genes with the 25 most significantly altered genes is shown in Fig.\u00a0p\u2009<\u20090.01 padj and >3, <3 Log2FoldChange ranked on baseMean values), the top 30 upregulated and downregulated genes are shown in Fig.\u00a0ICOS, CXCR6, TNFSRF4, TNFSRF9, TNFSRF18) this suggests endometrial Treg, from patients with RPL, have a heightened regulatory profile when compared to peripheral blood counterparts. Therefore we focused further studies on proteins involved in immune regulation, tissue residency and antigen presentation. We used flow cytometry to explore the endometrial phenotype, in RPL patients. Whilst there was no difference in FOXP3 nor HELIOS expression levels, endometrial Treg expressed higher levels of key regulatory, residency and antigen presentation molecules: CXCR6, ICOS, CTLA4, TIGIT, PD1, IL18R, CD39 and TIM3, but not LAG, than were expressed on Treg in the peripheral blood (p\u2009<\u20090.05), Fig.\u00a0Within a limited subset of samples with both endometrial and PB available, we asked whether the Treg transcriptional profile differed in the two compartments. The peripheral blood Treg were very similar with only 45 genes found to be differentially expressed between the PRPL (p\u2009=\u20090.0351), SRPL patients had experienced fewer miscarriages than PRPL (SRPL 3.43\u2009\u00b1\u20090.20) versus PRPL (4.33\u2009\u00b1\u20090.48) (mean\u2009\u00b1\u20091 S.E.M.) however this was not significant and no correlation could be found between the total number of miscarriages experienced and Treg counts, Fig.\u00a0As exposure to term pregnancy affected Treg transcription, we next compared cells between nulliparous controls and PRPL and parous controls versus SRPL, to remove potential confounding cellular changes resulting from this exposure, to determine if the CD4T or Treg compartments are altered in RPL. First we assessed the proportion of Tconv and Treg within the endometrium between nulliparous and parous controls versus primary and secondary RPL, Fig.\u00a0Next we compared gene signatures of endometrial Treg, comparing PRPL to nulliparous controls, the majority of genes overlapped, however unique genes were identified in nulliparous controls and PRPL, Fig.\u00a0Comparing SRPL to parous controls, few genes were unique to SRPL compared to the parous controls, Fig.\u00a0S1PR1; Sphingosine-1-Phosphate receptor-1 is a G-Protein coupled receptor involved with lymphocyte activation, migration and trafficking.14 We analysed mRNA expression of this factor, along with other factors identified by DESeq2 between groups and also known Treg factors identified as core signature genes, such as transcription factors, regulatory molecules, tissue residency/chemoattraction/exit molecules,15 between all Treg and Tconv cell subsets, Supplementary Fig.\u00a0Tbet and RORyc than Treg, whereas endometrial Treg have greater FOXP3, HELIOS and BLIMP1. CTLA4, TIGIT and CD39 were greatly enhanced in endometrial Treg compared to Tconv and also peripheral blood Treg. S1PR1 was higher in PRPL Treg than both control groups and also SRPL (2/6 expressed S1PR1) but genes for other tissue residency factors such as CD103 or the linked CD69 did not follow this trend. CCR7 and CXCR3 are likely Tconv chemoattraction markers, whereas CXCR6 and CCR8 were higher in Treg. We further analysed protein expression levels of several of these identified factors between controls and RPL. PRPL patients had higher levels of Treg S1PR1 than both control groups and SRPL patients, confirmed by flow cytometry, when considering both the mean fluorescence intensity of expression and the proportion of Treg cells present expressing S1PR1, however due to small sample sizes statistical analysis is not performed, Fig.\u00a0The second highest upregulated gene in PRPL was In conclusion, we have identified that endometrial and peripheral blood Tregs have altered regulatory transcriptomic and protein signature profiles, and that these profiles are altered by exposure to term pregnancy. In addition, we have identified not only changes to Treg between RPL and controls at the transcriptomic level, but also provide evidence of post-translational proteomic changes to Treg in RPL patients that would lead to potential Treg change of function.16 RNA sequencing analysis of endometrial CD4+ Tconv and Treg cells was performed and differential gene expression analysis highlights the core transcriptome, Fig.\u00a017 Tconv also expressed higher levels of CCL4 (MIP1\u03b2) and CCL5 (RANTES) indicative of capacity to recruit immune cells to the endometrium, which help facilitate embryo implantation through tissue remodelling.18 Endometrial Treg, had enriched regulatory molecules such as CTLA4, TIGIT and CD39, which were also validated via flow cytometry. CCR8 expression was highly enriched in Treg, the CCL1-CCR8 axis is known to potentiate Treg suppressive function19 which is in line with the phenotype noted here.The endometrial CD4T cell compartment occupies a lower proportion of the total CD3+ cell population than that in the PB; however 5% of CD4+T cells are FOXP3+/CD25hi/CD127lo Treg both in the endometrium and PB, a similar Treg proportion was reported in other mucosal tissues isolated from adults.GATA, BLIMP1, STAT3. The Tconv population contained cells expressing Tbet, GATA and also some RORC, the Tconv population are therefore as expected a likely combination of Th1, Th2 and Th17 cells. In addition, Treg expression of IKZF2 (HELIOS) was also detected; HELIOS, along with Neuropilin, has been suggested by some researchers to be expressed by peripheral Treg (pTreg) as opposed to thymic derived Treg, however controversy exists as to their true value as such markers,20 here we find low endometrial Treg gene transcripts for Neuropilin and higher HELIOS with variable protein levels.We targeted key T cell transcription factors was also highly upregulated in endometrial Treg, in vitro ICOS+ Treg produce abundant IL-1021 and ICOS expression is closely linked to Blimp1 expression in Treg located in mucosal sites22 as we have detected in these endometrial Treg. Immune-checkpoint molecules are a key target for immunotherapy, upon ligation inhibitory signals limit immunity;23 family members include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) and lymphocyte-activation gene 3 (LAG-3), all of which have been studied here. Of note the PD-1/PD-L1 pathway is critically important in murine pregnancy as blockade causes foetal resorption, due to an imbalance in Th17/Treg cells which is diminished with Treg cell adoptive transfer.24 The phenotype identified here indicated endometrial Treg have enhanced expression levels of several immune checkpoint molecules, indicating endometrial Treg cells have differing phenotypic functions to those found in the blood.The endometrial specific tissue Treg signature has not yet been described, but it is to be expected as Tregs have non-lymphoid tissue specific local adaptations.25 and in mice Tregs expand during pregnancy forming a memory response that is recalled in subsequent pregnancy.26 Previous studies on decidua from term placenta show that Tregs undergo clonal expansion throughout pregnancy, of note this cannot be detected in the peripheral blood Treg,27 but peripheral blood Treg numbers increase in human pregnancies as gestation progresses. Our observation that RPL Treg have significant phenotypic differences between PB and endometrium, suggests Treg tissue specificity, this was also observed by a study analysing the T cell receptor beta variable (TRBV) repertoire that found most variation in the Treg population between cells derived from the blood or decidua.28 Of note, we were unable to obtain peripheral blood samples from control patients due to ethical constraints, further analysis of Treg from peripheral blood between controls and RPL is required in future studies. IFN\u03b3 was identified as the most upregulated gene in endometrial Treg from parous women compared to nulliparity. Interestingly, parous women have been shown to harbour a subpopulation of NKG2Chi dNK cells thought to be produced during their first pregnancy, which also have increased capacity to produce IFN\u03b3 along with VEGF\u03b1, both are known regulators\u00a0of angiogenesis and the vascularisation required for efficient placentation.13 Future work to assess if Treg from endometrium secrete IFN\u03b3 and contribute to placentation is required. Indeed Treg are altered during pregnancy: decidual Treg have the ability to supress both foetus-specific and non-specific responses30 and Treg (FOXP3+CD25hi) in first trimester decidua, compared to peripheral blood counterparts, express higher CCR5, ST2, CD25, BATF, IL10, GITR, GARP and CCR8.31 Here we describe that prior to pregnancy, the RPL endometrial Treg already express this enhanced regulatory phenotype, and that changes to the population in pregnancy are long lived in the endometrium.This is the first description of the CD4T and Treg compartment in human endometrium comparing parity and controls. Treg have fundamental importance for materno-foetal tolerance: CNS1 dependent extrathymic differentiation of Treg occurred concurrently with placental evolution,32 there is limited evidence of increased FOXP3+ cells in the endometrium of RPL patients,33 but Tconv and Treg proportions and populations have mainly been studied in in the peripheral blood or decidua obtained after miscarriage. Fertile women have expanded peripheral blood CD4+CD25+FOXP3+ Treg in the late follicular phase, this expansion is not seen in RPL which also have reduced functional capacity to inhibit proliferation35 and inhibit NK cell cytotoxicity.36 In general, CD4T cells numbers are lower in RPL decidua than controls37 and in normal early pregnancy Tconv were not clonally expanded.38 CD4+CD25bright cells were a higher proportion of the CD4T cell compartment in early pregnancy decidua obtained after induced abortion (21.84\u2009\u00b1\u20092.92) than after sporadic miscarriage (7.14\u2009\u00b1\u20091.85),39 and Treg numbers are reportedly reduced in decidua from RPL patients compared to normal first trimester pregnancy.42 Here we find that the numbers of cells present are similar prior to pregnancy, in RPL and controls, therefore the observed reduction in Treg numbers in decidua in women suffering miscarriage is likely a consequence of failure to recruit or expand Treg populations upon establishment of pregnancy.Given therefore that the exposure to pregnancy has a significant impact on endometrial Tregs, we compared nulliparous women to patients with PRPL, and correspondingly parous women to SRPL. Using flow cytometry to quantify the proportions of Tconv in the T cell population and Treg in the Tconv populations, we found no significant differences in the cell proportions between groups Fig.\u00a0. Treg/CD14 Mice with S1PR1 deficient Treg develop autoimmunity, Treg display an activated phenotype prone to apoptosis.43 The functional role of endometrial S1PR1 Treg expression remains to be determined, and if the expression indicates a subset of Treg in PRPL which can exit the tissue. In addition, we studied key Treg immune checkpoint molecules, and found that endometrial Treg from PRPL had significantly lower TIGIT protein expression levels that corresponding controls. This is the first observation of altered Treg populations in RPL, phenotypic changes indicate reduced inhibitory capacity in PRPL. Whether patients with PRPL would benefit from therapies enhancing tolerance through this pathway merits further investigation.PRPL Treg had altered Rassf2, a novel tumour suppressor gene with associated K-Ras pro-apoptotic effector functions and the second highest differentially expressed gene was S1PR1, Fig.\u00a0The study was approved by the Oxford Research Ethics Committee C (ref:08/H0606/94). All participants gave written informed consent in accordance with the Helsinki Declaration of 1975.44 Control endometrial samples were taken in the cycle prior to IVF. Controls for SRPL patients were taken from women who had previous live birth pregnancy outcomes. Women had experienced none or no more than 1 miscarriage and patients with endometriosis or known autoimmunity were excluded. Control samples for RNAseq or matched control to RPL flow cytometry experiments were from patients who are proven fertile; they either have had a previous live birth but were undergoing IVF for male factor infertility , or had a live birth in the IVF cycle following endometrial biopsy .All women were <40 years of age. Blood or endometrial samples were taken in the mid-luteal phase of the menstrual cycle, through analysis of cycle length and/or lutenising hormone surge testing to remove variability due to changes in menstrual cycle/hormonally regulated cellular fluctuations. Participants were recruited at least three months post miscarriage or hormonal treatment. Women included in this study had experienced at least 3 consecutive miscarriages. All women with RPL had normal thyroid function, negative anti-phospholipid screen , negative thrombophilia screen and no evidence of uterine structural abnormalities (identified by ultrasound scan or hysteroscopy). Maternal and paternal karyotypes were only carried out if an unbalanced translocation was identified in karyotyped miscarriage tissue as per the Royal College of Obstetrics and Gynaecologists guidelines.7 Peripheral blood was collected into sodium heparin anti-coagulant (10\u2009U/ml) and peripheral blood mononuclear cells (PBMC) isolated using lymphoprep (Axis Shield Diagnostics). Single cell suspensions were frozen in 10% DMSO/ FCS using a Nalgene Mr. Frosty Freezing chamber (Thermo Fisher), before being transferred to liquid nitrogen for storage prior to use.Endometrial samples were obtained using an Endocell disposable endometrial cell sampler and digested using 1X Liberase (Roche Life Sciences) as previously described.n\u2009=\u20095) were taken from women with an average age of 36.2\u2009\u00b1\u20094.1 years , 2 were nulliparous (Primary RPL) and 3 were parous (Secondary RPL), 4 patients had experienced 3 miscarriages and one patient had 7 prior miscarriages. Table\u00a0Matched blood and endometrial samples . Briefly, both PBMC or endometrial cells were stained with Zombie AquaTM Live/Dead Viability dye then incubated with antibodies 650, CD4-FITC, CD25-PE, CD8-BV711, CD19-PeCy7, CD127-APC), all reagents were from Biolegend unless otherwise stated.Frozen endometrial cell digests or isolated PBMC were thawed into HS-media at 37\u2009\u00b0C and centrifuged at 300\u2009\u00d7\u2009CD4T cells were isolated as singlet cells (FSC-H vs FSC-A), Zombie Aqua-, CD45+, CD3+, CD4+, CD8\u2212, CD19-, CD25lo cells and Treg were Zombie Aqua\u2212, CD45+, CD3+, CD4+, CD8\u2212, CD19\u2212, CD25+ and CD127lo. Immediately after sorting, cells were pelleted by centrifugation and resuspended in RLT (Qiagen) and RNA was extracted using QIAGEN RNeasy Micro Kits according to the manufacturer\u2019s protocol. RNA quantity and quality were accessed on Agilent 4200 TapeStation System.Sorted cell populations (mean\u2009\u00b1\u20091.S.D.) were 2555\u2009\u00b1\u20092254 for endometrial Treg, 36373\u2009\u00b1\u200937819 for endometrial CD4T cells and 34450\u2009\u00b1\u200914531 blood Treg. No significant differences were detected within sorted cell population counts between nulliparous controls, parous controls, primary RPL and secondary RPL.45 the library was prepared using a Standard Nextera Illumina Library Prep kit using a unique dual- index strategy to barcode cDNA fragments. DNA samples were amplified for 16 cycles and the cDNA quantities assessed using a 2100 Bioanalyzer with Quant-iT PicoGreen . Clustering and sequencing was carried out by Novogene Co., Ltd. Briefly, clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) and paired end RNA sequencing was performed using the Novoseq 6000 platform, with 125 bp/150 bp read length and ~25 million reads per sample. Quality control was performed by Novogene Co., Ltd and reads mapped to Ensemble Homo Sapiens cDNA database release 94. 1 NulC and 1 SRPL Treg sample did not pass QC and were excluded from analysis.Samples were processed following Smart-Seq 2 protocols,46 Heatmaps were generated using p\u2009<\u20090.01 padj and >3, <3 Log2FoldChange values and top 30 changes in expression value shown ranked by baseMean expression levels. PCA plots were generated using vst transformation. Gene Set Enrichment Analysis (GSEA)47 was performed using GSEA software (version 4.0.2) and results visualized in Cytoscape software (version 3.7.2).48 Venn Diagrams were generated using genes with reads >10 and calculated using Bioinformatics & Evolutionary Genomics tool hosted by the University of Ghent, Belgium (http://bioinformatics.psb.ugent.be/webtools/Venn/)Data analysis was performed using R (version 3.5.3) and RStudio, differential gene expression analysis was performed using DESeq2.All flow cytometry reagents were from Biolegend unless otherwise stated. Samples were incubated with Zombie-Aqua Fixable Viability kit for 15\u2009min in the dark, then cells washed in PBS/2%\u00a0FCS and antibodies towards cell surface markers were added for 20\u2009min at 4\u2009\u00b0C in the dark. Antibodies used were CD4-FITC(OKT4), CD8a-PE(HIT8a), CD8-PeCy7 (RPA-T8), CD3-PeCy5(UCHT1), CD56-PeCy7, CD16-APCCy7, CD45-AlexaFluor700 or CD45-APC (HI30),CD45RO-APC-Cy7(UCHL1), CD127-AlexaFluor647(A019D5), PD-1(CD279)-APC(EH12.2H7), CXCR6-PECy7 (K041E5), CD25-PE/Dazzle594 (M-A251), CCR8(CD198)-PE (L263G8), CD3-APC/Fire750 (SK7), CD45- AlexaFluor700 (HI30), CD127-BV711 (A019D5), ICOS-BV650 (C392.4A), CTLA-4(CD152)-BV786 , TIGIT-BV421, (A15153G), ENTPD1(CD39)-BV421 , LAG-3(CD223)-PE (113C65), TIM-3(CD366)-BV785 (F38-2E2), IL18R-PE (H44), S1PR1-eFluor550 . Afterwards, cells were washed in PBS/2% FCS and intranuclear antibody staining was performed using \u2018True-Nuclear Transcription Factor Buffer Set\u2019 (Biolegend), cells were fixed for 1\u2009h then transferred into PBS/2% FCS overnight, then permabilisation performed before addition of antibodies FOXP3-AlexaFluor647 (259D), IKZF2(HELIOS)-PeCy7 (22F6) and additional CTLA-4 antibody for total cellular CTLA-4 measurement, for 45\u2009min incubation.Data was acquired using an LSR-II flow cytometer (BD Biosciences) and data analysed using FlowJo software (Tree Star Inc.), fluorescence Minus One (FMO) controls established gating strategies. Graphs were plotted and statistics generated in GraphPad Prism 8.4.3.Supplementary figures"} +{"text": "FOXL2 mutations in human cause Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). While type II BPES solely features eyelid abnormality, type I BPES involves not only eyelid but also ovary, leading to primary ovarian insufficiency (POI) and female infertility. Current mainstream reproductive option for type I BPES is embryo or oocyte donation. Attempts on assisted reproductive technology (ART) aiming biological parenthood in this population were sparse and mostly unsuccessful.Two Chinese type I BPES patients with low anti-m\u00fcllerian hormone (AMH) and elevated follicle stimulating hormone (FSH) presented with primary infertility in their early 30s. Genetic studies confirmed two heterozygous duplication mutations that were never reported previously in East Asian populations. They received in vitro fertilization (IVF) treatment and both exhibited resistance to gonadotropin and difficulty in retrieving oocytes in repeated cycles. Doubled to quadrupled total gonadotropin doses were required to awaken follicular response. Patient 1 delivered a baby girl with the same eyelid phenotype and patient 2 had ongoing live intrauterine pregnancy at the time of manuscript submission.This is the second reported live birth of biological offspring in type I BPES patients, and first success using IVF techniques. It confirmed that ART is difficult but feasible in type I BPES. It further alerts clinicians and genetic counsellors to type female BPES patients with caution in view of the precious and potentially narrowed reproductive window.The online version contains supplementary material available at 10.1186/s13048-021-00900-2. FOXL2 rather than from contiguous gene syndrome. 67% to over 80% patients were detected mutation-positive in FOXL2 gene after clinical diagnosis based upon four typical eyelid features: blepharophimosis, ptosis, epicanthus inversus, and telecanthus [Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is a rare genetic disease involving predominantly the eyelid and ovary. The prevalence of this disease is estimated to be 1 in 50,000 births [ecanthus . Patientecanthus \u20136. PatieEfforts are being made to specify the phenotype-genotype correlation in type I BPES, in order to predict ovarian risk and guide long-term follow-up strategy for affected females at an early age. Unfortunately, unlike the eyelid feature, variable interfamilial and intrafamilial expressivity of ovarian function and fertility was observed . The onsFOXL2 duplication mutations that were published for the first time in East Asian populations. The duplication in patient 2 has not been reported to have caused type I BPES previously. More importantly, it was the first reported attempt of IVF on type I BPES patients, which revealed characteristic profile including discordance between antral follicle count (AFC) and anti-m\u00fcllerian hormone (AMH), gonadotropin resistance, low follicular output rate (FORT) as well as low oocyte retrieval rate (ORR). By sharing our challenge and success, we wish to provide heads-up, hope and help to BPES patients.Here we describe two Chinese type I BPES patients with heterozygous Both patients had typical eyelid features and had received plastic surgery in childhood. They presented to our clinic at the age of 32 for infertility. Patient 1 had menarche at the age of 12, maintaining overall regular menstrual cycle thereafter, except for one episode of menorrhagia which revealed complex hyperplasia and was treated with Mirena. She had a spontaneous pregnancy when she was 30 after actively trying to conceive for one and half years, but unfortunately miscarried at 7\u2009weeks of gestational age. Patient 2 had menarche at the age of 13, followed by oligomenorrhea ever since. Ovulation induction with gonadotropins failed at the dose up to hMG 150\u2009IU/day.On clinical examination, both patients exhibited well-developed secondary sexual characteristics. Body mass index (BMI), Tanner staging of pubic hair and breasts were summarized in Table\u00a0Antral follicle count (AFC): number of follicles with diameter between 4milimetre (mm) to 9\u2009mm seen at day 2\u20133 of menses.Preovulatory follicle count (PFC): number of follicles with diameter between 14\u2009mm to 22\u2009mm.Follicular output rate (FORT)\u2009=\u2009PFC/AFC\u2009\u00d7\u2009100% .Oocyte retrieval rate (ORR)\u2009=\u2009number of oocytes retrieved/PFC\u2009\u00d7\u2009100% .Both patients received two cycles of controlled ovarian hyperstimulation (COH) and obtained only one embryo. Patient 1 received first cycle in another centre and second cycle in our centre. Details and outcomes of COH in her first cycle were obtained from her outpatient medical record with authorization. Patient 2 received both cycles in our centre.In first COH cycle for patient 1, unexpected gonadotropin resistance was observed under GnRH agonist protocol, rendering her a long hyperstimulation course with increasing dose of hMG up to a maximum of 450\u2009IU per day. Not one oocyte was harvested from the six preovulatory follicles. GnRH antagonist protocol was used for her second cycle in our centre. COH was started at a dose of rFSH 150\u2009IU plus hMG 150\u2009IU per day, and was increased to rFSH 150\u2009IU plus hMG 300\u2009IU per day at the sixth day of COH due to poor response Fig.\u00a0. This maIn first COH cycle for patient 2, combined use of hMG and clomiphene citrate was administrated simultaneously at a starting dose of 225\u2009IU/day and 100\u2009mg/day from day 3 of menses. Both ovaries were barely responsive to serum FSH at 20 mIU/mL achieved by hMG stepped-up to 450\u2009IU/day. HMG was eventually increased to 525\u2009IU/day, and serum FSH reached 28.8 mIU/mL was carried out to cover all exons and\u2009\u00b1\u200910\u2009bp of neighboring introns, with depth at 122.15\u00d7 and average coverage at 99.36%. All candidate variants were annotated and filtered against public databases including 1000 Genome, gnomAD, ClinVar, dbNSFP, LOVD and ZJU-DB. PCR and Sanger sequencing was performed to confirm variants within patients and other members in the family. In-silico analysis with the online tool PROVEAN was implemented. Mutation nomenclature was made according to Human Genome Variation Society (HGVS). Integrated interpretation was made according to American College of Medical Genetics and Genomics (ACMG) guideline.FOXL2 duplication mutation (c.843_859dupGGCCGCACCCCCGCCTC) resulting in frameshift and premature termination resulting in insertion of five hydrophobic amino acids, Val-Ala-Leu-Ile-Ala, between the 64th and 65th amino acids . The baby weighed 2600\u2009g at birth, and manifested the same eyelid features as her mother . Only 1 case of type I BPES was reported to have achieved live birth by AID with monofollicular ovulation induced by high dose of rFSH Table\u00a0. No natuFOXL2 gene belongs to the winged-helix/forkhead transcription factor family, members of which are highly conservative. Pathogenic variants lead to either disordered subcellular distribution or dysregulation of downstream genes [FOXL2 is most abundantly expressed in the ovary [am genes . Apart fhe ovary , 12. It he ovary .FOXL2 mutation and BPES [Both of the two patients presented to our reproductive medicine clinic for infertility with underlying ovarian function defect. Since their eyelid abnormality occurred early at birth and were surgically corrected before good understanding of the relationship between and BPES , in addiDuring their fertility treatment, they were clinically resistant to gonadotropins, but not completely unresponsive. Furthermore, both exhibited paradoxically normal AFC despite low AMH, and thus did not fall into any of the four groups under Poseidon criteria . Beyond FOXL2 mutation [This is inspiring news considering the very limited successes reported in biological parenthood achieved by assisted reproductive technology (ART) for BPES patients Table\u00a0. In a lamutation .In patient 1 (c.843_859dup), insertion of 17 nucleotides Fig. resultedFOXL2, with either loss-of-function or dominant- negative effect [FOXL2 mutants lost the direct binding ability to SF-1 in vitro, which mediated its repression on steroidogenic genes CYP17 [et a l. [The rarer c.178_192 duplication found in patient 2 leads to an elongated protein. Noteworthily, the insertion of five hydrophobic amino acids occurs in the regulatory forkhead domain (FHD) antral follicles in both of our patients at their age, the mechanism of low AMH in type I BPES patients may differ from patients with non-genetic causes of ovarian insufficiency, such as advanced age, or acquired pathology . AMH is AMH and FOXL2 at different stages, is there imminently exhausted primordial follicle pool behind the ultrasonographic normal AFC? We do not have ovarian tissue histology to uncover the real situation in our two patients, but previous studies suggested risk of follicular depletion at an alarming rate, which eventually led to the well-known final stage of POI in type I BPES. Two siblings with a strong family history of autosomal dominant inheritance of BPES were reported in 1988, before establishment of genetic causation. The elder sister aged 28 and the younger sister aged 20 presented with secondary amenorrhea and hypergonadotropic hypogonadism. Ovarian pathological sections obtained from laparoscopy respectively showed true premature menopause with total absence of primordial follicles in the elder sister, and normal number of primordial follicles in the younger sister [Foxl2lacZ homozygous murine ovaries, the presence of defected squamous-to-cuboidal transition and premature expression of Gdf9 indicated accelerated depletion of primordial follicles and oocytes atresia, providing plausible explanation for the elder sister\u2019s histological finding. In another paper, two patients with FOXL2 deletions after PAT and resultant putative elongated proteins were reported. The 19-year-old patient suffered from primary amenorrhea with underlying low AMH and abnormal ovarian histology . The 25-year-old patient presented with infertility, but had normal AMH and normal ovarian reserve clinically, ultrasonographically, and histologically. But there was also evidence of uncommon advanced expression of Ki67 in transitional follicles implying accelerated recruitment [2)Gonadotropin resistance: Our patients were resistant to gonadotropins, yet responsive at high doses with extended courses. Target serum FSH seemed to be higher than 25 to 30 mIU/mL. Doubled to quadrupled total gonadotropin doses in comparison to general population was required in various COH protocols in order to awaken follicular response, and clomiphene citrate was added to patient 2 for enhancement.Without the guarding by properly-expressed r sister . In FoxlClomiphene citrate is a selective estrogen receptor modulator. It was utilized in ovulation induction or stimulation by preventing estrogen negative feedback and thereby stimulating endogenous gonadotropin release. It was shown that supplement of clomiphene improved ovarian stimulation outcomes in IVF treatment and decreased total gonadotropin dosage, which applied to women with advanced age and/or poor response , 30. We FOXL2 gene localization, also described the phenomenon of ovarian resistance to gonadotropins in BPES patients [3)Abnormal ovulation and low ORR: Less studies focused on ovulation in BPES patients. As described, patient 2 was oligomenorrheic secondary to anovulation. Patient 1 maintained overall natural normal cycle with sonographically confirmed ovulation. It was at least once followed by successful natural fertilization and implantation but unluckily ended up with first-trimester miscarriage. However, she also reported to have experienced cycles judged as luteinized unruptured follicle syndrome (LUFS). Furthermore, the history of endometrial hyperplasia implied possible preceding anovulation. There might be some mechanism behind the disordered ovulation of graafian (preovulatory) follicles as well as the difficulty in harvesting oocytes from them in IVF. Hypotheses include hampered oocyte maturation, abnormal expression of Luteinizing hormone receptor (LHR) , defecteAs a result of the augmentation of hyperstimulation, FORTs could be improved but still low compared to general IVF patients. Apart from the AID case mentioned above, who succeeded by using high dose rFSH, few case reports, before and after patients , 31\u201334, Currently mainstream recommendation at genetic counseling for type I BPES patients includes donation of eggs or embryos, adoption or fostering, sacrificing biological parenthood. This is probably based on the following considerations: 1) missed reproductive window at presentation; 2) limited translation of in vitro maturation (IVM) into clinical use; 3) ethical concerns about precautionary ovarian tissue cryopreservation or oocyte cryopreservation for young girls with unclear ovarian phenotype; 4) risk of inheritance of pathogenic mutations.Noteworthily on the other hand, patients manifesting either type of BPES usually have normal menarche and secondary sex characteristics. Association with major defects including intellectual disability were rare with inconclusive causation. Even type I BPES phenotypes with primary or secondary amenorrhea possess potential capacity of natural conception and deserve a childbearing opportunity. Early intervention may rescue their threatened fertility.Clinicians and scientists were making efforts to specify the phenotype-genotype correlation in ovarian function . StudiesFOXL2 mutations were new to East Asian population database. Their ovarian profiles seem to differentiate type I BEPS from either pure resistant ovarian syndrome (ROS) or pure POI, and offers materials for further basic research on FOXL2 gene and type I BPES. Of course, due to the rarity of the disease, we look forward to more cases to reproduce the success and the observed phenomena. Nevertheless, our report confirmed that IVF in type I BPES is difficult, but still feasible with individualized treatment. It also serves as a stepping-stone to future attempts on IVM and PGT in this population.In summary, we described detailed IVF treatment to two Chinese type I BPES patients, whose Additional file 1. Summary of genetic analysis findings (table).Additional file 2. Patient 1\u2019s daughter-facial manifestations."} +{"text": "Coronavirus disease 2019 (COVID-19) has caused a global public health crisis. Preventive measures to tackle the deadly virus are influenced by people's knowledge, attitude, and practice (KAP) toward COVID-19. This study aimed to assess the level of knowledge, attitude, and practice toward COVID-19 among Nepalese residents in Nepal.A web-based cross-sectional survey was conducted among 755 Nepalese residents across all seven provinces of Nepal. The questionnaire used to determine the KAP of the participants was derived from a previous study conducted in Nepal. Descriptive analysis was done to identify the distribution of socio-economic and demographic characteristics of participants. Factors associated with residents\u2019 KAP regarding COVID-19 were examined using Chi-square tests at the significance level of 0.05.ka\u00a0= 0.184, p\u00a0<\u00a00.001), attitude-practice and knowledge-practice were positively correlated in this study.The mean age of the participants was 24.6 years. At the time of data collection, 8.2% of the participants had their families in isolation or quarantine center. In this study, 76.4%, 58.0%, and 63.6% of the participants had a good knowledge level, attitude level, and practice level respectively regarding COVID-19. Occupation and marital status were significantly associated with knowledge, attitude, and practice level. Age was significantly associated with knowledge and attitude level. Those participants who had their family members in quarantine were found to have a good level of preventive practice. The knowledge-attitude , good attitude (A), and good practice (P) level respectively regarding COVID-19.\u2022Occupation and marital status were significantly associated with KAP of participants. Similarly, those who had their family members in quarantine were found to have a good level of preventive practice.\u2022This study included participants from all of the seven provinces of Nepal during the peak of the COVID-19 pandemic. Study objectives were explained in the Google forms, and e-informed consent was taken from all the participants before the data collection and females (49.4%) participated in the study. The majority of the participants (68.6%) belonged to the age group below 25 years. Half of the participants were Brahmin/Chhetri (49.4%) ethnic group followed by Janajati (30.6%). In this study, more than one third of the participants were from Province One (38%) followed by Bagmati Province (23.6%) .Table 1C3.23.3In this study, 58% of the participants had a positive attitude level towards COVID-19. The attitude level of participants towards COVID-19 was found to be significant with age , marital status , and occupation .Table 3F3.4In this study, almost two-thirds (63.6%) of the respondents demonstrated a good practice level towards COVID-19. Marital status , occupation and family member in quarantine/isolation were significantly associated to the practice level. Table 4Fa3.54This study showed that 76.4% of the Nepalese residents had a good knowledge level regarding COVID-19, which is consistent with the findings of previous studies conducted in Malaysia , EthiopiParticipants in the lower age group (<25 years) had higher odds of having good knowledge and attitude regarding COVID-19 compared to participants in the higher age group. This is consistent with the findings of the study conducted in Nepal , and ChiFemales had 1.46 times higher odds of having a good knowledge level compared to their male counterparts. Studies conducted in Jordan , Saudi AIn our study, participants belonging to the health sector had significantly higher odds of having good knowledge, attitude, and practice toward COVID-19. The findings of previous studies conducted in Nepal , VietnamThis study showed a better attitude level among Nepalese residents than the previous study conducted in Nepal , EthiopiAlmost 64% of the participants showed a good practice level in our study which is better than the study conducted among residents of Ethiopia . It may This study showed a significant positive correlation between knowledge-attitude, knowledge-practice, and attitude-practice supporting the findings of similar studies conducted in Nepal , Banglad5This study included participants from all seven provinces of Nepal with a relatively bigger sample size (n\u00a0=\u00a0755). Another strength of this study is that it was conducted during the peak of the COVID-19 pandemic in Nepal.Despite its strength, this study has a few limitations. Firstly, all the measurements in this study were based on self-reports, which may have been prone to response and information bias. Secondly, this study was cross-sectional and, therefore, cannot demonstrate causality between the variables. Lastly, as this was a web-based survey, poor internet connection might have discouraged some participants to fill out the online questionnaire.6This study revealed a satisfactory level of knowledge, attitude, and practice toward COVID-19 during the pandemic. Based on these findings targeted community awareness intervention programs and effective health education that are aimed at improving knowledge, attitude, and practice toward disease outbreaks during the earliest phase to prevent further transmission might be useful. The findings may help local health authorities and policymakers to identify the target populations to conduct awareness programs in a future infectious disease outbreak. Collaborative efforts between health services providers, the ministry of health and population, local governments, development partners, and the media should be implement as an effective tools that increase the KAP of individuals and households regarding infectious diseases like COVID-19.The approval for this study was taken from the Institutional Review Committee, of Institute of Medicine (Ref no: 109(6-1) E2077/078. Study objectives were explained in the Google forms, and e-informed consent was taken from all the participants before the data collection.No funding was received for the study.MR conceived the study, administered the project, conducted formal analysis, and wrote the first draft of the manuscript. SB and KP contributed in the methodology, formal analysis, writing original draft, reviewing, and editing. BP, EK, BRK, BA, AG, and GS wrote the first draft of the manuscript. NK supervised the whole study. All authors reviewed and approved the final version of the manuscript.Study objectives were explained in the Google forms, and e-informed consent was taken from all the participants before the data collection.1. Name of the registry: None.2. Unique Identifying number or registration ID: None.3. Hyperlink to your specific registration (must be publicly accessible and will be checked):Sandesh Bhusal.Not commissioned, externally peer-reviewed.Authors have no conflict of interest to declare."} +{"text": "The global orthopedic market is forecasted to reach US$79.5 billion by the end of this decade. Factors driving the increase in this market are population aging, sports injury, road traffic accidents, and overweight, which justify a growing demand for orthopedic implants. Therefore, it is of utmost importance to develop bone implants with superior mechanical and biological properties to face the demand and improve patients\u2019 quality of life. Today, metallic implants still hold a dominant position in the global orthopedic implant market, mainly due to their superior mechanical resistance. However, their performance might be jeopardized due to the possible release of metallic debris, leading to cytotoxic effects and inflammatory responses in the body. Poly (ether-ether-ketone) (PEEK) is a biocompatible, high-performance polymer and one of the most prominent candidates to be used in manufacturing bone implants due to its similarity to the mechanical properties of bone. Unfortunately, the bioinert nature of PEEK culminates in its diminished osseointegration. Notwithstanding, PEEK\u2019s bioactivity can be improved through surface modification techniques and by the development of bioactive composites. This paper overviews the advantages of using PEEK for manufacturing implants and addresses the most common strategies to improve the bioactivity of PEEK in order to promote enhanced biomechanical performance. Population aging, sports injuries, traffic accidents, and overweight are some of the many factors that lead to increasing demand for orthopedic implants provoking public health concerns . For exaBone is a dynamic tissue that undergoes a continuous remodeling process. However, spontaneous healing and repair may fail in the case of large bone defects or pathological fractures ,8,9. ThePoly(ether-ether-ketone) (PEEK) was proposed as a biomaterial in 1998 by Invibio Ltd. . Since then, PEEK-based materials have become an important group of biomaterials used in orthopedic and spinal implants owing to their outstanding properties . Other cThis overview presents and discusses the reported strategies used to modify PEEK properties in order to mimic the biomechanical properties of bone. The content summarized herein aims to highlight future directions for manufacturing PEEK implants.Over the past decades, metals have dominated the orthopedic implant market ,17. NoneA common problem related to metallic implants in load-bearing applications is stress shielding. This phenomenon occurs because metals, such as cobalt-chromium-molybdenum alloy, 316L grade stainless steel, titanium-aluminum-vanadium alloy, and titanium, are much stiffer than the host bone, as shown in Another problem commonly observed in patients with metallic implants is metallosis, a medical condition characterized by the release of metallic wear debris into periprosthetic tissues and blood . MetalloMetallic devices are prone to corrosion due to the harsh body fluid environment they are exposed to. In vivo corrosion resistance in implants is a key factor in assuring their functionality and biocompatibility. Depending on the level of corrosion, the biomaterial might lose its mechanical properties. Furthermore, cytotoxic and carcinogenic metal ions may be released during corrosion, triggering allergy, inflammation, and even metal poisoning ,41. CompAnother advantage of polymer-based devices is their radiolucency, which allows for improved X-ray and computed tomography (CT) imaging compared to radiopaque metals . In the Metallic implants are also known to create artifacts in magnetic resonance imaging (MRI), which may significantly hinder the ability of researchers and clinicians to visualize tissue proximal to the implant . Figure Besides the aforementioned advantages of polymeric materials over conventional metallic orthopedic materials, PEEK devices are comparatively lightweight, offering ergonomic benefits to the patient. Furthermore, the high melting temperature of metals makes any melting processing step extremely energy-intensive and expensive. Regarding the materials\u2019 synthesis, there are two main PEEK polycondensation processes: the nucleophilic and the electrophilic routes. Most PEEK used in industrial applications is synthesized by the nucleophilic route patented in 1977 and commercialized by the brand Victrex PEEK . This meThe manufactured PEEK is a rigid high-performance semicrystalline engineering thermoplastic. It is known for its outstanding thermal stability and high mechanical strength, as well as for its wear and chemical resistances. Besides, PEEK is radiolucency, bioinert, and has radiation stability. PEEK also has an average melting temperature of 343 \u00b0C and a glass transition temperature of 143 \u00b0C ,58,59,60As aforementioned, PEEK is a bioinert material with poor bonding to the surrounding tissues leading to unsatisfactory bone\u2013implant integration. The lack of osseointegration along the implant\u2013bone interface can lead to implant loosening due to its encapsulation by fibrous tissue and/or colonization by bacteria caused by the foreign body reaction that happens after the surgery, as schematically illustrated in When a biomaterial is placed in a biological environment, the first molecules that reach the implant surface are those of water. Following that, proteins interact with the biomaterial, and this contact is affected by the adsorbed water molecules. Subsequently, the adhesion of cells interacting with the adsorbed proteins takes place, influencing tissue growth as schematically shown in Two methods are proposed in the literature to overcome PEEK\u2019s hydrophobicity and its lack of bioactivity: (i) the development of composites with bioactive fillers, and (ii) PEEK\u2019s surface treatment. This second method can be divided into direct surface modification and surface coating techniques 65]..65].After surgery, bone implants are directly in contact with bone tissue. Therefore, the biological properties of their surface are important for osseointegration. In this concern, the surface modification technique aims to alter the surface characteristics of PEEK without affecting its bulk properties .4H2. These groups were studied because they are capable of inducing apatite layer growth on their surface in the presence of simulated body fluid. The subsequent tests showed that these species presented beneficial properties supporting cell adhesion, spreading, proliferation, and higher osseointegration compared to pure PEEK. Sulfonation of the PEEK chain is another treatment that improves the polymer hydrophilicity and bioactivity [Chemical modifications introduce chemical groups into PEEK\u2019s surface, creating a series of surface-functionalized PEEKs. It changes the surface chemical structure of PEEK to generate an environment with a favorable cellular response ,69. Zhenactivity . Concentactivity proposedactivity introducactivity combinedactivity .The commonly used physical treatments to modify PEEK\u2019s surface are plasma, laser, accelerated neutral atom beam (ANAB), and ultraviolet (UV) irradiation. The plasma treatment was used to alter the surface chemistry of the material. Nitrogen ,75, oxygSome drawbacks in the treatments reported above lie in the recovery of the initial PEEK hydrophobicity ,92. This2) [3) [2 can also be coated into PEEK through the arc ion implanting process. The TiO2-PEEK substrate studied by Tsou et al. [The deposition of a bioactive layer on PEEK\u2019s surface is another modification process to improve its cell affinity. Several techniques are feasible for applying bioactive coatings, such as electron beam evaporation, arc ion plating, plasma spraying, plasma immersion ion implantation, chemical deposition, and spin coating. The deposition of bioactive materials such as titanium dioxide (TiO2) and calc2) [3) by e-beau et al. showed au et al. . After t3H groups, responsible for the increase in the surface roughness, and then HA was deposited. The obtained material presented an increase in wettability, which is an indication of improved bioactivity. The deposition of HA through the spin-coating technique was also reported by Johansson et al. [Furthermore, the stiffness at the bone\u2013implant interface with Ti-bond was significantly greater than in uncoated PEEK. Tantalum (Ta) and calcn et al. . Their rAs evidenced, the bioinertness of PEEK can be greatly enhanced by means of the coating of bioactive materials. Nonetheless, some difficulties are associated with this method. For example, coating PEEK with metallic materials may trigger problems already associated with the use of metallic implants. For instance, stress shielding, due to an increase in the elastic modulus and the release of metal ions, can increase the risk of inflammation and implant loosening ,104. FurIt is worth mentioning that even thin coatings can interfere with the clinical analysis of the bone\u2013implant interface owing to artifacts produced during medical imaging techniques . Among tAn alternative to overcome the aforementioned surface modification shortcomings is to tailor PEEK\u2019s properties by compounding it with nanoparticles. The melt-processing temperature of PEEK ranges between 360 and 400 \u00b0C, and it can be processed using all of the typical thermoplastic processes, such as injection molding, extrusion, and compression molding. The high processing temperature and inertness in most solvents hinder PEEK processability, making it a challenging procedure. In contrast, its high thermal and chemical stability provides remarkable resistance to sterilization by gamma and electron beam radiation, an important attribute in biomedical applications, among other advantages .2 [Different bioactive materials such as TiO2 , bioglas2 , calcium2 , \u03b2-trica2 ,112, nat2 , HA, and2 , as well2 , have al2 ,120,121.2 . TherefoOne way to compound PEEK and HA is by using ball milling and injection molding processes, as reported by Ma and Guo . The ten2CO3, and Na2CO3 were used for PEEK synthesis, and different contents of commercial HA powders were introduced to the reaction medium. Lower HA contents (2.6 and 5.6 vol%) increased the tensile strength of the composites. The composite reached the higher tensile strength of 106 MPa for 2.6 vol% of HA. However, higher HA contents decreased the strength, probably due to filler agglomeration. Despite the decrease in the tensile strength, the composite with 8.7 vol% of HA displayed a tensile strength of 75 MPa, which is in the range of the tensile strength of the cortical bone (50\u2013150 MPa) . AlthougA current proposal by Zhong et al. to produAnother way to prepare these composites is first to disperse PEEK and HA in a solvent to prevent the agglomeration of HA particles in the PEEK matrix. In Li et al.\u2019s study, PAn attempt to improve PEEK-HA bonding is to modify the HA surface. Ma et al. reportedAs seen in the reviewed studies ,118,119,One of the first fillers added to PEEK to improve its stiffness and strength was carbon fiber reinforcement (CFR). For instance, PEEK with CFR has an elastic modulus in the range of the cortical bone, i.e., around 20 GPa , while tThe biofunctionalization of PEEK is recommended to enhance its bioactivity. For example, in an attempt to obtain a composite with proper biomechanics, HA was added to the PEEK/CFR composite . The preDespite the improved mechanical properties promoted by the CFR, clinical concerns have already been reported in the literature regarding composites with CFR for biomedical applications. For example, a clinical study reported the failure of the PEEK/CFR tibial intramedullary nail 10 weeks after its placement . Another2 into PEEK can simultaneously promote reinforcement and antibacterial activity in the matrix [2 are generally recognized as safe and effective components by the Food and Drug Administration (FDA) [The incorporation of zinc oxide (ZnO) and TiOe matrix ,132,133.on (FDA) .2-ZnO), which was subsequently compounded with PEEK in a mini-extruder at 380 \u00b0C. The produced PEEK/masterbatch composites showed a higher stiffness, strength, and ductility than both the pure PEEK and the PEEK/ZnO composites prepared for comparison. The antimicrobial behavior of PEEK/masterbatch composites was similar to the one observed in their previous study, i.e., the antimicrobial effect increased by raising the amount of the nanoparticles and was found to be higher for PEEK/masterbatch composites. Monta\u00f1o-Machado et al. [2 composites using a single screw extruder was performed by Bragaglia et al. [2 slightly increased the stiffness of the material and barely affected the elongation at break and the tensile strength, not turning the material brittle. Although the antibacterial activity was not tested in the aforementioned study [2 in the blend PEEK/PEI promoted antibacterial activity in the material.D\u00edez-Pascual, Xu, and Luque incorporated ZnO and o et al. prepareda et al. . The preed study , in a pred study it was c\u00aeEPOCH\u00ae [In the orthopedic segment, a commercial femoral stem containing PEEK in its current architecture is the VerSysIN, USA) . It is aIN, USA) ,140. In IN, USA) which isIN, USA) . In the IN, USA) . HoweverThe growing need for implant customization leads to additive manufacturing (AM) as an emerging technique to fulfill this demand. This technology has attracted the attention of surgeons and patients, and its application is growing steadily. AM is a process used to make 3D-printed objects from a digital model by depositing successive layers of the material . Despite2, can significantly increase the mechanical strength of PEEK composites. However, the reinforced composite lacks bioactivity, a property that is a key factor in providing direct bone\u2013implant bonding, avoiding implant failure. Therefore, it is still a great challenge to produce a PEEK-based material that can be used in load-bearing conditions. This overview intends to point out the drawbacks of using metals in implant devices and highlight that it is important to concentrate efforts on producing a tough and bioactive non-metallic material in an attempt to improve the quality of life of people all around the world.The clinical interest in developing a material with adequate biomechanics is well recognized. PEEK has drawn considerable attention to being used in load-bearing biomedical applications. As extensively discussed in this work, its bioactivity should be enhanced. The surface modification techniques usually used for these purposes face more drawbacks than bulk modification. The main problem of the chemical and physical treatments relies on the recovery of the hydrophobicity of PEEK, jeopardizing the cell\u2019s interaction. Furthermore, the grafting of polar groups commonly used may create an unstable surface. Moreover, the delamination that a surface coating material may undergo inefficient stress transference between the bone and the implant. Therefore, bulk modification emerges as a better route to enhance PEEK\u2019s bioactivity. As evidenced, the addition of HA to PEEK, widely studied in the literature, turns the material brittle but improves its bioactivity. On the other hand, adding reinforcing fillers, such as CFR, ZnO, and TiO"} +{"text": "Polyether-ether-ketone (PEEK) biomaterial has been increasingly employed for orthopedic, trauma, spinal, and dental implants due to its biocompatibility and in vivo stability. However, a lack of bioactivity and binding ability to natural bone tissue has significantly limited PEEK for many challenging dental implant applications. In this work, nanocomposites based on PEEK reinforced with bioactive silicate-based bioceramics (forsterite or bioglass) as nanofillers were prepared using high energy ball milling followed by melt blending and compression molding. The influence of nanofillers type and content on the crystalline structure, morphology, surface roughness, hydrophilicity, microhardness, elastic compression modulus, and flexural strength of the nanocomposites was investigated. The scanning electron microscopy images of the nanocomposites with low nanofillers content showed a homogenous surface with uniform dispersion within the PEEK matrix with no agglomerates. All nanocomposites showed an increased surface roughness compared to pristine PEEK. It was found that the incorporation of 20 wt.% forsterite was the most effective in the nanocomposite formulation compared with bioglass-based nanocomposites; it has significantly improved the elastic modulus, flexural strength, and microhardness. In vitro bioactivity evaluation, which used biomimetic simulated body fluid indicated the ability of PEEK nanocomposites loaded with forsterite or bioglass nanofillers to precipitate calcium and phosphate bone minerals on its surface. These nanocomposites are expected to be used in long-term load-bearing implant applications and could be recommended as a promising alternative to titanium and zirconia when used as a dental implant material. Missing teeth with their supporting oral tissues are considered one of the major concerns in modern dentistry. Substituting single missing teeth, particularly in the anterior region, has always been an encounter for dentists . With inTraditionally, metals or ceramics are the most prevalent dental materials for use in dentistry. In particular, titanium (Ti) and its alloys were typically used over the past decades as orthopedic/dental implants due to their low density, high mechanical strength, excellent corrosion resistance, and cytocompatibility ,6. NeverPolymeric materials have been widely used for various applications in clinical dentistry. They offer a unique combination of properties and potentials that address fundamentally important issues of dental implants. Polyether-ether-ketone (PEEK) and polyether-ketone-ketone (PEKK) are both parts of the polyaryl-ether-ketone (PAEK) family of ultra-high performance thermoplastic polymers. PEEK is a semi-crystalline organic polymer exhibiting a highly stable chemical structure, which has been progressively employed for orthopedic and dental implants ,16. From10(PO4)6(OH)2, HA) because HA is a constituent of living bone [Although PEEK implants have been used frequently in modern medical operations, PEEK itself is chemically hydrophobic and biologically inert ,26,27. Sing bone . PEEK/HAing bone .Silicate-based bioceramics, such as bioglass (BG), forsterite (FT), calcium silicate, bredigite, and akermanite have received increasing attention to treat bone defects. BG has excellent bioactivity and biocompatibility. It is used in several biomedical applications, for example, non-load-bearing implants, bone cement, tissue engineering scaffolds, drug delivery systems, and bioactive coatings ,32. In pFT is a new bioceramic that has demonstrated good biocompatibility. Researches show that FT also possesses mechanical properties superior to those of calcium phosphate ceramics. Additionally, nanoscale FT has superior bioactivity in the biological environment to bulk-form FT . TherefoThe present study reports the fabrication of either BG- or FT-reinforced PEEK nanocomposites via melt blending followed by compression molding, a common and versatile method easy to scale up. A comprehensive characterization was performed to evaluate in detail the influence of the types and content of nanoparticles on the surface morphology, crystallization behavior, mechanical performance, and bioactive properties in vitro of these biomaterials that are very promising for use in dental implant applications.\u00ae PEEK polymers, Victrex Technology Centre, Lancashire FY5 4QD, UK) with an average particle size of 50 \u03bcm for compression molding was used as the polymer matrix. The density of PEEK is 1.3 g/cm3, and its melt viscosity is 350 Pas. Two types of bioactive inorganic nanoparticles, 45S5 bioglass (BG) of chemical composition and forsterite (FT) of chemical structure (Mg2SiO4) were purchased from NanoTech Egypt Co., Giza, Egypt. The BG nanopowder had an average particle size of 40 \u00b1 4 nm while the average particle size of the FT nanopowder was 30 \u00b1 5 nm. The chemical reagents for the preparation of simulated body fluid (SBF) are sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydrogen carbonate (NaHCO3), potassium chloride (KCl), hydrated dipotassium hydrogen phosphate (K2HPO4\u00b73H2O), hydrated magnesium chloride (MgCl2\u00b76H2O), calcium chloride (CaCl2), sodium sulphate (Na2SO4) and Trisma\u00ae base (NH2C(CH2OH)3) \u2265 99.9% and were purchased from Sigma-Aldrich Chemical Co., Darmstadt, Germany.Semi-crystalline PEEK fine powder . The X-ray beam was Cu K\u03b1 radiation (\u03bb = 0.1542 nm), operated at 40 kV and 30 mA. The XRD pattern was recorded in the 2\u03b8 range from 10\u00b0 to 50\u00b0 with a scanning rate of 5\u00b0 per min. The identification of phases was accomplished by comparing the obtained sample diffraction pattern through standard cards in the International Centre for Diffraction Data database and the Joint Committee on Powder Diffraction Standards (ICDD-JCPDS) database .The surface morphology of micro-roughened PEEK/BG and PEEK/FT nanocomposites was characterized by a scanning electron microscope (SEM) using a JEOL instrument , which was run at 25 keV. Prior to SEM observation, all samples were ultrasonically washed for 1 min. and sputter-coated with gold to a thickness of 30 nm.n = 6 per group) was evaluated (according to ISO 4287-1:1997) by an optical profilometer . Four different locations perpendicular to the surface of each sample were recorded. Mean Ra values were statistically analyzed and used as the final Ra score for each sample [The surface roughness (Ra) of the samples were used to determine the surface hydrophilicity of the samples. The static sessile drop technique was performed using the video contact angle method. Six samples in each stage were used to provide an average and standard deviation .The microhardness of the samples was measured as a Vickers\u2019 hardness number (VHN) . The indentations were made using a diamond pyramid micro-indenter with a 136\u00b0 angle between the opposing faces under a load of 200 N applied for a dwell time of 10 s.Compression analysis was carried out by using a universal testing machine according to ISO 604: 2002 (Plastics\u2014Determination of compressive properties). The test was performed with a cylindrical sample of dimensions (10 mm diameter \u00d7 6 mm height) at a crosshead speed of 1.0 mm/min until the specimen failed. Flexural properties were measured using a three-point bending test method in the same universal testing machine according to ISO 178: 2010 . The test was carried out with a rectangular bar sample of dimension (80 mm \u00d7 6 mm \u00d7 6 mm) at a crosshead speed of 1.0 mm/min at room temperature. A total of six samples were tested for each material group to obtain the average value .The pristine PEEK, PEEK/BG, and PEEK/FT samples were immersed in a simulated body fluid (SBF), almost equal to that of human blood plasma at 37 \u00b0C by water bath to examine the bioactivity. After 7, 14 and 28 days, the specimens were removed from the given solution, gently rinsed with distilled water, and quickly dried and kept in a drying oven at room temperature. The chemical composition of surface deposits was characterized by energy-dispersive X-ray (EDX) microanalysis attached to the SEM. Analysis was performed on uncoated samples at 15 kV for 60 s and the Ca/P ratio on the surface of the samples was determined. After sputter-coated with gold, the microstructures of specimens were examined by SEM equipped with EDX .p = 0.05, Tukey\u2019s post hoc test, and Student\u2019s t-test.Statistical Package for the Social Sciences (SPSS) software version 20 was used to analyze the data. The Kolmogorov\u2013Smirnov (K\u2013S) test was applied to assess the assumptions of normal data distributions and homogeneity of variances. Multiple comparison analyses were performed using a one-way analysis of variance (ANOVA) analysis with PEEK/BG and PEEK/FT nanocomposites were successfully fabricated by the dry mixing of PEEK and bioactive nanofillers using high energy ball milling, followed by melt blending and compression molding. Indeed, the uniform dispersion of the nanofillers particles within the PEEK matrix is crucial for improving the mechanical properties. However, this is laborious in the case of PEEK nanocomposites, as they cannot be processed by solution methods due to the insolubility of PEEK in most organic solvents except concentrated hydrofluoric and methyl sulfonic acids. Hence, the ball milling technique was used to reduce the particle size of pristine PEEK from the millimeter to micrometer scale (~5 \u03bcm) and disperse nanofillers particles into the PEEK matrix . Moreove\u03b8 = 18.5\u00b0, 21\u00b0, 22.5\u00b0 and 28.6\u00b0, corresponded to the characteristic peaks of PEEK [\u03b8 of around 29.24\u00b0 and 33.75\u00b0, there appeared peaks that were typical amorphous structure characteristic peaks of silicate glass, according to ICDD [ of PEEK . The XRD to ICDD . The res\u03b8 = 42.80\u00b0, suggesting that the high crystalline FT phase was formed within the PEEK/FT nanocomposites [The crystalline phases of the as-milled PEEK and PEEK/FT were also examined, as presented in mposites .The PEEK/FT nanocomposites showed higher crystallinity in comparison with the PEEK/BG nanocomposites and pure PEEK. The reason behind this result is that the introduction of FT nanofillers to the PEEK matrix can provide additional nucleation sites within the polymer and thereby increase the overall crystallization of PEEK/FT nanocomposites .However, the addition of BG nanofillers into the polymer matrix decreases the overall crystallinity of the PEEK/BG nanocomposites in comparison to the pure PEEK, which could be attributed to the amorphous nature of the BG nanofillers, which is not expected to show any changes in the crystallinity of pure PEEK. It was reported that the amorphous nanoparticles could, more or less, hinder the mobility of the polymer chain segments during crystallization, especially when the particle concentration is high enough ,41,42.SEM micrographs of the pure PEEK and PEEK nanocomposites loaded with different bioactive nanofillers contents were captured to investigate the exact microstructure, as shown in Regarding the SEM images of PEEK/FT (PKFT group), the surface appeared more compact and smoother in comparison to that of the PKBG group, and white particles appear randomly distributed on the surface of the PEEK matrix with no agglomerates shown on the surface, as illustrated in p < 0.05) the surface roughness of the PKBG group by 31%, 43%, and 56%, respectively. However, the increase in the surface roughness of the PKFT group was less than that of the PKBG group. It can be noticed that the interactions of BG nanofillers with PEEK are not strong enough to enable good dispersion within the polymer matrix. Furthermore, FT nanofillers are relatively more compatible with the PEEK matrix. These results are in agreement with previously reported data [Average values of surface roughness for the pure PEEK and PEEK nanocomposites loaded with different BG and FT nanofillers contents are summarized in ted data ,45,46.It is worth mentioning that surface roughness is thought to be beneficial for the bioactivity of dental implants. Several authors stated, that adding surface roughness will enhance the wettability or hydrophilicity of PEEK dental implants, which in turn, exerts a positive influence to overcome the limited bioactivity of PEEK ,48. For p < 0.05) the contact angle in comparison to pure PEEK. For example, loading 20 wt.% BG and FT reduced the water contact angle to 66.7\u00b0 and 70.4\u00b0, respectively, revealing the increased hydrophilicity by the addition of nanofillers. It is remarkable that the contact angle of PEEK/BG nanocomposites is significantly less than that of PEEK/FT nanocomposites. The reason behind this result is the relatively more hydrophilic nature of BG in comparison to FT [The bioperformance of dental implants is affected by surface hydrophilicity, as it promotes the interaction between the dental implant material and the surrounding tissue . Thus, ion to FT .p < 0.05) when BG nanofiller was incorporated into the nanocomposite formulation. For example, the addition of 20 wt.% BG decreased the microhardness value from 35 to 26 Kg/mm2, that is, 26% lower than pure PEEK. No significant difference was observed within the PKBT group with increasing the amount of BG. The remarkable decrease in hardness could be explained by the widening of the silicate network reducing the glass density and thus, affecting the mechanical properties of the glass, especially the microhardness. Moreover, the intrinsic brittleness of the BG can negatively affect microhardness [p < 0.05) by 23% with the incorporation of 20 wt.% FT nanofillers into the PEEK matrix but did not increase further when the FT content was increased to 30 wt.%. This may be due to the higher stiffness of the FT nanofillers. Moreover, the relatively uniform distribution of the FT nanoparticles, as seen in the SEM image, and the decrease in the interparticle distance with increasing the particle loading in the matrix results in an increase in the resistance to indentation of the PEEK matrix.The surface microhardness assessment could be considered a crucial property when testing and comparing different dental implant materials. This is mainly owing to the critical demand for different dental implants for variable surface treatments to achieve adequate osseointegration and to gain long-term success . The reshardness ,53. On tThe compression elastic modulus of pure PEEK and its nanocomposites, fabricated by loading different contents of BG and FT nanofillers, are shown in The flexural strength of pure PEEK and its nanocomposites exhibited similar trends, as displayed in From these results, we can conclude that the PKFT-20 sample has the highest flexural compression elastic modulus and strength among all groups, which could be explained by the more uniform and homogenous dispersion of the FT nanofillers when compared to BG. In addition, the intrinsic brittleness of the BG fillers can negatively affect the mechanical properties ,53,54,55Regarding PKFT-20 nanocomposite, few crystals of apatite islands formed on the surface on day 7, as seen in The EDX results acquired from the immersed specimens revealed that the formed particle contains Ca and P and their ratio was displayed in The trend of the increase in apatite formation on the surface of the PKBG-20 sample is due to the presence of Ca and P ions within the composition of the BG, which increases the nucleation rate of HA in SBF. This finding agrees with a previous study, which concluded that the composition features of BG are responsible for making bioglass highly reactive to an aqueous medium ,60. On tIn this study, by evaluating the microhardness, elastic modulus, and flexural strength of PEEK/BG and PEEK/FT nanocomposites with different BG and FT contents (0\u201330 wt.%), the 20 wt.% FT was considered to be an optimal content. Compared with pure PEEK, both PEEK/BG and PEEK/FT nanocomposites induced apatite formation after immersion in SBF. More importantly, the rough PEEK/FT nanocomposite loaded with 20 wt.% FT exhibits improved bioactivity by immersing the nanocomposite disks in SBF for 28 days due to the co-effects of rough structure and nano-FT crystals. These results have paved the way for the PEEK-based nanocomposite to be used as a potential substitute for metal implant material in orthopedic and dental applications. However, experiments on the in vivo bioactivity and osteogenic differentiation capacity have to be conducted in order to confirm the bone formation around the PEEK-based nanocomposite implants."} +{"text": "In vitro biological evaluations showed that MC3T3-E1 osteoblast responses including adhesion, spreading, proliferation, alkaline phosphatase activity, extracellular matrix mineralization, collagen secretion, and osteogenesis-related gene expression exhibited dose-dependent enhancement depending on the density of phosphonate groups. Most importantly, histological analysis and biomechanical tests showed that in a rat femur implantation model, PEEK bearing phosphonate groups had a better bone-to-implant contact ratio and corresponding bone-to-implant bonding strength at 12 weeks post-implantation than unmodified PEEK. Thus, this work provides a simple method to boost the osteogenic activity and osseointegration ability of PEEK, which has potential clinical applications in orthopedic and dental implants.Polyetheretherketone (PEEK) is a promising alternative for biomedical metallic implants in orthopedic and dental applications because its elastic modulus is similar to that of bone. However, PEEK is a bioinert material that cannot be integrated with host bone. Our previous study showed surface phosphonation enhanced the osteogenic activity of PEEK. The purpose of this study was to evaluate the effect of the density of phosphonate groups on the bioactivity of PEEK. X-ray photoelectron spectroscopy and water contact angle measurement confirmed the successful grafting of different densities of phosphonate groups to the PEEK surface using a one-step ultraviolet-initiated graft polymerization method. Atomic force microscopy revealed that the surface treatment did not significantly alter the surface topography and roughness. Polyetheretherketone (PEEK) is a promising alternative for biomedical metallic implants in orthopedic and dental applications because its elastic modulus is similar to that of bone. The modulus can be tailored to closely match that of cortical bone by preparing carbon-fiber reinforced composites, and thus the extent of stress shielding can be reduced or thoroughly eliminated. Additionally, its radiolucency facilitates the monitoring of bony fusion during the postoperative follow-up period.3 However, PEEK presents inferior osteogenic activity and bone-implant integration capability because of its bioinertness,4,5 and thus its clinical applications as orthopedic and dental implants are limited.Polyetheretherketone (PEEK) is a promising alternative to biomedical metal for orthopedic and dental implants due to its outstanding chemical resistance, satisfactory mechanical properties, and excellent sterilization resistance.6\u20139 However, the poor physical bonding between PEEK and ceramics results in a trade-off between mechanical properties and bioactivity. Alternatively, various techniques have been developed to deposit bioactive ceramics coatings on PEEK surfaces, such as microwave assisted coating,10 thermal plasma spray,11 spin coating,12 cold spray,13 ion beam assisted deposition,14 electron beam evaporation,15 and biomimetic.16,17 However, the ceramics coatings are apt to delaminate due to the weak bonding strength, and may induce severe inflammation and bone resorption.18 Additionally, layer-by-layer self-assembly and mussel-inspired surface chemistry strategies have been employed to prepare multi-functional PEEK surface with enhanced bacteriostasis and anti-inflammatory and osseointegrative properties.19,20 Surface chemistry is a pivotal factor in regard to cell and tissue response.21,22 As such, the tailoring of surface chemicals such as in the case of covalent grafting of functional groups or biomolecules without the hindrance of the detachment is a promising alternative to coating techniques. Our groups and others have reported that implants with incorporated functional groups such as carboxyl,23,24 sulfonic groups,25 amino26 and phosphonate groups,27,28 or biomolecules such as RGD peptides29 and antimicrobial peptides30 exhibit up-regulated bone cells osteogenic activity in vitro and osseointegration in vivo.To address this deficiency, numerous research groups have exploited various strategies to tailor PEEK to enhance its osseointegration activity. One such strategy is to incorporate bioactive ceramics component including hydroxyapatite, calcium silicate and bioactive glass into the PEEK matrix to prepare a composite.31 Recently, it has been shown that the diphenylketone moiety in the PEEK backbone acts as a photo-initiator, similar to that of benzophenone.32 When the PEEK surface is subjected to ultraviolet (UV) irradiation, semibenzopinacol-containing radicals of benzophenone units in the PEEK molecular structure can be self-induced, which initiates a polymerization reaction with functional monomers. As such, the PEEK surface can be modified with functional groups under UV-irradiation via a one-step and simple self-initiated graft polymerization method. The grafting of poly(acrylic acid), poly(2-(methacryloyloxy)ethyltrimethylammonium chloride) and poly on PEEK surfaces has been achieved via photo-induced graft polymerization and the modified surfaces exhibited improved bio-tribological properties and aqueous lubrication.33,34 Our group has also used photo-induced graft polymerization to incorporate sulfonic groups and phosphonate groups on the surface of PEEK, which elevated the proliferation and osteogenic differentiation of osteoblast in vitro, and the bone-implant contact ratio in vivo.25,28In comparison to other surface grafting approaches such as plasma- or radiation-induced grafting, photo-induced grafting has several advantages such as low cost, simple equipment, mild reaction conditions, easy of industrialization, weak penetrability, and long-term stability of the grafted chains without affecting the bulk materials.35,36 Whereas, it is difficult to acquire the optimal content of phosphonate group because these investigations used copolymer containing other functional groups. In this investigation, different densities of phosphonate group were incorporated on a PEEK surface via a one-step UV-initiated graft polymerization method to screen out optimal surface-phosphorylated PEEK implants. The effects of surface phosphonation on the osteogenic activity of MC3T3-E1 osteoblast, including proliferation, mineralization, and osteogenic differentiation, were then examined. Moreover, in vivo osseointegration ability, including bone-implant contact ratio and bonding strength, was also assessed after implantation of the surface-phosphorylated PEEK into a rat's femur.Previous studies have indicated that phosphonate groups enhance adhesion and differentiation of osteoblasts in a dose-dependent manner.2.2.1\u03a615 mm \u00d7 1 mm) for surface characterization and in vitro studies, or a plate format (6 mm \u00d7 2.8 mm \u00d7 1 mm) for in vivo animal experiments. Prior to use, all the samples were washed sequentially with 2-propanol, acetone, ethanol and ultrapure water in an ultrasonic cleaner, and finally dried under vacuum overnight. Vinylphosphonic acid (VPA) was provided by TCI (Shanghai) Development Co., Ltd. \u03b1-MEM, fetal bovine serum, and penicillin/streptomycin were purchased from Hyclone. Cell Counting Kit-8 (CCK-8) was provided by Dojindo . BCA kit and ALP assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. Alizarin Red, cetylpyridinium chloride, and Sirius Red were obtained from Sigma. TRIZOL reagent was from Invitrogen Life Technologies. PrimeScript RT reagent kit with gDNA Eraser and TB Green premix EX Taq II PCR kit were obtained from Takara .Polyetheretherketone (PEEK) sheets were provided by Jiangsu Junhua High Performance Specialty Engineering Plastics (PEEK) Products Co., Ltd . The PEEK substrate was machined either into a disc format with a maximum intensity at approximately 365 nm was used to induce graft polymerization. The graft polymerization was performed for 20 min, 50 min and 90 min, and the corresponding poly(vinylphosphonic acid) (PVPA) grafted PEEK was abbreviated as PEPA20, PEPA50, and PEPA90, respectively. After the reaction, the PEPA samples were ultrasonically cleaned twice in ultrapure water for 20 min, to remove non-reacted monomers and non-grafted homopolymers on each occasion, and then subsequently dried at room temperature.Surface-phosphorylated PEEK was prepared as previously described.2.3The surface chemical composition of the substrates was investigated through X-ray photoelectron spectroscopy (XPS). XPS data were acquired using a Kratos Axis Ultra system with a monochromatized Al K\u03b1 source at a take off angle of 20\u00b0. The sample charging was calibrated to the reference of C1s set at 284.8 eV. Spectra data was analyzed using the analysis software XPS PEAK95 Version 3.1.via static water contact angle measurements using the sessile drop method with a goniometer . A volume of 3 \u03bcl ultra-pure water was placed on the surface of the samples using a motor driven syringe at room temperature. The mean value was calculated using four measurements.Surface wettability of the intact PEEK and PEPA was determined The surface topography of PEEK samples was detected by atomic force microscopy in the tapping mode at room temperature. An area of 20 \u03bcm \u00d7 20 \u03bcm was imaged, and the roughness was calculated using the software NanoScope Analysis.2.42.4.12 at 37 \u00b0C. The medium was refreshed every three days. All the samples were sterilized using epoxy ethane before cell seeding.Mouse MC3T3-E1 osteoblasts were cultured in \u03b1-MEM with 10% fetal bovine serum, 1% penicillin/streptomycin in a humidified atmosphere of 5% CO2.4.24/well. After incubating for 4 h and 24 h, the medium was removed, and the samples were washed twice with sterile PBS and fixed with 2.5% glutaraldehyde overnight. Then, the fixed osteoblasts were progressively dehydrated with graded ethanol solutions for 15 min each. The samples were dried and sputter-coated with gold prior to SEM observation of the osteoblast adhesion morphology.The MC3T3-E1 cells were seeded onto the samples at a density of 2.5 \u00d7 102.4.324 Briefly, the cells were seeded on each sample with a density of 1.0 \u00d7 105/well for 4 h. At a scheduled time, the specimens were rinsed three times with PBS and then incubated in 300 \u03bcl \u03b1-MEM with a supplement of 30 \u03bcl CCK-8 solution for 1.5 h. The solution was carefully transferred to a 96-well plate to measure the optical density using a microplate reader at 450 nm.The CCK-8 assay was used to determine the ability of the cultured osteoblast to adhere to the samples, as described elsewhere.2.4.44/well and cultured for 1, 4 and 7 days. The other detailed procedures were the same as those of the cell attachment test.The cell proliferation was also evaluated using the CCK-8 assay. The cells were seeded on each sample with a density of 1.0 \u00d7 102.4.54 cells per well for 7 and 14 days. At a prescribed time, the samples were washed thrice with PBS and cells were lysed using 1% Triton X-100 for 40 min at 4 \u00b0C. After centrifugation at 14\u2009000 rpm for 10 min, the supernatant was extracted for total protein and ALP activity assay using a BCA kit and ALP assay kit following the manufacturer's respective protocols. The ALP activity of the test samples was normalized to the total protein content.Cells were seeded on different samples placed in 24-well plates with a density of 1 \u00d7 102.4.6After MC3T3-E1 cells were cultured on the samples for 7 and 14 days, the samples were washed thrice with PBS and fixed with 75% ethanol for 1 h. They were then stained with 40 mM Alizarin Red water solution (pH = 4.2) for 10 min at room temperature. Afterward, the samples were washed with distilled water until the orange color was no longer observed. For quantitative analysis, the adsorbed Alizarin Red was dissolved with 10% cetylpyridinium chloride in 10 mM sodium phosphate (pH = 7.0) and the absorbance was measured using a microplate reader at a wavelength of 620 nm.2.4.74/well for 7 and 14 days. At a prescribed time, the samples were washed thrice with PBS and fixed in 4% paraformaldehyde. Following the three rinses in PBS, the samples were stained with a 0.1% solution of Sirius Red in saturated picric acid for 18 h. They were then washed with 0.1 M acetic acid until the red color was not observed. For quantitative analysis, the stain on the samples was dissolved using 500 \u03bcl eluent (0.2 M NaOH/methanol = 1\u2009:\u20091). The absorbance at 540 nm was then measured on a microplate reader.Collagen secretion of the MC3T3-E1 osteoblast on the specimens was quantified using Sirius Red staining. The cells were seeded on the samples with a density of 1.0 \u00d7 102.4.8The expression of osteogenesis-related genes, including alkaline phosphatase (ALP), osteocalcin (OCN), collagen type I (COL-I), and osteopontin (OPN) were quantitatively analyzed by RT-PCR using \u03b2-actin as the housekeeping gene for normalization. The sequences of the forward and reverse primers are listed in 2.52.5.1in vivo osseointegration study. Prior to surgery, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital solution (50 mg kg\u22121), and the medial side of the knees of both legs was shaved and depilated. Afterwards, a longitudinal incision of approximately 15 mm long was made down to the periosteum. A slit-like incision (2.8 mm \u00d7 1 mm) was prepared at the left and right distal tibiae parallel to the long axis of this bone using a dental drill. Subsequently, the implants were inserted into the slit in a press-fit manner. Finally, the wound was carefully closed layer-by-layer. After the operation, the rats received a subcutaneous injection of oxytetracycline (30 mg kg\u22121) for 3 days. They were euthanized 12 weeks postsurgery, and the femur bones containing the PEEK implants were retrieved and fixed in 10% formaldehyde.All animal procedures and experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of China. Male Sprague-Dawley rats (200 \u00b1 20 g) were used with the approval of the Institutional Animal Care and Treatment Committee of North Sichuan Medical College. A rat femur distal model was used for the 2.5.2via evaluation of bone-implant contact (BIC) ratios based on the acquired images using the Image-Pro Plus 6.0 software.The samples were dehydrated using increasing concentrations of ethanol, and embedded in polymethylmethacrylate resin. The embedded specimens were cut into 50 \u03bcm thick sections perpendicular to the femur axis using a Leica SP1600 saw microtome . The sectioned samples were stained using toluidine blue-fuchsine, and the integration of the bone-implant was observed using optical microscopy . Histomorphometric analysis was performed 2.5.3\u22121. The pull-out load was calculated by averaging the results from five pull-out tests.To investigate the bone-implant interface bonding, pull-out tests were performed on a biomechanical apparatus under a displacement speed of 1 mm min2.6in vitro evaluation, and paired t-test for in vivo evaluation was used to determine the statistical significance of observed differences using SPSS 16.0 statistical software. p < 0.05 was considered statistically significant.All experiments were performed at least in triplicate. One-way analysis of variance (ANOVA), Tukey's multiple comparison tests for 3.3.13/2 and P2p1/2 that originated from high oxidation state phosphorus, i.e. phosphate groups.37 Thus, the XPS results indicate that phosphonate groups were successfully introduced on the PEEK surface. The atomic percent of P and P2p/C1s ratio were quantified using the XPS survey scans and the results are depicted in To confirm the successful grafting polymerization of VPA on the PEEK substrate, PEEK, PEPA20, PEPA50, and PEPA90 samples were analyzed using XPS. via XPS, the static water contact angles of all the PEEK samples were measured and the results are summarized in After verification of the successful chemical modifications via AFM. AFM images of all PEEK substrates and the corresponding Ra roughness are shown in Ra roughness is in the range of 17 to 20 nm. This indicates that surface graft polymerization does not alter the surface topography and roughness.The surface morphologies of PEEK, PEPA20, PEPA50, and PEPA90 substrates were examined 3.23.2.1p < 0.01), indicating that surface phosphonation could enhance the attachment of MC3T3-E1 cells. Additionally, the adherent cell numbers on PEPA50 were higher than those of PEPA20 and PEPA90. However, cells adherent on PEPA50 exhibited no statistical difference compared to those of cells adherent on PEPA20 and PEPA90.p < 0.05) than that on PEEK, whereas this was not the case for PEPA20 and PEPA90. When the culturing time was extended to 4 and 7 days, the number of MC3T3-E1 cells on the surface-phosphorylated PEEK substrates was significantly higher than that on PEEK (p < 0.001). This indicates that surface phosphonation is more beneficial to MC3T3-E1 cells proliferation and causes no cytotoxic effects on cells. Additionally, the proliferation activity of MC3T3-E1 cells grown on PEPA50 was significantly higher (p < 0.05) than that of the cells grown on PEPA20 and PEPA90.The time-related proliferation of MC3T3-E1 cells cultured on all PEEK substrates surfaces was measured using CCK-8 assay as shown in 3.2.2The morphology of the MC3T3-E1 cells grown on all the PEEK substrates surfaces at different time points was observed by SEM and the images are presented in 3.2.3p < 0.05) than that of the unmodified PEEK. This indicates that surface phosphonation can improve the initial osteogenic differentiation of MC3T3-E1 osteoblasts grown on PEEK. Additionally, MC3T3-E1 osteoblasts cultured on PEPA50 displayed a relatively higher ALP activity compared to those of osteoblasts cultured on PEPA20 and PEPA90, although the difference between them was not significant.To evaluate the effect of surface phosphonation on the osteogenic differentiation of MC3T3-E1 osteoblasts, the ALP activity of the cells on all the PEEK substrate surfaces was measured after culturing for 7 and 14 days. As shown in p < 0.05) compared to those grown on the PEEK control. MC3T3-E1 osteoblasts cultured on PEPA20 and PEPA90 exhibited comparable ECM mineralization levels during the observation period. Moreover, ECM mineralization of MC3T3-E1 osteoblasts seeded on PEPA50 was higher compared to those on the PEPA20 and PEPA90 samples. Moreover, ECM mineralization of MC3T3-E1 osteoblasts seeded on PEPA50 was significantly higher (p < 0.05) compared to that on PEPA20.The quantitative results for the ECM mineralization of MC3T3-E1 osteoblasts cultured on all PEEK substrates surfaces for 7 and 14 days are depicted in p < 0.05) compared to that on the PEEK control. In addition, MC3T3-E1 osteoblasts grown on the surface-phosphorylated PEEK exhibited a comparable collagen secretion level throughout the observation period.The quantitative results for collagen secretion of MC3T3-E1 osteoblasts cultured on all the PEEK substrates are displayed in 3.2.4p < 0.05) than those grown on the PEPA20 at day 14. In addition, MC3T3-E1 cells cultured on PEPA50 exhibited obviously higher expression levels of OCN compared to those grown on PEPA90 at day 7 and 14.3.3in vivo implantation studies. To compare osseointegration capability, PEEK and PEPA50 were implanted into rat femurs, and the bone-implant contact ratio (p < 0.05) bone-implant contact ratio than PEEK. In accordance with this result, the bone-implant bonding strength was significantly higher (p < 0.05) in PEPA50 compared to that in PEEK. These results demonstrate that surface phosphonation improves the osseointegration activity of PEEK.The aforementioned results demonstrate that surface phosphonation affects osteoblast activity in a dose-dependent manner, and thus PEPA50 with an optimum phosphonate group concentration was used for the ct ratio and bonect ratio were det4.via chemical reactions. Nevertheless, many scholars have pursued some exploratory work using wet chemistry.16,23,29,37,38 A modified PEEK surface via wet chemistry has been reported to improve the bioactivity of PEEK with respect to the enhancement of the bone-implant interface. However, there are several disadvantages such as multi-step procedures and time-consuming reactions. Fortunately, the benzophenone groups in the PEEK backbone generates active radical on its surface under UV-irradiation. And the radicals directly initiate functional monomer polymerization and surface-functionalized PEEK can be obtained in a relatively short time period. In this case, PEEK surfaces with different content of phosphonate groups were prepared via UV-initiated graft polymerization to identify optimal surface-phosphorylated PEEK implants based on previous reports where it was shown that phosphate modification ameliorated the bioactivity and osseointegration capability of PEEK implants both in vitro and in vivo.23,28,37Although the resonance stabilized chemical structure of PEEK confers its outstanding chemical resistance and biocompatibility, the structure also makes it extremely difficult to functionalize PEEK 39\u201341 In this work, XPS confirmed that different amounts of phosphonate groups are incorporated onto the PEEK surface via one-step UV-initiated graft polymerization fumarate hydrogel containing 620 \u03bcmol bis(2-(methacryloyloxy)ethyl)phosphate showed the optimal human fetal osteoblast proliferation and ALP activity.35 Gemeinhart et al. created a phosphonate-containing copolymer-modified surface and observed that surface modified with 30% vinyl phosphonic acid in the feed exhibited a maximal cell adhesion, proliferation, and calcification.36 Some explanations can be found in the literature to explain why surface phosphonation influences osteoblast activity. Phosphonate groups are negatively charged under physiological conditions, and thus are capable of chelating calcium ions from media, which is a propitious feature for transducing osteogenic cues.42,43 In fact, polymers and metal bearing phosphonate groups initially attract calcium ions and then induce bone-like apatite deposition on their surface.16,44,45 Then the polymers and metal directly bond to living bone via the bone-like apatite layer. Recently, accumulating evidence suggests that bone regeneration is strongly influenced by the cross-talk between bone-forming cells and immune cells, particularly macrophages.46,47 An efficient and timely switch from pro-inflammatory M1 to anti-inflammatory M2 macrophage phenotype results in an osteogenic cytokine release and the corresponding formation of new bone tissue.46 In addition, the physicochemical properties of the materials play prominent roles in macrophage plasticity. A recent study demonstrated that more macrophages might be polarized toward an M2 macrophage phenotype on surface-phosphorylated PEEK than pristine PEEK.27 Additionally, it has been reported that the surface chemistry was able to influence the type, quantity and activity of adsorbed matrix proteins, including vitronectin, collagen I, fibronectin and laminin.48 In addition, osteoblast adhesion, spreading, proliferation and differentiation have been reported to depend on these proteins. A study by Tan et al., demonstrated that protein adsorption peaked in a dose-dependent manner during the investigation of protein adsorption on substrates containing phosphonate groups.49 As the concentration of the phosphonate group on the substrate surface exceeds a specific threshold, there is greater repulsion between the substrate and the negatively charged proteins, which consequently leads to a decrease in protein adsorption and a decrease in cell response.50 Therefore, MC3T3-E1 osteoblasts on the PEPA50 surface exhibit optimal overall responses in terms of adhesion, spreading, proliferation and osteogenic differentiation. These reasons partially account for the excellent osteogenic activity of MC3T3-E1 osteoblasts observed on the surface-phosphorylated PEEK, however, the underlying mechanism remains to be further elucidated.To evaluate the influence of surface phosphonation on the osteointegration of PEEK, both honation and 6 an control and 8. TFinally, we compared the osseointegration capabilities of PEEK and PEPA50 implants in terms of the bone-implant contact ratio and bonding strength using the rat femur implantation model. In this study, we determined that the surface-phosphorylated PEEK exhibited significantly higher bone-implant contact ratios and bone5.via a one-step UV-initiated graft polymerization of vinyl phosphonic acid. The in vitro osteoblast response results indicate that phosphonate groups are competent to enhance cell adhesion, cell spreading, cell proliferation, alkaline phosphatase activity, extracellular matrix mineralization, collagen secretion, and osteogenesis-related genes expression of MC3T3-E1 osteoblast in a dose-dependent manner. And the surface-phosphorylated PEEK generated after 50 min of UV induced grafting shows the optimal osteoblasts responses. Moreover, the in vivo results show that the surface-phosphorylated PEEK is more beneficial for bone tissue growth and osseointegration than intact PEEK surfaces. It was determined that the surface-phosphorylated PEEK provides a more favorable surface for bone regeneration compared to that of the bare PEEK surface, which boosts the potential for future clinical applications as orthopedic and dental implants.PEEK surface bearing various phosphonate groups content was fabricated There are no conflicts to declare."} +{"text": "In the domain of dentistry, PEEK materials have been utilised as removable partial denture frameworks, implant substances, and fixed dental prostheses. Even though PEEK polymer has a low modulus of elasticity, its mechanical attributes could be fine-tuned by integrating various inorganic filler materials. The study intends to characterise and assess Nano SiO2 and PEEK composite made using the melt blend technique and studies their characteristics with reference to pure PEEK. The following assessments are conducted: SEM and EDX assessment with AFM investigation in addition to tensile strength test, transverse strength test, and wettability test. All data were scrutinised by SPSS software version 24 and the statistical analyses included the mean, standard deviation, and the independent sample t-test. The design of this study uses 3% SiO2/PEEK compared with pure PEEK. The outcomes indicate that there is a statistically highly significant increase in the mean value of transverse strength was seen with the PEEK/SiO2 composite (3503.02\u2009MPa) versus PEEK (2694.61\u2009MPa), while there is a statistically significant decrease in the mean value of the tensile strength for PEEK/SiO2 (63.69\u2009MPa) versus PEEK (97.62\u2009MPa). Moreover, improvement in hydrophobic characteristics and surface roughness of PEEK/SiO2 (81.78\u00b0), (0.66\u2009nm) versus PEEK (71.01\u00b0), (1.23\u2009nm), respectively, thus giving more chance to composite to be investigated in human bone/implant substitution. Furthermore, the results of EDX and SEM images exhibited adequate distribution of Nano SiO2 within the PEEK matrix. There was also a statistically substantial decrease in the surface roughness and tensile strength obtained from the AFM investigation. The outcomes of this investigation pertain to the differences in characteristics of a composite of SiO2 to prepare a composite of SiO2/PEEK by using the melt blend approach. Nano SiO2 can alter the SiO2/PEEK composite's transverse strength and reduce the hydrophobic characteristics of the surfaces with proper distribution of nanoparticles within the matrix of PEEK with less surface roughness. As far as this study is concerned, a conclusion can be made that we can use 3% Nano SiO The PEEK (poly ether-ether-ketone) is derived from poly aryl-ether-ketones. PEEK is identified chemically as a linear configuration of poly aryl-ether-ketone. PEEK is a high-performance, semicrystalline thermoplastic polymeric substance and it is a melt processable aromatic polymer; the Tm (melting point) lies between 330\u00b0C and 385\u00b0C, which depends on the relative number of ether-ketone groups connecting the phenylene rings. The extent of crystallinity is dependent on the thermal histories as well as the processing conditions, like annealing treatments and cooling rates. PEEK has attracted interest as a medical transplant material since it has excellent biocompatibility, dimensional stability, resistance to chemicals, and mechanical attributes and displays a low modulus like that of human bone .In the dentistry domain, PEEK materials have been examined for use as fixed dental prostheses or detachable partial denture frameworks. Even though PEEK is a polymer with low modulus, its mechanical qualities can be regulated by including inorganic fillers and modifying the filler content. Furthermore, bioactive materials have been recently introduced to combine the strength of composites and the benefits of their bioactivity. Moreover, for commercial uses, the primary aim of including inorganic fillers into polymers is to reduce cost and improve stiffness \u20134.3). In the dentistry field, the implants must have greater elastic modulus, which is provided by PEEK material, specifically for abutments and superstructures [Cortical bone, titanium (Ti), and ceramics have greater elastic modulus compared to PEEK material. PEEK exhibits elastic modulus (3\u20134\u2009GPa), high stability, and low density (1.32\u2009g/cmructures .A number of strengthened composites have been formed using PEEK: CFR (carbon-fibre reinforced) PEEK and GFR (glass-fibre reinforced) PEEK. The value of the elastic modulus of both these materials can reach up to 18\u2009GPa. Osseointegration targeted studies compare PEEK and conventional implant candidates, such as titanium and zirconium. Research studies report insignificant differences , 7.3 density [PEEK is classified as a poly-aryl-ether-ketone (PAEK) polymer. It is used as a substitute of metal implants for orthopaedic and trauma-related uses. It is an aromatic compound containing ketone (-CO-) and ether (-O-) groups in the centre of the aryl rings. It is a white radiolucent material that stays thermally stable up to temperatures of 335.8\u00b0C. It does not affect oral mucosa and demonstrates a low plaque affinity. Its attributes include heat conductivity of 0.29\u2009W/mK, 1140\u2013170\u2009MPa flexural modulus, and 300\u2009kg/mCook et al. fortified the PEEK material implant with titanium and carbon fibre and then placed it into femurs. After the evaluation of 8 weeks, similar contact ratios for bone-implant were reported. [Nanocomposites of this polymer have attracted substantial interest and attention globally during the previous decade. Among the various fabrication techniques for polymer Nanocomposites building, the sol-gel technique seems to be the most beneficial one.Initially, the Nanoparticles were dispersed and further combined at molecular or near-molecular level with the polymer gel .Two primary approaches have been used to address the issue of PEEK's inert nature. One method is a surface adjustment to activate PEEK using only the surface treatment or treatment combined with a coating on the surface. Another approach is to prepare bioactive composites of PEEK by introducing bioactive substances into PEEK material. Researchers believe that altered bioactive PEEK material will have a broad range of orthopaedic uses .2 particles improves the heterogeneous crystallisation of the PEEK matrix. The substantial improvement in thermal and mechanical properties of the PEEK composites indicates that these composites can prove to be useful for uses in electronic packaging using Nanosubstrates. Most PEEK materials used in dentistry are reinforced by inorganic filler material to enhance their rigidity [The addition of SiOrigidity , 12.2 in the PEEK material affected the bonding between resin cement and PEEK. The study assessed TBS (tensile bond strength) prior to and after 10,000 thermal cycles besides flexural strength, flexural modulus, and crystallinity. Furthermore, the tensile bond strength was increased and the greatest flexural strength was obtained when SiO2 content was increased in PEEK material at 40 wt. % SiO2 [In 2019, Rikitoku et al. carried out research to evaluate how the different levels of SiO. % SiO2 .2O3 and SiO2 particulates indicate an enhancement in the rigidity, tensile strength, and transverse strength. The addition of the inorganic filler material into the PEEK matrix can enhance the thermal stability of the resultant Nanocomposites [Further research using composites of PEEK material reinforced by Nanosized Almposites .2. The null hypothesis entails that the addition of Nano SiO2 will not affect PEEK properties.The aim of this study is to modify and improve the physical and mechanical properties of PEEK by an additional 3% wt. Nano SiO2) in pulverised form, coated with 3\u20134% KH570 Silane coupling agent. Nanomaterials particle size 20\u201330\u2009nm Silane coupling agent KH570 is gamma Methacryloxy propyl trimethoxy silane (CAS No: 2530-85-0). Houston Inc., USA. This Nano will be added to PEEK powder (China type KLC607) of microsized with an average size of 68.35\u2009\u03bcm. Based on the research strategy, a mould was formed, as displayed in This research used Nanosilicon dioxide powder is added to alcohol and dissolved using a probe sonicator with 80\u2009pw and frequency of 000.5\u2009s for 15 minutes with constant cooling. Then, powdered PEEK (97% wt. 67.9\u2009gm) is gradually added to alcohol which contains SiO2. This concoction is then blended by a rotatory mechanical stirrer for 15 minutes at room temperature [2/PEEK blend was dehydrated in a hot air oven for 2 hours at 150\u00b0C as per Kurtz and Devine, 2007 [At the beginning of this stage, Nano SiOperature . Further2/PEEK composites were prepared. Usually, an extruder instrument is used to melt the polymer and mix SiO2. The chamber is filled gradually with powder. The blended powder of SiO2/PEEK is gradually filled into the chamber until it melts and blends completely, which further gets converted into a brown-coloured homogenous blend at 320\u00b0C with 125\u2009rpm. At this point, the rpm is decreased to 70\u2009rpm and run for 5 minutes for uniform blending of the mixture.A thermal compression and melt blend technique was an appropriate method using which Nano SiOThen, the dough of the PEEK material is collected using sharp knives and placed in a metal container. An electronic balance is used to measure the weight of the collected PEEK material dough before loading it into the moulding chamber.A heat press was used for the heat compression process. The heat component thermostat was set at 380\u00b0C. When the heat reached 200\u00b0C, the moulded polymer was introduced into the heat press chamber so that the heat was absorbed gradually. When the target temperature of 380\u00b0C was achieved, the pressure that started with 5\u2009MPa increased gradually every 10 minutes until it reached 30\u2009MPa finally. The mould was kept in the hot press chamber for 5 minutes. Afterward, the mould was taken out from the press chamber and kept in a cooling equipment at 30\u2009MPa pressure and kept there until its temperature dropped to 200\u00b0C. Then, the pressure was released and the mould was put on a bench at 25\u00b0C\u00b0\u00b1\u00b02 room temperature to achieve gradual cooling. After cooling, reconstruction of the mould and extraction of the sample from the frame was carried out and prepared for pretest calibrations.S\u2009=\u20093PL/2bd2S\u2009=\u2009stress MPaP\u2009=\u2009load at a given point on the load-deflection curve, NL\u2009=\u2009support span, mmb\u2009=\u2009width of beam tested, mmd\u2009=\u2009depth of beam tested, mmThe samples were constructed from a sheet with a length of 65\u2009mm, a width of 10\u2009mm, and a thickness of 3\u2009mm). The test was carried out as per ASTM D790-03 by using a universal testing machine Instron with a velocity of (2\u2009mm/min) at room temperature. The strength was computed as per the following formula.The samples were formed and cut as per ASTM D 638-Type3 (Standard 2011). The tensile test was carried out using a universal testing machine Instron 5567 tester at room temperature, with a gauge length of 25\u2009mm and a cross-head speed of 5\u2009mm/min.An accelerating voltage of 10\u201320\u2009kV of SEM type is employed for microstructural analysis, while the energy dispersive X-ray spectroscopic analysis (EDX) is employed for determining the elemental composition of the materials. The main principle applied in spectroscopy is that each element possesses a unique atomic structure that can present a unique set of peaks with regards to its electromagnetic emission spectrum. EDX analysis relies on the interaction between a specimen for the elemental analysis and some source of X-ray excitation .FTIR is employed for determining polymeric materials and it gives information based on the entire sample's physical state and chemical composition .The AFM depends on the scanning technique and offers a high-resolution 3D image from the sample's surface. It is usually employed for identifying the surface morphology, topography, roughness, and distribution of particle size. A sharp tip that is present towards the end of the cantilever is employed in contact with the surface as well as the sample that has been displaced with piezoelectric scanners. The force present on the tip results in generating deflection that can be measured with the tunnelling capacitive or optical detectors. Zero standard pressure is used for the joint (to avert the chances of surface deformation).A goniometer along with a charge-coupled-device camera and an image capture program employing Lab VIEW software are utilised to record contact angles.t-test was used for comparison.SPSS version 24 software was utilised in the statistical analysis of the data. Means and standard deviations were obtained for each test and unpaired 2 Nanoparticles tend to have optimum characteristics that can be used in 3% with PEEK through the hot press method. This study aims to characterise PEEK as well as SiO2/PEEK composites, which can be prepared through melt blending procedures and hot press.In this study, fillers are added to improve the PEEK characteristics. SiO2/PEEK composite showed changes in the tensile strength and transverse strength versus PEEK alone. As per 2 composite versus PEEK alone. With regards to the tensile strength, a statistically significant decrease in mean value was observed.The SiO2 composite, respectively. These figures clearly show the elemental composition pertaining to the PEEK and SiO2/PEEK composite.Figures 2 was noted in nanometres within the matrix based on the SEM image at MAG 35.00\u2009kx pertaining to PEEK/composite and PEEK. As represented in 2 particle within the PEEK matrix which is related to thorough and even mixing to the Nano by sonication then mechanical mixing for both Nano and powder then end with melt blending procedure.As presented in 2 Nanoparticles is confirmed by the FTIR transmittance spectrum (400 to 4000\u2009cm\u22121). To determine the presence of the O-H group, a broad peak in the range of 3000\u22123700\u2009cm\u22121 was assigned. Similarly, at 1649\u2009cm\u22121, a peak was seen that corresponded to vibration bending, which signifies the presence of the O-H stretching bond. Furthermore, the strong bands seen at 1093, 459, and 798\u2009cm\u22121 were related to Si-O-Si stretching vibration bonding. Si\u2010OH (960\u2009cm\u22121 and 1630\u2009cm\u22121) or Si-O-Si were also detected.As per the FTIR analysis result in 2 composite and PEEK only showed marked differences. Between the two studied groups, there was a difference in the distribution and size of the spike.As per the results of the AFM analysis seen in 2/PEEK composite versus PEEK alone.2 in Nanoparticles via the hot press method. The study aims to characterise PEEK as well as SiO2/PEEK composite prepared via melt blending and hot press methods.This study aims to enhance PEEK's properties with the addition of fillers. An optimum property was seen for PEEK with the addition of 3% SiOThe melt blending method is generally opted for synthesising Nanocomposites of a polymeric matrix. Usually, the polymer is melted first that let it to blend well with the required amount of fillers.2/PEEK composite was carried out by complying with ISO178:2010. [2 was seen to clearly impact the transverse strength pertaining to PEEK. The addition of 3% SiO2 to PEEK is regarded to be optimum for fortifying PEEK and giving a clear enhancement in transverse strength versus PEEK alone which comes in line with Rikitoku et al. [In this study, testing of the flexural strength pertaining to PEEK and SiO78:2010. As per tu et al. .2 which facilitates the creation of a large amount of interphase to offer a broad interfacial interaction as well as enhance the mechanical strength. The SEM images showed this and indicated the presence of SiO2 particles that are evenly distributed while maintaining minimal particle agglomerations.This was possible due to the large surface area pertaining to Nano size SiO2 resulted in a decrease in tensile strength mean value, which is in line with the study of Wang et al. (2011), wherein the tensile strength relied on factors, such as the added concentration of Nanofillers to PEEK, the particles' dimension, and the way in which stress is transferred from the filler to the matrix [With regards to the tensile strength, the addition of Nano SiOe matrix .This aspect can be related to the specific Nanomorphology particles that, beyond a concentration percentage in this study seems to be near to 4%, led to a dramatic toughness reduction and mechanical performance decline .2/PEEK Nanocomposites. With regards to SiO2/PEEK Nanocomposites added with 3% of hydrophobic Nano SiO2, there was an increase in the contact angle value versus PEEK alone. The surface roughness of the pure PEEK matrix could decrease when Nano SiO2 particles are added. This could be because of Nano SiO2 that are present on the surface of the Nanocomposites and are evenly dispersed within the PEEK matrix, as well as their small Nanoparticle size from 20\u201330\u2009nm [With regards to the Contact Angle Measurement, a variation was seen in the contact angle measurements pertaining to PEEK and Nano SiO20\u201330\u2009nm .The small size of filler, particularly Nanosize, allows adhering to the resin matrix, thus offering a smoother surface finish, this result comes in agreement with Senawongse and Pongprueksa .2 composite could be studied.The limitation of this study was the limited tests used so more tests are required like compression and fatigue tests and the addition of another bioactive Nano to the PEEK/SiO2 can be employed for synthesising SiO2/PEEK composite via the melt blend technique. Nano SiO2 can enhance the SiO2/PEEK composite's transverse strength while decreasing the hydrophobic characteristics pertaining to the surfaces with fair dispersion of nanoparticles within the matrix of PEEK.By keeping the study limitation in mind, it can be said that 3% Nano SiO"} +{"text": "Polyetheretherketone (PEEK) has been widely applied in fixed dental prostheses, comprising crowns, fixed partial dentures, and post-and-core. PEEK\u2019s excellent mechanical properties facilitate better stress distribution than conventional materials, protecting the abutment teeth. However, the stiffness of PEEK is not sufficient, which can be improved via fiber reinforcement. PEEK is biocompatible. It is nonmutagenic, noncytotoxic, and nonallergenic. However, the chemical stability of PEEK is a double-edged sword. On the one hand, PEEK is nondegradable and intraoral corrosion is minimized. On the other hand, the inert surface makes adhesive bonding difficult. Numerous strategies for improving the adhesive properties of PEEK have been explored, including acid etching, plasma treatment, airborne particle abrasion, laser treatment, and adhesive systems. Polyetheretherketone (PEEK), a member of the polyaryletherketone (PAEK) family, has recently emerged as a promising alternative material for fixed dental prostheses. PEEK is a high impact polymer material that is made from fluorine benzene ketone, hydroquinone, and sodium carbonate or potassium carbonate, dissolved in diphenyl sulfone. PEEK consists of an aromatic nucleus linked by ketone and ether groups, providing it with a superior chemical stability that seems to be a double-edged sword ,2. Regar3/MC) than metal and its alloys (1.6 \u00b1 2.0 mm3/MC), which should lead to significant improvement for crown restorations [3), which can result in favorable stress distribution for lightweight framework restorations [PEEK exhibits excellent mechanical properties, including a low Young\u2019s modulus (3 to 4 GPa), which, compared to metal alloys 110 to 130 GPa), is closer to that of human cortical bone 14 GPa) . Given i0 GPa, is GPa . Giorations . Additioorations .PEEK exhibits superior biocompatibility . It is nPEEK is a metal-free material with a gray color. It provides aesthetic improvement for implantology when compared with metal and its alloys , whereasThe success of a fixed dental restoration depends on three key factors: biomechanical behavior (wear resistance and fracture resistance), marginal fit, and aesthetics, generating extremely strict demands for the restoration material. Zirconia has become a popular alternative to metal in fixed dental prostheses, known for its excellent aesthetics ,33,34. MThis study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Medical Ethics Committee of the Hospital of Stomatology of China Medical University . Patients provided written informed consent to publish case details and any images.2) crowns demonstrated the pivotal role of crown geometry in crown preservation [2 after thermal loading [Regular mastication and progressive erosion result in unavoidable wear of the crown, and the crown material must possess considerable wear resistance . Numerouervation . The PEE loading .Regarding fracture resistance, PEEK exhibits superior flexural strength (140 to 170 MPa) compared to conventional materials, protecting restorations from bulk fractures ,42. ShetPrecise margins are crucial to successful crown restorations, with failure resulting in adhesive dissolution, dentin hypersensitivity, secondary caries, and periodontitis. Crowns with PEEK coping had better margin fit and internal adaptation than crowns with zirconia coping, and both were clinically acceptable . VariatiStress distribution, fracture resistance, and fracture pattern are primary considerations for fixed partial dentures (FPDs) . The You2 while others [2, 11.3 mm2). Other factors in FPD fracture include the presence or absence of veneer, aging, abutment models, and so on. Even with the varying degrees of difference in the design of the above-mentioned studies, PEEK can still be regarded as a viable alternative material for FPDs.Rodr\u00edguez et al. examined the potential of PEEK as an alternative FPD material along wie others have advIn terms of its clinical utility for FPDs, Rauch et al. have noted that PEEK requires less fabrication time and is lighter than zirconia, and although zirconia has exhibited a better aesthetic result than veneered PEEK, both are aesthetically acceptable . PEEK FPCekic\u2013Nagas et al. compared the load bearing capacity of inlay-retained FPDs fabricated from PEEK vs. other resin-based materials and founPost-and-core material requires high fracture and fatigue resistance, accurate matching with the morphology of the root canal, and more importantly, a Young\u2019s modulus similar to dentin (18.6 GPa) . The elaFEA consistently confirms the potential of PEEK as an alternative material to FRC or glass fiber in post-and-core restoration. In terms of prefabricated posts, PEEK and glass fiber posts show similar intensity and stress distribution when trialed with an occlusal load , and PEERegarding the influence of the PEEK manufacturing technique, FEA is useful for predicting the mechanical behavior of PEEK in post-and-core restorations and for evaluating the accuracy of PEEK fabricated by different methods. The work of Lalama et al. has predicted higher accuracy of heat-pressed PEEK posts in comparison to CAD/CAM PEEK posts .The superior properties of PEEK as post-and-core material are also evidenced in vitro and in vivo. PEEK posts showed the highest fracture resistance in comparison to polymer infiltrated ceramic (PIC) posts and FRC posts, but teeth had a less favorable fracture result with PEEK than with FRC . PEEK poIn addition to common fixed restorations, PEEK has also been tested in vitro for possible use in endocrowns and inlays. Because of decementation, PEEK endocrowns had the lowest retention force in comparison to infiltrated ceramic, partially stabilized tetragonal zirconia, and lithium disilicate ceramic endocrowns . AlthougIn conclusion, because of the deformability related to its lower elastic modulus, PEEK can provide favorable stress absorption for abutment teeth, adjacent tissues, and the cementation layer in fixed dental prostheses when compared with metal alloys and zirconia . PEEK noThe superior mechanical properties of PEEK can be offset by its aesthetic limitations. PEEK requires composite veneering to enhance its aesthetic properties. However, PEEK has an inert surface that makes adhesion difficult, and this is an important hindrance to its potential for widespread application in prosthetics. Numerous techniques have been tested for improving the adhesion of PEEK, including acid etching, plasma treatment, airborne particle abrasion, laser treatment, and adhesive systems.Accumulating evidence has demonstrated that sulfuric acid etching can significantly improve the SBS of PEEK. Much attention has been devoted to discovering the ideal acid concentration and etching duration . In termp < 0.05) [Recent studies indicate that combined methods can have a synergistic effect for improving bonding strength of PEEK. While acid etching improves the SBS of PEEK, acidic adhesive conditioning (pH = 2.5) failed to form favorable adhesion regardless of the processing time . However < 0.05) . Convers < 0.05) based on < 0.05) .Sulfuric acid etching considerably increases the SBS of PEEK, but the residual sulfuric acid is proven to trigger damage to human cells . A variePlasma treatment\u2014comprised of helium (He), argon (Ar), oxygen (O), hydrogen (H), nitrogen (N), and their mixed plasma\u2014has shown great promise as an excellent surface modification method for PEEK . Plasma The effect of He plasma modification of PEEK remains controversial. Okwa et al. treated fiber reinforced PEEK with He plasma, which significantly increased the surface energies and generated OH functional groups . CFR- anZhou et al. have reported satisfactory adhesion with Ar plasma-treated PEEK, noting that surface changes including cracks, grooves, and deposits were associated with significantly higher SBS (4.0 \u00b1 0.2 MPa) compared to an untreated surface (0.0 \u00b1 0.0 MPa) . Lu et aBesides O, H, He, and Ar plasma treatments, N plasma treatment can provide markedly enhanced adhesiveness to PEEK. Younis et al. reported that N plasma treatment resulted in higher PEEK SBS (10.04 \u00b1 1.84 MPa) than Ar (9.56 \u00b1 1.35 MPa), air (9.27 \u00b1 1.33 MPa), and O (8.59 \u00b1 1.64 MPa) plasma treatment, all of which were significantly higher than the SBS in the untreated group (5.38 \u00b1 2.90 MPa) . In addiAbrasion by sandblasting can increase the surface roughness and wettability of PEEK, subsequently improving the bonding between PEEK and veneering composites . GouveiaAlthough a majority of studies to date have supported the efficacy of sandblasting, there are a few that have disagreed. Adem et al. found no significant difference in SBS between untreated PEEK (5.39 \u00b1 1.36 MPa) and PEEK treated with 50-\u03bcm particles for 10 s at 2 MPa (6.43 \u00b1 1.05 MPa) and reported that the bonding performance (SBS) after sandblasting was much worse than the bonding obtained after 1-min 98% sulfuric acid etching (13.43 \u00b1 1.42 MPa) . Parkar The use of silica-modified alumina particles in sandblasting is an innovative surface modification method that promotes both mechanical and chemical connection between PEEK and the veneering composite, thereby more effectively enhancing the bonding strength . SpyropoIn conclusion, sandblasting shows promise as an easy, safe, and efficient chairside surface modification method for PEEK, although some results differ. The grain size of the aluminum oxide particles and the duration and pressure of abrasion are determinants of the efficiency of sandblasting for improving the bonding performance of PEEK . Because2) laser, erbium-doped yttrium aluminum garnet (Er:YAG) laser, neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, potassium titanyl phosphate (KTP) laser, and neodymium-doped yttrium orthovanadate (Nd:YVO4) laser laser . However2 laser treatment was not successful in creating mechanical interlocking, resulting in little significant improvement of SBS for PEEK, GFR-PEEK, or CFR-PEEK; the adhesive performance of CFR-PEEK was diminished by the CO2 laser treatment, whereas GFR-PEEK displayed no significant change [2 laser treatment resulted in a lower SBS (10.6 \u00b1 1.9 MPa) than Er:YAG laser (14.4 \u00b1 1.7 MPa), but both treatments were associated with significantly enhanced bonding strength of PEEK [COt change . In anot1.8 MPa) . Taha et1.8 MPa) . Notably1.8 MPa) . This is1.8 MPa) . However1.8 MPa) .4 laser groove treatment can enhance the SBS not only between PEEK and veneering composite resin, but also between PEEK and various resin-based luting agents, and the improvement of SBS is directly related to the depth of penetration of the Nd:YVO4 laser [Elsewhere, Nd:YAG laser resulted in the highest SBS (16.35 \u00b1 0.63 MPa), followed by Er:YAG laser (14.29 \u00b1 0.49 MPa) and then KTP laser (11.3 \u00b1 0.41 MPa); all had significant effect in improving adhesion , and PEEK exhibited better bonding performance than zirconia after laser treatment . Nd:YVO42.9 MPa) .Various combinations of surface modification methods and adhesive systems have been advanced to improve the bonding strength of PEEK. Resin luting cement, comprising universal resin cements and self-etch resin cements, has provided promising adhesion improvements for PEEK. Sole application of resin based luting cement often fails to provide sufficient SBS for PEEK. It is typically accepted that SBS should not be lower than 10 MPa . Tsuka eAlongside various luting cements, bonding primer has also provided improvement of PEEK bonding behavior. A satisfactory bonding strength can be achieved by Visio.link because of its specific composition of pentaerythritol triacrylate (PETIA) in solution, MMA monomers, and additional dimethacrylates, which causes micro-interlocking between resin cement and PEEK and increases the SBS of PEEK . Caglar In conclusion, a single application of luting cement often fails to provide satisfactory SBS for PEEK, but the performance improves when the luting cements are combined with appropriate bonding primers and surface modification methods. The available body of research is still insufficient, requiring further study to establish an optimal combination for the adhesion of PEEK.As described in this article, because of its excellent mechanical, chemical, biocompatibility, and aesthetic properties, PEEK is regarded as a promising alternative to conventional materials for fixed dental prostheses. PEEK prostheses have exhibited comparable or better clinical performance than metal or zirconia. However, PEEK also has some disadvantages when applied in fixed prosthodontics. Because of its grey color, PEEK fails to achieve the aesthetic effect of zirconia unless it is veneered with composite resin; however, the inert and hydrophobic surface of PEEK makes PEEK bonding with composite resin and abutment teeth difficult. This is a known obstacle to wider adoption of PEEK over conventional prosthetic materials. A variety of techniques for surface modification and improvement of the adhesive properties of PEEK, including acid etching, plasma treatment, airborne particle abrasion, laser treatment, and adhesive systems, are described in this report.As noted, neither lone application of luting cement, nor any single surface modification method, has provided satisfactory adhesive behavior for PEEK ,105. ThiThe restoration and bonding behavior of PEEK is also known to depend on the fabrication technique, whether 3D-printed, milled, or heat-pressed. The fabrication method affects the mechanical properties of PEEK and has also been associated with restoration outcomes ,73,108."} +{"text": "To compare mechanical properties of 3D-printed and milled poly-ether-ether-ketone (PEEK) materials. To define post-production treatments to enhance biocompatibility of 3D-printed PEEK.Standardised PEEK samples were produced via milling and fused-deposition-modelling 3D-printing. To evaluate mechanical properties, tensile strength, maximum flexural strength, fracture toughness, and micro-hardness were measured.3D printed samples were sandblasted with 50 or 125 \u03bcm aluminium oxide beads to increase biocompatibility.Scanning electron microscopy (SEM) evaluated microstructure of 3D-printed and sandblasted samples, estimating surface roughness at scales from 1mm-1\u03bcm.Cell adhesion on 3D printed and sandblasted materials was evaluated by culturing primary human endothelial cells and osteoblasts and evaluating cell growth over 48 h.3D printed materials had lower tensile strength, flexural strength, and fracture toughness, but higher micro-hardness.SEM analysis of 3D-printed surfaces showed sandblasting with 125 and 50 \u03bcm silica particles removed printing defects and created roughened surfaces for increased HUVEC and HOBs uniform cell adhesion and distribution. No cytotoxicity was observed over a 48h period, and all cells demonstrated >95% viability.3D-printing of PEEK is an emerging technology with clear advantages over milling in maxillofacial implant production. Nonetheless, this manufacturing modality may produce 3D printed PEEK devices with lower mechanical resistance parameters compared to milled PEEK but with values compatible with natural bone. PEEK has poor osteoconductivity and ability to osseointegrate. Sandblasting is an inexpensive modality to remove irregular surface defects and create uniform micro-rough surfaces supporting cell attachment and potentially enhancing integration of PEEK implants with host tissue. Biomaterials, Polyetheretherketone, CAD-CAM, Biocompatibility, Surgery. The gold standard reconstructive treatment is autologous bone transplantation. However, autologous grafts have drawbacks such as limited supply, donor site comorbidity, and unpredictable resorption during healing. There is also no aesthetically satisfactory donor site for critical-sized, geometrically complex craniofacial defects. These challenges encouraged the development of tissue engineering by utilising alloplastic grafts to address the problems , 2.Poly-ether-ether-ketone (PEEK) is a high-temperature thermoplastic polymer which demonstrates excellent chemical resistance to solvents except highly concentrated acids and is considered insoluble in biological fluids. PEEK\u2019s implant radiolucency inside the body is beneficial as it demonstrates high resistance to electron beam and gamma radiation . This me3, and strong strain and fatigue resistance , Mechanical testing demonstrated that 3D printed materials had lower tensile strength or large aluminium particles smoothened and abated the irregularities creating a rough micro topography less than 100 microns . However, other studies have used 40, 50 and 60 mm/s. Wang \u201380 mm/s ; and the\u201380 mm/s . This ca4.3Both FDM PEEK and Milled PEEK were tested and evaluated for tensile strength, 3-point flexural strength, fracture toughness and Vickers hardness.et\u00a0al. have suggested that this difference is due to injection moulding introducing external pressures which reduces internal defects and improves the density of parts [As Tables\u00a0of parts . Other sof parts , 51. OurBy observing the path of the crack propagation using numerical simulation specific FDM processes can increase the fracture toughness of PEEK. This is achieved by depositing filament along the same trajectories that mechanical stresses are applied to; providing the material with extra reinforcement . FDM proBoth FDM PEEK and milled PEEK demonstrate tensile strength, flexural strength, fracture toughness and Vickers hardness values close to those of natural cortical bone, supporting PEEK\u2019s applications as a bone replacement material. This includes applications in clinical dentistry, including different types of crowns, maxillofacial prosthesis, fixed partial dentures and removable partial dentures . PEEK ca4.4et\u00a0al. pointed out the importance of eliminating the printing patterns as cells tend to reside in the deep valley of the printing patterns and lead to a heterogeneous cell distribution [Han ribution . The eliAn evenly distributed surface roughness was also created through sandblasting. Sandblasting has demonstrated the ability to produce an enhanced surface roughness which promotes osteoconduction, bone to implant contact and increased removal torque; thus supporting sandblasted PEEK\u2019s viability as a bone implant .et\u00a0al. demonstrated that sandblasted ceramic filled PEEK dental implants were able to osseointegrate in 9 experimental dogs; concluding that the aluminium oxide particle blasting created a PEEK surface topography which can promote the migration, distribution, and proliferation of osteoblasts to the surface-a crucial aspect to osseointegration. The study suggested this can create contact osteogenesis which could give potential to continuous bone remodelling under cyclical loading, with bone density statistics comparable to those of Titanium [Previous studies confirmed the cytocompatibility of sandblasted PEEK , 69. TheTitanium . MoreoveTitanium . Other sTitanium , 75. Thiet\u00a0al. showed grooves and increased surface roughness have shown to be factors that support the adhesion and accumulation of cells, further emphasising the benefits of sandblasting and FDM production [As seen in the SEM analysis , the FDModuction .5In this current study it can be concluded that the data obtained revealed that 3D-printing of PEEK generated objects with substantially lower mechanical properties compared to Milled PEEK. However, FDM PEEK mechanical properties remain in a range very similar to natural cortical bone, and thus both types of PEEK can be considered a valid replacement to other bone substitute biomaterials such as Titanium. The closer mechanical strength values of both types of PEEK with bone increases the viability of PEEK\u2019s clinical applications with potential for less stress shielding effects, better osseointegration as a scaffold, and less chance of implant failure. The current lack of significant studies evaluating the mechanical properties of FDM PEEK and PEEK produced by Milling processes is an aspect that this research adds value too and has furthered.FDM PEEK was successfully 3D printed with promising results in accuracy, diameter, and thickness, increasing the potential of FDM printing for designing complex patient-bespoke objects with increased efficiency and less waste of material. The high print accuracy demonstrated in this study is an important aspect when considering the potential of complex geometric PEEK implants. It is also beneficial to consider the outcomes of utilising different FDM printing parameters when printing PEEK devices, and how to apply these to different clinical applications when balancing mechanical strength and interconnected porosity.The SEM image confirmed the effect of sandblasting treatment on eliminating the printing patterns and creating homogenous roughness, thus proving us a useful, cost-effective method of increasing PEEK biocompatibility. This study also signified the advantages of sandblasting as a form of mechanical treatment enhancing surface topography, as the results showed increased osteoblast and HUVEC cell adhesion with treated surfaces when compared to untreated. This can again give a potential cost-effective method of enhancing PEEK surface biocompatibility which may give clues to potential osseointegrative capabilities for PEEK scaffolds.It is worth considering the limitations of this study with guide to future work. Furthermore, future studies may consider adding FTIR or X-ray diffraction analyses to the 3D-printed and Milled PEEK specimens to understand the differences in crystallinity in the two production methods and how this can affect the mechanical strength of the material , 77, 78 Despite a small sample size, we have standardised in-vitro experimental conditions to minimise this issue. The relatively small standard deviation observed in most groups seems to confirm this and thus we have assumed that the data are likely normally distributed as typical in in-vitro setups .Further detailed examination of surface chemical compositions will be necessary to ensure no contamination was caused by the surface treatment. The successful cell adhesion and zero toxicity suggests there were no toxic contaminants present. However, it may also be beneficial in future studies to undertake more comprehensive industrial cleaning processes post-surface treatment and consequent biocompatibility assessment to ensure the absence of surface contaminants.Finally, in vivo studies under physiological load using 3D printed PEEK will be necessary to evaluate osseintegration and long-term success of implants.In conclusion, PEEK has many current applications in dentistry and maxillofacial reconstructive surgery and 3D printing significantly expands the repertoire of possible designs with great promises for the manufacture of bespoke medical devices and implants.Dr Neil Limaye: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Dr Lorenzo Veschini: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Professor Trevor Coward: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data included in article/supp. material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper."} +{"text": "Background: During the last decade, there has been an increased demand for non-metallic materials in orthodontics due to allergies, compatibility with medical imaging devices such as MRI, and aesthetic reasons. Monolithic poly-ether-ether-ketone material could address medical issues such as allergies and MRI compatibility. Moreover, nickel\u2013titanium (NiTi) archwires covered in PEEK, either by a tube or electrophoretic deposition, could address esthetic concerns. This scoping review aims to summarize the available evidence in the literature to provide an overview of the applications and material properties of PEEK in orthodontics. Methods: This scoping review was conducted according to the Joanna Briggs Institute Manual for Evidence Synthesis for scoping reviews and the Preferred Reporting Items for Systematic Review and Meta-Analyses Protocols extension for Scoping Reviews (PRISMA-ScR). We searched for relevant publications in MEDLINE (via PubMed), Embase, Web of Science, Cochrane Library, CENTRAL, ProQuest, and SCOPUS. A gray literature search was conducted on Google Scholar. Results: Six studies were included. In three studies, the authors investigated the feasibility of developing a composite PEEK-NiTi wire, while in two other studies, the authors investigated the feasibility of monolithic PEEK wires. In the final study, the authors investigated the feasibility of PEEK as a bonded retainer. Conclusions: The included studies show promising results in developing monolithic and composite (PEEK-NiTi) materials. Further research on the robustness of PEEK composites in the oral cavity, the status of cytotoxicity and roughness values, and the (bio)-mechanical behavior of the composites is needed. A homogenously set up comparative study of clinically relevant, evenly sized, monolithic PEEK wires versus conventional orthodontic wires for their biomechanical, mechanical, and material properties would clarify the possibilities of developing monolithic PEEK wires. Missing data in the retainer study suggest more research on the mechanical properties and points of failure of PEEK-bonded retainers, and a comparative study comparing the failure and mechanical properties of PEEK-bonded retainers to flat braided metallic bonded retainers is needed. A growing interest in developing substitute materials with comparable mechanical characteristics to human bone, so-called \u201cisoelastic\u201d materials, started in the mid-1980s. During this time, researchers in the field of orthopedic surgery, who were looking for possible new biomaterials for use in spinal trauma and hip stems, became aware of the existence of polyaryletherketones (PAEKs) , a familDue to its chemical structure, PEEK is highly stable at low and high temperatures exceeding 300 \u00b0C and is resistant to chemical and radiation damage, allowing its sterilization prior to medical application. PEEK is compatible with reinforcing agents , which enables tailoring of the elastic modulus . BecauseThere has been an increased demand in orthodontics for non-metallic materials during the last decade . To imprSeveral manufacturers have developed PEEK-coated wires in recent attempts to enhance the biocompatibility of NiTi wires. The PEEK coating was applied by either a tube covering the wire or electrophoretic deposition ,14. ElecSeveral investigators evaluated the load-deflection and friction properties of orthodontic wires made from PEEK to assess their feasibility as a replacement for (elastic) NiTi wires ,15. TheyThis scoping review protocol followed the Joanna Briggs Institute Manual for Evidence Synthesis for scoping reviews ,17 and tIncluded in this review is any study regarding the application of PEEK in orthodontics or fundamental research leading to possible applications in orthodontics, including in vivo studies and/or clinical trials. Exclusion criteria are the application of (part of) restorative treatment in dentistry, expert opinions or case reports/series, book chapters, papers published as manufacturers\u2019 statements, and non-indexed journals.The search was conducted in May 2022. Studies published between 2010 and May 2022 were included using combinations of terms in MEDLIStudies were selected in two phases. One reviewer (TN) searched the electronic database with a pre-determined syntax and set the search to titles, abstracts, and keywords when applicable. The process was documented, and a second reviewer (BA) re-ran the search to verify the results. Duplicates were removed by hand. Both reviewers performed TIAB screening on the unique records extracted from the primary search and assessed the eligible full-text records for inclusion. Differences in opinion concerning eligibility for inclusion were discussed between the reviewers. Patients and the public were not involved in this research project\u2019s design, conduct, reporting, or dissemination plans.Any relevant data concerning (bio)mechanical behavior, material properties, or biocompatibility was recorded.The primary outcomes were defined as any relevant data concerning (bio)mechanical behavior, material properties, and biocompatibility. Secondary outcomes were not defined.The high heterogeneity of the reported outcome measures did not allow the quantitative synthesis of the data retrieved from the included studies. Therefore, a qualitative synthesis of the results was performed.Of the 635 unique records, six studies were included in this review . Three sBoccaccini et al. assessed the feasibility of sintering a PEEK layer to NiTi wires . Shape mSheiko et al. assessedThe investigators showed a 10\u2013100 fold smaller release of Ni-ions compared to passivated NiTi. Moreover, mechanical stress in continuous cycles of axial compression/stretching at 20% strain for 7 million cycles showed no substrate delamination. However, locally applied large pressures (>2 GPa) might fracture the PEEK film, which could lead to exposing the NiTi wire.Shirakawa et al. assessed the feasibility of covering orthodontic archwires in PEEK tubes by sliding the tube over the wire . DiffereFurthermore, friction and surface roughness was tested in an unspecified conventional bracket. Archwires were ligated in the bracket with an elastomeric module. Uncoated and PEEK-coated 0.018-inch archwires showed no difference in friction during sliding, while the PEEK-coated 0.017 \u00d7 0.025-inch archwires showed a 50% reduction in friction values compared to their uncoated counterparts. Analysis of the base surface of the bracket slot showed no surface irregularities (scratches) after sliding any of the PEEK-coated wires. However, the uncoated wires showed significant surface irregularities of the bracket slots after sliding.Mechanical properties of wires fabricated from PEEK were investigated by Maekawa et al. , compariA comparison of mechanical characteristics of clinically relevant-sized PEEK wires to NiTi wires (0.016 inch) was described by Tada et al. . The invThe PEEK wires showed comparable load-deflection values to the 0.016-inch NiTi wire. Permanent deformation of an unspecified \u201cgreater degree\u201d was observed in the PEEK wire compared to the NiTi wire. The investigators concluded the 0.019 \u00d7 0.025-inch PEEK wire to be the most comparable to the 0.016-inch NiTi wire when comparing the load values. In the stress-relaxation test, the wire was deflected at 2 mm and fixated for 24 h. The investigators determined the amount of stress (or force) exerted by the wire after 24 h. The NiTi 0.016-inch wire retained 78% of its initial force, while 0.016-inch PEEK, 0.016 \u00d7 0.022-inch PEEK, and 0.019 \u00d7 0.025-inch PEEK wires retained 77%, 67%, and 69% of their initial force, respectively. No statistically significant difference was found in the amount of static friction between the PEEK wires and the NiTi wire.Kadhum and Alhuwaizi investigIn the bonding failure test, the investigators observed the comparable failure of the PEEK wires to the dead-soft metallic wires (both suffered from wire rupture). Only the braided wire was observed to fail at the bonding interface. The results of the pull-out performance test showed that the PEEK wires pre-treated by air abrasion or air abrasion/monomer conditioner suffered from wire rupture. Moreover, all metallic and non-treated PEEK wires were pulled out of the bonding interface.In recent years, the demand for non-metallic orthodontic appliances has increased, mainly to improve esthetics and avoid complications due to allergies and interferences with magnetic resonance imaging (MRI) ,24. PolyThus, HPPs such as PEEK have allowed researchers and developers to apply new solutions to old problems. Two main approaches for using PEEK have been investigated: the development of composite wires and the development of monolithic wires as a substitute for metallic wires. In developing composites, the good mechanical properties of metallic materials (NiTi) are combined with the biocompatible and esthetic properties of non-metallic materials (PEEK). In contrast, monolithic wires are fabricated solely from PEEK and are supposed to act as a substitute for NiTi wires.Boccaccini and Sheiko investigated the development of composites by coating NiTi wires with PEEK. Moreover, Shirakawa investigated the feasibility of covering NiTi wires with a PEEK sleeve (sleeving), while Boccaccini et al. developed a PEEK-NiTi composite using electrophoretic deposition (EPD). To improve adherence of the coating (PEEK) to the substrate (NiTi) after the EPD process, the investigators applied a sintering process with a temperature of 340 \u00b0C for 20 min . HeatingDip-coating, as used by Sheiko et al., could solve the potential deterioration adhesion of the coating to the substrate by not having any adhesion, allowing the coating to flex with the substrate in its martensitic state. Electrochemical polishing and dip-coating a NiTi wire could result in a 10\u2013100-fold decrease in Ni-ion release compared to passivated or chemically etched NiTi . Data onCovering over-the-counter orthodontic archwires with a PEEK tube (by sliding the tube over the wire) has shown to be a feasible and cost-effective way to fabricate an esthetically pleasing archwire . Its friAnother (non-composite) approach has also been investigated, fabricating orthodontic wires solely from PEEK or monolithic PEEK wires ,15. AccoIn contrast, applying PEEK in the passive (or less-active stage) of orthodontic treatment as a bonded retainer has shown favorable results . The bonThe included studies show promising results in developing monolithic and composite (PEEK-NiTi) materials. However, several questions have remained unanswered and provide a basis for future research. For instance, it is unknown: how robust the PEEK-NiTi composites are in the oral cavity; the status of cytotoxicity and roughness values; and the (bio)-mechanical behavior of the composites. Thus, a homogenously set up comparative study of clinically relevant, evenly sized, monolithic PEEK wires versus conventional orthodontic wires for their biomechanical, mechanical, and material properties would clarify the opportunities in developing monolithic PEEK wires. Missing data in the retainer study suggest the need for more research on the mechanical properties and points of failure of PEEK-bonded retainers, and a comparative study of the failure and mechanical properties of PEEK-bonded retainers to flat braided metallic bonded retainers is needed.By design, the goal of this review was to scope a wide body of literature to identify knowledge gaps. The shortcomings of this study are found in its design; only studies published in indexed journals were included. As a consequence, a number of studies not published in indexed journals were excluded ,33,34.Roughness values, robustness in the oral cavity, cytotoxic values, and (bio)-mechanical behaviors of (PEEK)-coated NiTi wires;Homogenously set up comparative studies, comparing clinically relevant-sized PEEK wires to conventional orthodontic wires;Mechanical properties and points of failure in PEEK-bonded retainers compared to braided flat metallic retainer wires.The development of monolithic and composite PEEK materials shows promising results. Mapping the available evidence about the applications of PEEK polymers in orthodontics, we identified several opportunities for future research:"} +{"text": "This comprehensive review focuses on polyetheretherketone (PEEK), a synthetic thermoplastic polymer, for applications in dentistry. As a high-performance polymer, PEEK is intrinsically robust yet biocompatible, making it an ideal substitute for titanium\u2014the current gold standard in dentistry. PEEK, however, is also inert due to its low surface energy and brings challenges when employed in dentistry. Inert PEEK often falls short of achieving a few critical requirements of clinical dental materials, such as adhesiveness, osseoconductivity, antibacterial properties, and resistance to tribocorrosion. This study aims to review these properties and explore the various surface modification strategies that enhance the performance of PEEK. Literatures searches were conducted on Google Scholar, Research Gate, and PubMed databases using PEEK, polyetheretherketone, osseointegration of PEEK, PEEK in dentistry, tribology of PEEK, surface modifications, dental applications, bonding strength, surface topography, adhesive in dentistry, and dental implant as keywords. Literature on the topics of surface modification to increase adhesiveness, tribology, and osseointegration of PEEK were included in the review. The unavailability of full texts was considered when excluding literature. Surface modifications via chemical strategies and/or physical approaches discussed in the literature are summarized and compared. Further, approaches such as the incorporation of bioactive materials, e.g., osteogenic agents, antibacterial agents, etc., to enhance the abovementioned desired properties are explored. This review presents surface modification as a critical and essential approach to enhance the biological performance of PEEK in dentistry by retaining its mechanical robustness. In general, high-performance polymers are polymeric materials that can retain their desirable mechanical, thermal, and chemical properties under harsh environmental conditions, such as high temperature, high pressure, and corrosive chemicals . In dentAn excellent polymeric material for dental applications is PEEK, a high-temperature semi-crystalline, synthetic thermoplastic polymer . Some liWe applied the keywords PEEK, polyetheretherketone, osseointegration of PEEK, PEEK in dentistry, tribology of PEEK, surface modifications, dental applications, bonding strength, surface topography, adhesive in dentistry, and dental implant to Google Scholar, Research Gate, and PubMed database. Duplicate literature was sorted manually. Literature considered eligible for review if complied with inclusion criteria: English-written, with the topic on surface modification to increase adhesiveness, tribology, and osseointegration of PEEK. Pieces of literature were excluded if the full text was unavailable. Additional literature searching using keywords surface sulfonation of PEEK, PEEK plasma treatment, PEEK UV treatment, surface coating of PEEK, surface polymerization of PEEK, PEEK sandblasting, PEEK laser treatment, accelerated neutral atom beam PEEK, layer by layer PEEK, particle leaching of PEEK was performed. This step allowed us to do a thorough review of each strategy published in the literature.Practically it is difficult to achieve all the desired properties for a given application using one single polymeric material, and PEEK is no exception. PEEK provides several exceptional properties that provide necessary reliability for dental materials, but it also suffers from other drawbacks, which, unless addressed, cannot be widely employed in the market. This section briefly explains the general attributes of PEEK and their impacts on dentistry.PEEK architecture consists of a linear aromatic backbone composed of functional ketone and ether groups . PEEK haFurther, the studies also indicate the superior biocompatible nature of PEEK compared to Ti. For example, higher osteoblast viability and proliferation were detected on PEEK surfaces than on Ti6Al4V surfaces. When the PEEK was modified with 5% beta-tricalcium phosphate, an enhanced osteoblast differentiation was observed . Peng etPEEK, unfortunately, is also accompanied by several drawbacks when employed in dentistry. For instance, PEEK has been reported to be biologically inert due to its low surface energy challenging its clinical applications ,42,43. PIn addition, PEEK is also inert to several chemical treatments (except for sulfuric acid) , renderiIn addition to PEEK\u2019s biocompatibility, superior mechanical, thermal, and chemical properties, it is desired of PEEK to have exceptional adhesiveness , bioactivity , and friction-wear resistant when employed in dentistryAs mentioned in Adhesives function simultaneously as a bonding agent between composite resin and dental hard tissue , and as a shielding component to protect collagen fibers at the resin-dentine interface from deterioration through remineralization . AdhesivThe ability of an adhesive system to stand mechanical load during mastication is crucial in determining successful PEEK-supported implant prostheses. Good retention between PEEK and titanium bases requires strong adhesive and cohesive strength of the adhesive system. Yilmaz et al. addresseDoping is another strategy to minimize bonding-related deterioration in restorative dentistry. Dentine demineralization caused by acid etching and increased resin infiltration promotes bonding between resin and dentine. Following the elimination of mineral ions during dentine demineralization, the collagen matrix becomes visible and exposed. The hybrid layer is then produced where the resin adhesive penetrates and polymerizes within the demineralized collagen. A fraction of demineralized and exposed collagen is prone to deterioration by host-derived matrix metalloproteinases and bacterial collagenases, which are present at the bottom of the hybrid layer, worsening the restorative bond. The bottom of the hybrid layer thus presents the weakest bond in the composite restoration . For thiResilient liners are another example of soft and elastic adhesive agents, which have wide-range applications in the field of maxillofacial prostheses. They have been proven as an excellent clinical adjunct in patient care with persistent denture pain . PlasticSurface conditioning is an approach where the surface is pre-treated with an adhesive layer to achieve durable bonding. Stawarczyk et al. explored the tensile bond strength between veneering compounds and PEEK at various pretreatment conditions and conditioning systems . The pre2 for 60 s). The results showed that shear bond strength was increased as conditioning duration was increased, i.e., a ca. 4.95 MPa for 0 min to ca. 21.43 MPa for 5 mins, respectively.Rosentritt et al. studied shear bond strength between PEEK and veneering composites after surface roughening and conditioning. Enhanced bonding of the composites on PEEK surfaces was achieved when the surfaces were conditioned with either acetone- or phosphate-based methacrylate primers or were tribochemically treated. In addition, it was revealed that application of opaque materials increased the shear bond strength . EscobarThe bonding strength between polymeric dental devices is also influenced by their fabrication techniques. Ultrasonic welding is one of the practical methods employed to permanently join various dental PEEK pieces together in a denture structure . The ultOsseointegration is when the implant directly interacts with the surrounding host bone until they are entirely stabilized. This process is necessary for a successful prosthesis . ClinicaIt is then clear that implanted dental devices should stimulate osseointegration. However, considering that PEEK is bioinert, osseointegration is challenging. Moreover, PEEK is not intrinsically osseoconductive . EffortsCandida species are prone to adhere onto acrylic dentures . . 181]. K E. coli c 186]. . graftin E. coli d 187]..grafting2) nanofibers. Methacrylated hyaluronic acid (MeHA) was then deposited and polymerized using UV irradiation. Electrospun TiO2 provides surface roughness and a more favorable elastic modulus, while MeHA polymer backbone provides interaction sites for mesenchymal stem cells (MSCs). Biological characterizations showed increased cell adhesion, proliferation, and osteogenic differentiation . . 211]. Ia = 4.63 nm to Ra = 3.45 nm). The bioactivity was assessed using dental pulp stem cells where their attachment and proliferation were increased. In addition, accelerated osteogenic differentiation was also observed [S. aureus, E. coli, and S. epidermis bacteria adhesion [ANAB is a surface treatment where the surface is bombarded with energetic neutral atoms produced by a gas cluster ion beam . As a reobserved . ANAB-trobserved . Anotherobserved . Remarkaadhesion .LbL technique employs the deposition of oppositely charged polymers (polycations and polyanions) on a charged surface via electrostatic interactions . Other f3)2\u00b74(H2O), followed by immersion in gentamicin sulfate-containing (NH4)2\u00b7HPO4 to assemble Ca2+ ions and antibiotic gentamicin sulfate, respectively. The deposition of Ca2+ ions and gentamicin sulfate layer were repeated 3, 6, and 9 times to obtain different layer thicknesses of composite containing Ca2+ ions and gentamicin sulfate on the activated PEEK. From this LbL process, PEEK surfaces functionalized with brushite (CaHPO4.2H2O) layers containing antibiotic were obtained. Both in vitro (using S. aureus and E. coli) and in vivo (in rats) experiments showed that the brushite-gentamicin-modified PEEK surfaces exhibited antibacterial and osseointegration properties solution followed by immersion in poly(styrene sulfonate) solution. The activated PEEK was immersed in Ca and polycation poly(styrene sulfonate) on PEEK surfaces. Cell activity was assessed in vitro using bone marrow stromal cells, and the results showed that cell adhesion, cell proliferation, cell growth rate, and ALP activity were significantly improved. Animal studies using a rabbit model and 20 LbL layer PEEK demonstrated improved osseointegration. In addition, samples with 20 LbL layers also presented the lowest water contact angle .E. coli and S. aureus, and the results showed that the presence of Ag ions effectively inhibited the growth of the tested bacteria. Surface biocompatibility was also improved due to the presence of porous structures (from sulfonation process) and Zn ions, which included an enhanced cell viability, spreading, and proliferation, as well as accelerated osteo-differentiation, and osteo-maturation [Deng et al. combined sulfonation and LbL techniques to incorporate antibacterial properties and enhance biocompatibility of PEEK. The LbL multilayers consisting of Zn ion-containing chitosan (as positively charged polyelectrolyte layer) and Ag ion-containing sodium alginate (as negatively charged polyelectrolyte layer) were deposited onto sulfonated PEEK. Antibacterial assay was conducted against turation .2 and 390 \u00b0C for 20 min. After cooling, the substrates were immersed in water for 72 h (room temperature), followed by immersion in stirred boiling water for 1 h. The produced pores were uniform with a defined pore interconnectivity and an average diameter of ca. 270 \u00b5m. The mechanical properties of PEEK after NaCl leaching were, unfortunately, decreased. Biological characterization using fibroblast cell line resulted in 86% of cell viability [Particle leaching is a method to introduce porosity on a surface by leaching the particles embedded either in bulk or on the surface of a material. Common particles include NaCl salt, sucrose, etc., with NaCl as the most used particle. Leaching is executed by solving the particles in respective solvent, leaving behind a porous surface . Torstriiability . Evans eiability . In the iability . A ratheiability .In dentistry, polyaryletherketones (PAEKs) are a class of high-performance polymers employed for numerous clinical cases. This class includes numerous members with various chemical structures, among which PEEK is the most widely used in dentistry due to its similar bulk properties as bone. Our review addresses various aspects of surface modification and treatment methods to generate PEEK as a desirable alternative to metal- and ceramic-based dental materials. The surface-related properties such as adhesiveness to other materials, osseointegration, antibacterial, and tribological (friction-wear) are critical and uncompromisable in a material when employed in dentistry. Hence, various physical and chemical surface treatment processes, such as air abrasion, laser treatment, sulfonation, plasma treatment, etc., present effective approaches to address the requirements. Literature showed that the surface treatment approach could simultaneously impact several properties, both in desired or undesired ways; hence a careful optimization of surface parameters or combinatorial modification approaches are often required.a > 0.2 \u03bcm) can also assist bacterial adhesion and biofilm formation, which is shown to be overcome by incorporating various antibacterial compounds into the PEEK surface. Furthermore, various growth factors and cell-attractive biomolecules attached to the PEEK interface are shown to enhance the desired cell growth and proliferation.This review suggested that highly durable and resilient polymeric adhesives, liners, and conditioners, when added to the PEEK (pre-treated) surface, successfully bond PEEK with tissue, veneer materials, or titanium implant bases. To this end, a section in this review discusses the strategies for improving the performance of PEEK using acrylic- and silicon-based polymers. In addition, the review presented that surface roughness, controlled via various surface treatment approaches, is a critical parameter that can enhance PEEK adhesion to other materials via enhanced surface energy and/or mechanical interlocking. Both in vitro and in vivo studies showed this approach to be effective in enhancing the interactions with tissue cells. Nevertheless, higher surface roughnesses and surface physical topography play a critical role in influencing the attachment and bioactivity of PEEK to other materials, whether it is biological entities like bacteria and tissue cells or other materials like titanium, veneer composite, adhesive cement, etc. In general, the presence of PEEK on dental materials like titanium helps in reducing frictional wear in artificial saliva, hence presenting fewer health concerns from debris ingestions. This review sheds light on the design rules of high-performance polymers, especially PEEK, in dentistry."} +{"text": "The incorporation of bioactive ceramic into polyether ether ketone (PEEK) was expected to improve the bioinertia and hydrophobicity of pure PEEK, further facilitating osseointegration and bone ingrowth. However, the addition of bioceramic also changes the anisotropy of mechanical properties and failure mechanism of composite. Therefore, three-dimensional printed (3D-printed) PEEK/hydroxyapatite (HA) composite filaments with differing proportions (HA content: 10\u201330 wt%) were prepared using physical mixture and melting extrusion processes. The tensile elastic modulus and tensile strength of composite filaments were tested experimentally. These microscopic models, with multiple diameter variations and differing dispersity of HA particles, were built to estimate mechanical properties using finite element analysis. Based on a generalized version of Hooke\u2019s Law, the influence of diameter variation and particle clustering on the elastic modulus was evaluated. The mathematical relationship between the elastic modulus and volume fraction of the bioceramic was established using the Halpin\u2013Tsai model. The results showed that with an increase in HA content from 10 wt% to 30 wt%, the elastic modulus of the composite increased from 2.36 GPa to 2.79 GPa, tensile strength decreased from 95 MPa to 74 MPa, and fracture elongation decreased from 63% to 23%, presenting brittle fracture failure. When the dispersion of particles was uniform, the elastic modulus was less affected by diameter variation, but the modulus anisotropic coefficient was greatly affected by the composition ratio, particle diameter, and dispersity. Hence, 3D-printed PEEK/HA composite filaments can meet the strength requirements of human bone, and understanding the influence of mechanical anisotropy plays a very important role in the design, manufacture, and clinical application of medical implants. Polyether ether ketone (PEEK), a material with excellent biocompatibility and anti-corrosion, similar density and mechanical properties compared to natural bone tissue, and high radiographic penetrability, has been widely used to fabricate medical protheses using additive manufacturing technology, such as thoracic or rib prosthesis ,2, interCurrently, modification strategies for PEEK material mainly include surface modification and material hybrid modification. For surface modification, physical or chemical treatments have often been used to improve the surface roughness or microporous structure of material, as well as the deposition of bioactive materials to alter bioactivity and osseointegration . The surDespite an improvement in bioactivity, the addition of bioceramics also affects the overall mechanical properties of the composites to some extent. Zheng et al. fabricatHence, this study fabricated 3D-printed filaments of PEEK/HA composite with differing HA contents. According to the distribution of particle diameter observed using a scanning electron microscope, a numerical model was established to analyze the mechanical properties of the composite. The effects of material proportion, particle diameter variation and dispersion uniformity on mechanical properties were investigated. Tensile testing of PEEK/HA composite filaments was performed. Finally, the mathematical relationship between elastic modulus or tensile strength and volume fraction of HA was built. Through comprehensive analysis of mechanical properties and anisotropy of PEEK/HA composite filaments, this study lays a solid foundation for the design and evaluation of 3D-printed PEEK/HA composite medical implants.PEEK powder and HA powder were used to fabricate the PEEK/HA composite. The microscopic morphology of two powders was observed using a scanning electron microscope . As shown in According to the ASTM D4018\u201311 standard, the tensile testing of PEEK filament and PEEK/HA composite filament was performed using a universal testing machine at room temperature. Both ends of each filament specimen with a length of 250 mm were adhered to cardboard for a length of 50 mm. A crosshead separation rate of 2 mm/min was set, and an extensometer with a gauge length of 100 mm was used to measure deformation. Thereafter, tensile elastic modulus and tensile strength were calculated. For each group, four duplicate specimens were tested to ensure repeatability. Finally, the fracture surface of composite filaments was observed by SEM.Cijkl is a fourth-order stiffness matrix with 81 components. Owing to the inherent symmetries, the coefficients of the stiffness matrix can be reduced to 21, and the matrix format is given as Equation (2).According to the generalized Hooke\u2019s Law, the tensor form of stress\u2013strain relationship during the elastic deformation stage can be expressed as follows:In order to calculate each stiffness coefficient, different loading and boundary conditions were set using a multi-level hypothesis. The specific solution process was mentioned in our previous study . The spaA micro-geometrical model of the PEEK/HA composite was built by randomly distributing spherical HA particles into the PEEK matrix. Next, the geometrical model was imported into Abaqus software . The material properties of each component of PEEK/HA composite were set, respectively, with an elastic modulus of 1.97 GPa and a Poisson ratio of 0.3 for the PEEK matrix, and corresponding parameters of 100 GPa and 0.28 for HA particles . AccordiBased on the above analysis method, three kinds of PEEK/HA composites with HA contents of 10, 20, and 30 wt% were analyzed according to the fabrication of the composite filament. As shown in The tensile mechanical properties of PEEK and PEEK/HA composite filaments are given in For the PEEK/HA composite filaments, when HA content increased from 10 wt% to 30 wt%, the elastic modulus increased from 2.36 \u00b1 0.20 GPa to 2.79 \u00b1 0.16 GPa, while the ultimate tensile strength decreased from 95 \u00b1 3.18 MPa to 74 \u00b1 3.46 MPa, and the percentage of total extension at fracture decreased from 63% to 23%. According to Rodzen\u2019s study ,23, whicAfter observing the fracture surface by SEM, the failure mechanism of PEEK/HA composites changed from a ductile fracture to a brittle fracture. This is mainly attributed to the change in continuous stress distribution inside the composite, owing to the random dispersion of discrete ceramic particles in the PEEK matrix. In particular, some of the local stress concentrations at the contact interface of different phases further aggravated the initiation and propagation of microcracks, which finally evolved into brittle fractures. Comparing the strength of cortical bone in different locations and orientations, usually ranging from 50\u2013151 MPa , the mecEmin/Emax fell between 0.6 and 0.7 within the set proportion range.The spatial distributions of elastic moduli derived from numerical analysis of the microscopic model for the PEEK matrix and PEEK/HA composites are shown in The influence of particle diameter variation and dispersion uniformity on the elastic modulus and anisotropy for PEEK/HA composites with differing proportions were plotted in E, EHA and EPEEK are the elastic moduli for the composite, HA material and PEEK matrix, \u03c6V is the volume fraction of HA content, and the factor \u03be is an empirical parameter determined by curve fitting of experimental data and used to quantify the geometry of the inclusion. As shown in \u03be was equal to 1.679 within the setting range of volume fraction.Moreover, the numerical analysis results were generally larger than the experimental results, with a deviation of about 20%. This difference may have resulted from the limitation of finite element analysis, causing difficulty in fully reflecting some problems in the actual preparation of composite filaments, such as the irregularity of micropore formation, particle dispersion uniformity or agglomeration, imperfect or interfacial bonding, etc. In order to establish a mathematical relationship between the elastic modulus of the composite and its internal composition, according to the Voigt model by assuming equal strain for longitudinal loading and the Reuss model with equal stress for transverse loading , the uppThe distribution of von Mises stress for three composite microscopic models under the unidirectional tensile load, and the statistical results of the von Mises stress distribution at the PEEK/HA interface nodes are shown in (1)The PEEK/HA composite filaments used for fused deposition modeling were fabricated. With an increase in HA content from 10 wt% to 30 wt%, the elastic modulus of the composite increased from 2.36 GPa to 2.79 GPa, while the tensile strength decreased from 95 MPa to 74 MPa, and the fracture elongation decreased from 63% to 23%, exhibiting brittle fracture failure under uniaxial tensile testing.(2)The influence of particle diameter and dispersity on the elastic modulus of PEEK/HA composite was estimated using finite element analysis. When HA particles were uniformly distributed, the elastic modulus was less affected by the change in particle diameter. However, the modulus and anisotropic coefficient increased with the increase in HA content and particle clustering. Therefore, during the preparation process of the PEEK/HA composite, it is necessary to ensure the uniformity of particle size and dispersion uniformity as far as possible, so as to ensure the stability of its mechanical properties.(3)\u03be was equal to 1.679 within the setting range of volume fraction.The mathematical relationship between the elastic modulus of a composite and the volume fraction of HA content was established on the basis of the Halpin\u2013Tsai model and experimental data. The empirical parameter In this study, the mechanical properties of 3D-printed PEEK/HA composite filaments were investigated. It can be concluded that:"} +{"text": "Evolutionary transitions in individuality (ETIs) involve the formation of Darwinian collectives from Darwinian particles. The transition from cells to multicellular life is a prime example. During an ETI, collectives become units of selection in their own right. However, the underlying processes are poorly understood. One observation used to identify the completion of an ETI is an increase in collective-level performance accompanied by a decrease in particle-level performance, for example measured by growth rate. This seemingly counterintuitive dynamic has been referred to as fitness decoupling and has been used to interpret both models and experimental data. Extending and unifying results from the literature, we show that fitness of particles and collectives can never decouple because calculations of fitness performed over appropriate and equivalent time intervals are necessarily the same provided the population reaches a stable collective size distribution. By way of solution, we draw attention to the value of mechanistic approaches that emphasise traits, and tradeoffs among traits, as opposed to fitness. This trait-based approach is sufficient to capture dynamics that underpin evolutionary transitions. In addition, drawing upon both experimental and theoretical studies, we show that while early stages of transitions might often involve tradeoffs among particle traits, later\u2014and critical\u2014stages are likely to involve the rupture of such tradeoffs. Thus, when observed in the context of ETIs, tradeoff-breaking events stand as a useful marker of these transitions. Evolutionary transitions in individuality (ETIs) are events of major significance in the history of life. They begin with lower-level entities (particles) and complete when higher-level entities (collectives) acquire properties sufficient to participate directly in the process of evolution by natural selection . ETIs of particular note include the evolution of chromosomes (from genes), the eukaryotic cell , and multicellularity (from single cells) . Here, w\u2014and guiding experimental analysis\u2014has been the concept of \u2018fitness decoupling\u2019, which posits that the fitness of a collective in the early stage of a transition is directly proportional to the fitness of its particles and that, as the evolutionary transition proceeds, collective fitness \u2018becomes decoupled from the fitness of its lower-level components\u2019 , could in reality be one where there is no difference between the two. In other words, it could be a spurious observation.To take an example where the two environments, rather than the times over which reproductive outputs are measured, are different, consider two identical plants receiving different quantities of resources. The plant receiving more resources produces more seeds. Yet, this difference in reproductive output cannot lead to the conclusion that these two plants have different fitnesses where \u2018fitness\u2019 is associated with natural selection. In our example, because the two plants are initially identical, they necessarily have the same fitness. This situation is depicted in A is in environment 1, and B is successively in environments 1 and 2, the two resulting fitness values are not comparable because they do not inform one of the potential outcomes of competition in environment 1, in environment 2, or in a temporal succession of the two. This invalid comparison is represented in In some situations where one wants to make fitness comparisons, the environment presents fluctuations in time. In this case too, to be comparable, they must refer to the same set of events. For instance, if If the environmental changes are not deterministic, a weaker condition than the same temporal succession of environments is that the two organisms experience the same distribution of environments and transition probabilities between environments (steady-state) see . This tyshort-term particle growth rate coinciding with increased collective-level performance , rather than in terms of transfer of fitness between particles and collectives. If it is true that the fitness of cells and collectives cannot decouple, where does this leave our understanding of dynamic processes that underpin ETIs? The mechanistic approach with an ecological focus is one route .We begin by elaborating on the notion of \u2018fitness decoupling\u2019 and related concepts such as \u2018export of fitness\u2019 and \u2018fitness transfer\u2019, showing how this idea has been used in both the theoretical and experimental literature (Section 1). In Section 2, we present a population projection model of an abstract proto-multicellular organism and show that particle and collective fitness are necessarily equal for all structured population dynamics models that reach a stable collective size distribution, generalising and unifying results from the literature e.g., . In SectOne classical way to characterise particle fitness is to measure long-term reproductive success under a given set of environmental conditions relative to other particles . In a moMichod and Nedelcu, 2003; populations, The concept of fitness decoupling has been regarded as one indicator for the ETI from cells to multicellular individuals . In a stMeasures of cell and collective fitness must derive from analyses performed in the same reference environment and over precisely the same timescale and the collective level and their deviations.To summarise, there are two ways in which the export of fitness has been used in the literature: first as a transfer between actual quantities, and second as a process concerning counterfactual quantities , but without the ability to produce multiparticle collectives. This is also close to the experimental measurements of cell density performed experimentally . Each can be adequate in different contexts. It is appropriate to measure particle fitness considering within-collective events , our mathematical proof applies to a larger class of population dynamics models\u2014it is not tied to an MLS1/MLS2 framework and does not require any assumptions regarding the relationship between particle and collective life history traits, such as the usual assumption that collective viability is a linear function of individual viability . It uses the Matpopmod library . Thus, as selection acts during the ETI and drives the trait values towards fitter collectives (towards Once a mutation (or change in the environment) makes the first multiparticle collective possible, the process that drives population dynamics is one associated with Let In this model, the optimal trait value for counterfactual . To explain this phenomenon, we present a modification to the model described in Section 3, which we call the \u2018tradeoff-breaking model\u2019.A tradeoff is essentially a constraint on the combined values of a set of traits; it prevents a given organism from simultaneously performing well in two or more functions\u2014for instance, growth and survival or photosynthesis and nitrogenase activity in cyanobacteria, as discussed below. Thus, if a mutant lineage is able to bypass the tradeoff and perform well in different functions, it is expected to be fitter than its ancestor and increase in proportion in the population. In some cases, mutations that lead to a multicellular morphology might be a necessary step towards bypassing or breaking the tradeoff. This provides an \u2018adaptive\u2019 explanation for the emergence of multicellular organisms. Collectives emerge by mutations constrained by the tradeoff, but their long-term persistence is rendered more probable by tradeoff-breaking mutations that would not have been possible, or would not have broken the tradeoff, had they occurred in their unicellular ancestors.2)\u2014in cyanobacteria. The tradeoff is caused by the oxygen sensitivity of the enzyme nitrogenase, which catalyses the process of reducing N2 to ammonia (NH3). This prevents cells from performing both functions simultaneously and has resulted in several morphological and physiological adaptations that only fix N2 and exchange the fixed nitrogen compounds against carbon products with the photosynthesising cells of the filament. This example seems to be compatible with the tradeoff-breaking framework\u2014both multicellular and differentiated species, (a) Anabaena oscillarioides and Trichodesmium sp., seem to have broken away from the tradeoff, which leads to tradeoff-breaking observations in 2-fixing cells allow the organisms to break away from the tradeoff that is present in the unicellular and physiologically undifferentiated phyla is given by Equation 2.3 from Libby et al., 2016The population dynamics of both types where We now recast this model within our framework. The whole life cycle fitness of the organism Let p increases to 0.2, corresponding to optimisation on the tradeoff leads to a fitness-decoupling observation when selection first acts along the switch probability tradeoff (leading to higher switching and an increase in multicellular types) because the optimal trait values for re as in .In the ratcheting model, proto-multicellular organisms are in an environment alternating between multicell-favouring and unicell-favouring. This yields a tradeoff between the two states and the selection for a high probability of multicells to revert to unicells. Some mutations (\u2018ratcheting (type 1) mutations\u2019) are assumed to be beneficial in a collective (multicellular) environment while deleterious in a unicellular context; thus, they play the tradeoff-breaking role. In the ecological scaffolding model, the environment, structured both spatially and temporally, allows for selection of collective-level properties without the need to assume anything about the particles other than that they reproduce at different rates. Both models illustrate the flexibility of our tradeoff and tradeoff-breaking approach. First, it allows multiple mechanisms of evolutionary transitions to be formalised in a unified way. Second, tradeoff-breaking observations can be used as a general marker across various mechanisms of evolutionary transitions.The trait-based approach we have described so far delineates an adaptive scenario for an ETI. This scenario is divided into several phases. These are, first, the formation of collectives, in which individuation mechanisms that might be adaptive are at play ; second,Before an ETI starts, the unicellular ancestors have been selected for traits that optimise growth rate in a unicellular environment under the constraints of their genetic constitution , and establishing how those collectives change and potentially overcome the constraints that historically bear on particles (phase 3) offers a good empirical handle to study ETIs. Conversely, the lack of any tradeoff-breaking observation in such an empirical system could be used as an indication that the transition is still in its early stages.Our main objective in advocating this trait-based approach is to offer a consistent framework for interpreting models, empirical observation, and evolutionary experiments of ETIs, which can accommodate the apparent fitness-decoupling observations, in addition to the tradeoff-breaking ones, without referring to the export-of-fitness metaphor. Provided this empirical objective, it is legitimate to ask to what extent the tradeoff-breaking scenario is representative of ETIs and can be used as a marker that an ETI is underway or has occurred. First, one must recognise that tradeoff breaking might occur outside ETIs. For instance, tradeoff breaking and, more generally, \u2018constraints breaking\u2019 are already considered key events in the evolution of body plans and are expected to be a widespread mechanism for the emergence of novelties see . Thus, aFitness-centred approaches to ETIs have been influenced by the concept of fitness-decoupling between lower-level particles and higher-level collectives. In this view, the fitnesses of particles and collectives are initially proportional to one another but diverge as an ETI occurs\u2014particle fitness decreases while collective fitness increases. This interpretation comes with some inconveniences. First, fitness is notoriously difficult to define and measure. This, in turn, makes fitness comparisons across levels difficult. Second, fitness values in and of themselves do not provide a mechanistic model of the system. Progress in understanding ETIs relies on our ability to circumvent limitations inherent in the currency of fitness. We suggest that, to study ETIs, focusing on traits and tradeoffs between traits, rather than focusing on fitness, is both more parsimonious and practically achievable. Finally, we propose that rare tradeoff-breaking events are a crucial part of ETIs and could be used experimentally for their detection.Our first main finding is a new formal argument cementing the position that decoupling between commensurable measures of fitness is impossible. Starting from the recognition that fitness is a concept difficult to define consistently , the proP. fluorescens and tradeoff-breaking deviations. In Section 3, we clarify the conditions under which a fitness-decoupling observation (between incommensurable fitness measures) could, in principle, be made\u2014using a simple tradeoff model between trait values, we find that one condition is that the optimal trait values for counterfactual particle fitness and whole life cycle fitness are different. In Sections 4 and 5, we show that if the tradeoff is relaxed through the existence of rare tradeoff-breaking events, fitness-decoupling observations may not hold for whole evolutionary trajectories. We suggest that an evolutionary trajectory can be divided into three phases: a first phase during which collectives are formed, followed by an optional second fast-paced phase during which optimisation \u2018on the tradeoff\u2019 occurs, and, finally, a slower phase driven by rare tradeoff breakings. Tradeoff-breaking mutations might result in lineages where both counterfactual and whole life cycle fitnesses are increased compared to the ancestor. We propose that departures in collective-level entities from ancestral tradeoffs\u2014tradeoff-breaking points\u2014are a mark of a key moment in ETIs and might be used to characterise them. This proposal is compatible with recent models found in the literature on ETIs\u2014namely, the ecological scaffolding model and the orescens .Our tradeoff-breaking framework could also serve as a springboard to generate new hypotheses. The study of tradeoff breaking requires estimating changes between the pre-ETI ancestral traits and post-ETI derived traits. Access to ancestral traits can be gained in multiple ways, depending on the system studied. The first is through phylogenetic reasoning, such as by reconstructing the sequence of ecological and phenotypic trait evolution during the evolution of cyanobacterial multicellularity as in . The secFitness-centred approaches to ETIs may have reached their limits. We propose to refocus the problem on tradeoffs between traits, thereby bypassing the difficulties inherent to fitness comparisons. The advantages of this move are multiple and range from allowing better experimental accessibility to producing a more mechanistic theory. The way the collective-level context affects the constraints that link traits together is the linchpin of our framework. In particular, we argue that tradeoff-breaking events represent a mark of significant evolutionary innovation towards individuality at the higher level that might be missed by fitness-centred approaches. This article makes two important, independent contributions to the multicellularity/major transitions literature. First, it sheds light on the concept of \u2018fitness decoupling\u2019, providing a strong mathematical foundation for the claim that the fitnesses of cells and groups cannot be formally decoupled during an evolutionary transition in individuality. Second, the article proposes using the evolution of tradeoff-breaking traits as an indication that an evolutionary transition has occurred. This dovetails well with existing fitness-based approaches, extending the toolkit of those studying major evolutionary transitions. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Patricia Wittkopp as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Mar\u00eda Rebolleda-G\u00f3mez (Reviewer #1); Matthew Herron (Reviewer #2); Corina E Tarnita (Reviewer #3).Thank you for submitting your article \"Beyond Fitness Decoupling: Tradeoff-breaking during Evolutionary Transitions in Individuality\" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Fitness decoupling is an important topic that has created a lot of confusion in the field over the last decade and a half. Work clarifying this topic, and moving beyond looking at fitnesses, is welcome and important. The authors are well-poised to write this paper, bringing together real strength in the philosophy of biology, mathematical modeling, and experimental evolution of multicellularity. The modeling framework used here is useful, showing that cell and group-level fitnesses cannot be decoupled when they are both assessed over the same time scale. I agree with referee 3 that it is great to see the authors wrestling with real world data, and not relying only on simple mathematical models, because a key piece of the utility of this kind of theory will rely on people being to apply it to empirical systems. I also agree with the authors that traits, not just fitnesses, can be useful in determining when an ETI has occurred. Moving into traits, the authors propose to focus on trade-off breaking as an indicator that an ETI has occurred, which is creative and potentially quite useful, though further clarification will be required.The referees raised several important points that will need to be addressed in the revision.1) As Referee 1 points out, the idea that cell and group-level fitnesses cannot be formally decoupled was noted by Michod in his 2005, 2014, and 2020 papers. While I do not think that Michod has been entirely clear on this, his 2020 paper makes this point unambiguously. I think there's room for grace here with respect to what Michod thinks, and I'd like to avoid un-necessary strawman arguments. I don't think we should conflate Michod's views on this with Okasha's, and should give credit to Michod for accepting that cell and group-level fitness are inexorably correlated. But perhaps more importantly, the conclusion that in simple mathematical models cell and group-level fitnessess cannot be decoupled is not itself novel: Dr. Bourrat himself published this result as the central message of his 2015 paper on fitness decoupling . So I'd recommend modifying the language in the manuscript here around whether this is a novel finding .2) I think the main contribution of this paper is the shift from focusing on fitnesses to traits. Specifically, the authors advocate for the concept of trade-off breaking as an indication that an ETI has occurred. I like this idea, in part because it is far easier to measure traits than fitnesses in real organisms. However, the referees have raised some important issues with this idea that should be addressed during revision. As Referees 1 and 2 pointed out, organisms evolve trade-off breaking traits all the time without affecting biological individuality. I thought Referee 1's example of the zebra herd foraging trait was quite effective here, illustrating how collective-behaviors that break trade-offs may be not meaningfully affect the level of biological individuality.Most importantly, I'd like to see this idea developed a bit more to describe the conditions under which trade-off breaking is an indication of an ETI. Specifically, I'd like the authors to clarify: (1) Is every case of trade-off breaking a case of an ETI? (2) If not, are all ETIs characterized by trade-off breaking? Or (3) Is trade-off breaking one possible way of driving an ETI, but not a universal trait of ETIs? (4) If the latter is true, and trade-off breaking is not necessary or sufficient to distinguish an ETI, then what else is needed to distinguish an ETI? The authors have made a compelling case that the fitness decoupling literature has significant problems, but it is not yet clear that trade-off breaking is the solution.To clarify my thinking on this a bit more: trade-off breaking seems like a useful tool when there are clear trade-offs that can be ameliorated by multicellular adaptation . But what about a case of simple multicellular adaptation in the absence of pre-existing trade-offs? For example, consider a simple organism like a snowflake yeast that is obligately multicellular, reproducing by group fragmentation. Over generations, cells evolve novel traits that improve the fitness of cells living in multicellular groups, but these traits reduce the growth rate of cells. It's easy apply the counter-factual approach and see how the same traits increase the fitness of groups and the cells living in these groups, while decreasing the counterfactual fitness of solitary cells (they grow slower). To me, this is a straightforward use of the counterfactual fitness approach, in line with what Michod suggests in their 2020 paper, and is indicative of an ETI in progress. This isn't hypothetical, it's precisely what we see in snowflake yeast . I don't think our model system is very unusual here, and I would think that this kind of simple group-level adaptation is common during the transition to multicellularity.Thus, the 3 referees and I agree that it is essential that the authors clarify whether trade-off breaking is a universal trait of ETIs, or is one potential diagnostic trait that an ETI has occurred. If it is not universal , then it would be useful to outline the conditions under which one might want to use a trait-based argument vs. a fitness-based argument . The referees and I generally thought that trade-off breaking could be a useful tool for determining that an ETI has occurred, even if it is not a diagnostic feature of all ETIs.3) Referee 3 points out that the model developed in this paper is not itself novel, being similar in structure and analysis to her prior work. Please cite/discuss this prior work appropriately.Reviewer #2 (Recommendations for the authors):Figure 2 would be more useful if it included a comparison of fitnesses, for example between different genotypes .Reviewer #3 (Recommendations for the authors):Let me start by saying the I am sympathetic to the authors' position on fitness decoupling, which I've always found to be so contrived as to be either non-sensical or meaningless, depending on how one defines fitness. I also quite like the simple proposals of sections 3 and 4 and appreciate that the authors engaged both with empirical evidence and with ways to see existing theoretical models through the lens of the tradeoff-breaking framework. This being said I have a few concerns that I would like addressed:1. the writing is fairly dense and convoluted: it took me several hours to go through the manuscript, and I'm still unsure that I'm fully grasping all details. If this is going to be engaged with by a broad audience, and especially a non-specialist one, the authors need to make more of an effort to clean up, streamline, and provide intuition. For instance,\u2013 I can't make sense of Figure 4 I don't know which ones of the dots are particles; I don't know which is the focal particle whose fitness we are tracing, I don't understand what the vertical dotted lines are, and I don't know why there are multiple horizontal lines, seemingly associated with both blue and orange dots. This lack of clarity around exactly how to measure f2 and F propagate, making it sort of hard to rigorously understand what the authors meant f2 to be in various of the examples .\u2013 I found it tedious to go through all the math derivations and references to theorems in the main text, with, often, little to nothing by way of biological intuition that would help ground some of those results. When some biological intuition is provided, it often left me wanting some more explanation. For instance, in discussing the example of section 3.3, the authors state: \"Note importantly that the opposite directions of the dynamics of F and f2 is a consequence not a cause of the underlying tradeoff mechanism that drives the evolutionary dynamics.\" It would be great if the authors could explain why this is; and, in general, this whole example could use a bit of biology on the very bare theoretical bones. For example, it seems that all collectives of size i >= 2 have the same survival probability and the same probability to grow to the next class size; if the authors can't contextualize this with a bit more biology, at the very least it would be nice to have a sentence that simply states this assumption in words. A non-mathematical reader might have a hard time inferring that from the fact that the expressions of p_i and g_i don't depend on i.\u2013 Although I understand the tradeoff in Black et al., I'm still not entirely sure I understand the tradeoff in the model in Box 2: is there supposed to be a tradeoff linking \u03b2 and q? It seems that \u03b2 is a growth rate whose increase would be good for both germ cells and soma cells. In both Boxes 2 and 3 it would be helpful to have it more clearly spelled out which modeling aspects are new to this paper and which ones are taken from the original papers, which were themselves modeling papers.\u2013 Simultaneously, some things that are simple to state intuitively, are presented in a somewhat convoluted and tedious way: e.g., the 1st paragraph of Box 1 and associated Figure B1a make overly complicated the story that any reader would appreciate with a simple example: a bacterium that doubles every hour will make more of itself in one year than a mouse that \"doubles\" every year . I also think that some such intuitive discussions should come earlier, as the authors set up the discussion of fitness in the main text .\u2013 In general, I'm not convinced that some of the mathematical detail needs to be in the main text as opposed to a detailed methods section.2. The model in Section 2 is not new. Tarnita et al. 2013 J. Theor Biol (Evolutionary construction by staying together and coming together) have used the same model to explore the origin of multicellularity. Even though that paper looks at competition of such collectives against the solitary ancestor, the ideas, setup and analyses are nearly identical to what the authors do in section 2.3. This brings me to my final and broadest points:\u2013 First, if I understand correctly, in this manuscript Bourrat et al. do not seem to consider competition against an extant unicellular ancestor anymore. But, it seems to me that one of the greatest challenges would've been the persistence of the multicellular lifestyle in the earliest stages, before rare mutations can break tradeoffs and whatnot. It would be useful to at least have some discussion of this early on, so that I can situate where exactly the modeled worlds are in the origin of multicellularity and transition to individuality .\u2013 Second, how should we think of free riders in this framework? They don't seem to be explicitly included in any of the modeling.\u2013 Third, much as in the original Michod thinking, it seems to me that the current framework also sort of assumes that the collective is a sum of what the component cells do and does not consider spontaneous emergent properties that simultaneously confer benefits of group living on the group and also on the parts, such as some degree of division of labor . Such spontaneous benefits could in themselves set the stage for an ETI without requiring fundamental reversals of tradeoffs (even though these of course could follow later). Again, I'm not suggesting that the current framework should be explicitly modified to account for such dynamics, but it would be informative to have at least some discussion of whether the authors believe that tradeoff-breaking by rare mutations is the only way to get an ETI or whether it's one possible thing to look for empirically, but not necessarily the only route.To contextualize this point further, it might be helpful to look at Ulrich et al. 2018 Nature (Fitness benefits and emergent division of labour at the onset of group living) and Brahma et al. 2017 . Both of these look at the evolution of eusociality rather than multicellularity, but the essence of the ETI can be captured by the same model . I'm not aware of similar empirical work in multicellularity, though see Gavrilets 2010 PLoS Comp Biol for theoretical work.eLife. Your revised article has been evaluated by Patricia Wittkopp (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled \"How Tradeoff-Breaking Events Go Beyond Fitness Decoupling During Evolutionary Transitions in Individuality\" for further consideration by The three referees and I have read your revised manuscript, and really appreciate some of the new additions. In particular, there is a lot of enthusiasm for the new Section 5. Everyone found the paper to be thought-provoking and thought that this paper will make an important contribution to understanding ETIs if suitably revised.The main thing we would like to suggest is a more inclusive tone for the paper. I do not think it takes away any of the novelty of the results, and indeed, can strengthen the scholarship of the work. It is critical that you revise the introduction to more fairly reflect Michod et al.'s stated meaning (not the authors' inferred meaning) about the concepts of export of fitness, fitness decoupling, and the counterfactual account. Second, the referees and I do not see why the counterfactual account is not a valid indicator that an ETI has occurred. This does not undercut the utility of your work, showing that trade-off breaking mutations can be an indication of an ETI. But all four of us are convinced that both approaches have merit.Referee 2 has left a detailed review, highlighting five major points. The referees and I agree that it is essential to address all five points with changes to the MS, not arguments for why revision is not necessary. Please respond to their review thoroughly. I have carried forward their five summary points and provided a bit of additional context on some below, reflecting our collective post-review discussion.1. Adequately address Michod and colleagues' \"counterfactual fitness\" interpretation of their own model or give a compelling reason not to in the manuscript.Despite pointing this out in the last revision, the paper still does not treat Michod et al.'s position on fitness decoupling and counterfactual fitness fairly. The paper is framed in such a way that it critiques a straw-man version of Michod et al's position on these topics , which stands in contrast to what Michod and colleagues have explicitly said they mean by these terms in their more recent papers.2. Make a compelling case for the lack of utility of the counterfactual fitness version of fitness decoupling in the manuscript or remove this claim from the manuscript.Given that you are using a Darwinian Individuality approach to define what is an individual , I do not see how one can dismiss the utility of the counterfactual fitness approach. Empirical measures consistent with the counterfactual fitness account can provide insight into whether groups have been acting as Darwinian Individuals. For example, consider a lineage evolving along a pre-existing trade-off between traits that improve the fitness of groups and those that improve the fitness of cells, like those identified in Figure 8's depiction of 'optimization on the trade-off'. Using the example given in this section, consider the mutations that make snowflake yeast cells elongate, which increase group size (and thus fitness) but decrease cell growth rates (and thus fitness). This would be detected in the counterfactual fitness account and would rightly be interpreted as a trait that evolved due to selection acting among groups, not among individual cells, demonstrating that groups are Darwinian Individuals. Trade-offs have not been broken, but nonetheless, groups replicate, have heritable variation in group-level traits, and selection acts on those traits, resulting in adaptations that would not have occurred among unicellular populations. I think we can all agree that in this case, measurements consistent with the counterfactual approach would indicate that groups are a Darwinian Individual according to PGS's criteria, right? On a related note, I couldn't figure out why the MS embraced the 'Ratcheting' model but rejected the counterfactual account since ratcheting is based on the same trade-offs that would be detected by the counterfactual fitness account.However, I think your paper raises very valid limitations on the scope of counterfactual fitness- not all multicellular adaptations will necessarily create a trade-off between fitness in groups and fitness in isolated cells, reverting lineages to unicellularity may often prove impossible, and measuring fitness can be difficult, etc. From my perspective, both the counterfactual fitness account and the trade-off breaking account may be useful indicators that an ETI has occurred. And importantly, I do not think either is necessary or sufficient to show an ETI has occurred without additional information about the traits themselves. They both require context to be informative.Since neither is necessary for an ETI, a lineage may evolve along either route . I could imagine cases where it is much easier to identify trade-off breaking mutations and others where it is easier to identify counterfactual fitness. Having both tools available is a net positive for our field, and I don't see the need to limit ourselves only to tradeoff-breaking mutations. All the more so if trade-off breaking mutations are not necessary or sufficient to show an ETI, making the decision to only consider them somewhat arbitrary.3. Consistently describe what is and is not novel about the finding of incommensurability.This is described clearly in the original review and I have no additional context to add.4. Consistently acknowledge that trade-off breaking is insufficient as a marker for an ETI.As acknowledged in the prior review, trade-off breaking is not sufficient to determine that an ETI has occurred. It is possible to have a trade-off-breaking mutation occur within a single level. The referees and I agree that the MS should be consistent about this limitation and avoid saying the existence of that trade-off breaking mutations mean an ETI has occurred. To this end, it would be very helpful if the authors could identify what additional information one would need to know in order to conclude that a trade-off breaking trait indicates that an ETI has occurred .5. Clarify, in the manuscript, how the dynamics of F and f2 fail to explain the dynamics described in lines 407-417.This is described clearly in the original review and I have no additional context to add.Reviewer #1 (Recommendations for the authors):This paper will be an important read for everyone interested in discussions about fitness, transitions in individuality, and innovations more broadly. It helpfully pushes the conversation away from fitness and into understanding the underlying traits.This revised version frames the argument better within its context in the previous literature, and it does a good job of clarifying central points that were hard to follow in the previous version. Figure 8, the new discussion of the snowflake yeast example, and the added text at the end of the trade-off breaking section add clarity and set the boundaries of what is and what isn't explained by this framework.Reviewer #2 (Recommendations for the authors):The revised manuscript largely fails to adequately address the central criticisms in my review of the original manuscript. Reasons for this are given in the authors' response, but these responses are inadequate for the reasons detailed below.1. In my review of the original manuscript, I suggested that the manuscript did not fully address Michod and colleagues' description of their fitness decoupling models in terms of \"counterfactual\" fitness . The revised version still does not. Counterfactual fitness is not introduced at all until Section 2.3, so the Introduction and much of the Results read as if Michod and colleagues had never clarified that this is how their fitness decoupling models should be understood, and the authors' preferred interpretation, was explicitly rejected by Michod and colleagues, is central to the manuscript's framing. The authors' response gives some (inadequate) rationale for largely ignoring Michod and colleagues' preferred interpretation of their own model, but this rationale is not included in the manuscript. The reason I say the rationale is inadequate is that it privileges the authors' interpretation (what the \"metaphor implies\") over Michod and colleagues' explicit, repeated, published explanations of their own model. If the authors wish to frame the manuscript in terms of a version of Michod and colleagues' model that Michod and colleagues themselves say is mistaken, an adequate justification for doing so should be included in the manuscript.2. In my review of the original manuscript, I said that \"the final assessment (Section 2.3) does not make a compelling case for its central argument, the lack of utility of the fitness decoupling concept.\" The relevant section still does not make a compelling case that the fitness decoupling concept lacks utility. The reason given is \"because they [F and f1/f2] are not commensurable.\" Since commensurability of F and f2 is not part of Michod and colleagues' formulation of fitness decoupling, this is not a compelling reason. As I said in my original review, \"\u2026'because they are not commensurable' does little to explain why the counterfactual interpretation of fitness decoupling 'does little on its own to clarify the process of an ETI,' since commensurability is not a claim that the counterfactual interpretation of fitness decoupling makes.\" The authors' response is inadequate for reasons I have touched on before (privileging the authors' interpretation over Michod and colleagues' explicit description of their own model), and the manuscript has not been modified to address this point.3. The Review Editor points out that \"the conclusion that in simple mathematical models cell and group-level fitness cannot be decoupled is not itself novel\", and the authors' response acknowledges \"this idea has already been developed in the literature\"; however:\u2013 The Abstract reads (lines 34-37) \"Using a mathematical approach, we show this concept to be problematic in that the fitness of particles and collectives can never decouple-calculations of particle and collective fitness performed over appropriate and equivalent time intervals are necessarily the same.\" The Abstract reads as if this were a novel finding of this work, which it is not.\u2013 Similarly, lines 95-97 and 109-110 suggest the novelty of this result.\u2013 Lines 190-191 read \"While fitness decoupling and the export-of-fitness model might seem useful concepts for understanding ETIs, we show formally here that commensurably computed fitnesses cannot generally be decoupled.\" Again, this sounds like a novel result, which it is not. The subsequent sentences provide some context but do not acknowledge that Michod derived this result in 2005. It may be that the authors intend some distinction from prior results as suggested in the authors' response , but this distinction is not clear in the manuscript. What is novel about this result and how it differs from prior results will need to be clarified in the manuscript.4. The authors' response acknowledges that \"tradeoff-breaking events cannot be regarded as sufficient conditions for an ETI\", and the revised manuscript acknowledges (line 654) that \"\u2026a tradeoff-breaking event in and of itself cannot be used as a marker of an ETI.\" However:\u2013 The Abstract (line 42) reads \u201ctradeoff-breaking stands as a useful marker for ETIs,\u201d.\u2013 Line 537 that \"\u2026tradeoff-breaking observations are the mark of significant genetic innovation and, thus, can be used as a hallmark of ETIs,\".\u2013 Lines 417-418 that \"\u2026one genuine marker for an ETI is the capacity for a lineage to break away from such a tradeoff,\".\u2013 Lines 669-670 that \"a general marker of ETIs to use in lieu of fitness decoupling is the emergence of rare tradeoff-breaking mutations.\"\u2013 Lines 687-689 suggest that trade-off breaking is superior to fitness decoupling because \"a fitness decoupling observation is not a reliable marker that an ETI is occurring\". Since trade-off breaking is also not a reliable marker that an ETI is occurring, the point of this contrast is unclear.5. I do not see how the conclusion that \"\u2026relative changes in fitness F and f2 do not explain the dynamics observed\" follows from the rest of the paragraph (lines 407-417).\u2013 For example, \"the opposite directions of the dynamics of F and f2 are a consequence of optimal trait values on a tradeoff being different in different environments, not an inherent relationship between them\" describes Michod's models neatly, with the dynamics of group fitness (F) and counterfactual fitness (f2) being a consequence of optimal trait values being different in different environments (within the group versus outside the group). This seems to be an example, not a counterexample, of relative changes in fitness in F and f2 explaining the dynamics observed.eLife cycle fitness F increases\" again seems to be an example of relative changes in fitness in F and f2 explaining the dynamics observed, but it is presented as a counterexample. Trade-offs and fitness decoupling in the counterfactual sense are not mutually exclusive explanations; the fact that trade-offs are involved, as in Michod's fitness decoupling models, does not mean fitness decoupling in the counterfactual sense is not occurring.\u2013 In the snowflake yeast example, \"f2 decreases\u2026when wholRecommendations for authorsThese have a 1:1 correspondence with the items above1. Adequately address Michod and colleagues' \"counterfactual fitness\" interpretation of their own model or give a compelling reason not to in the manuscript.2. Make a compelling case for the lack of utility of the counterfactual fitness version of fitness decoupling in the manuscript or remove this claim from the manuscript.3. Consistently describe what is and is not novel about the finding of incommensurability.4. Consistently acknowledge that trade-off breaking is insufficient as a marker for an ETI.5. Clarify, in the manuscript, how the dynamics of F and f2 fail to explain the dynamics described in lines 407-417.Reviewer #3 (Recommendations for the authors):I really enjoyed reading the revised manuscript, especially the newly-added Section 5. I think everything is much clearer now, and although parts and ideas have been discussed elsewhere, bringing them together in a coherent conceptual and theoretical framework is novel and useful. It certainly got me thinking in directions that I hadn't previously considered.I have only one final comment. I appreciate the idea of separating the stage of collective formation from the subsequent ones and the authors' clarification that they focus on the latter (and hence that they choose not to focus on the competition with unicellular ancestors). But what if some collective property is needed precisely to overcome the competition with the unicellular ancestor? That's certainly how the experiments in Ulrich et al. 2018 came about: because in a model similar to the one in Tarnita et al. 2013 but published in the context of eusociality in Nowak et al. 2010, we predicted that a necessary condition for eusociality to evolve was for per capita fitness to get a drastic boost at very small, incipient group sizes, without any time for additional mutations. And in 2010 we had no clue what that could be. In 2018 we showed experimentally that spontaneous, rudimentary division of labor could do it.In other words, one might sometimes be in a situation where Stage 2 is altogether skipped precisely because only collectives that snap right into some form of Stage 3 already during stage 1 can outcompete the ancestor. Put differently, Stage 1 = Stage 3.Again, it seems to me that there are many ways to skin a cat and the authors are making it clear now that rare mutation doesn't have to be the only route to an ETI, but that some overarching framework based on traits and tradeoffs might offer a more productive way to look broadly at ETIs. I just wanted to alert them to the possibility that tradeoff breaking might not necessarily always occur after Stage 1 (and the associated competition with the ancestor) but sometimes might even occur during it. Unless I'm missing something? Essential revisions:Fitness decoupling is an important topic that has created a lot of confusion in the field over the last decade and a half. Work clarifying this topic, and moving beyond looking at fitnesses, is welcome and important. The authors are well-poised to write this paper, bringing together real strength in the philosophy of biology, mathematical modeling, and experimental evolution of multicellularity. The modeling framework used here is useful, showing that cell and group-level fitnesses cannot be decoupled when they are both assessed over the same time scale. I agree with referee 3 that it is great to see the authors wrestling with real world data, and not relying only on simple mathematical models, because a key piece of the utility of this kind of theory will rely on people being to apply it to empirical systems. I also agree with the authors that traits, not just fitnesses, can be useful in determining when an ETI has occurred. Moving into traits, the authors propose to focus on trade-off breaking as an indicator that an ETI has occurred, which is creative and potentially quite useful, though further clarification will be required.The referees raised several important points that will need to be addressed in the revision.1) As Referee 1 points out, the idea that cell and group-level fitnesses cannot be formally decoupled was noted by Michod in his 2005, 2014, and 2020 papers. While I do not think that Michod has been entirely clear on this, his 2020 paper makes this point unambiguously. I think there's room for grace here with respect to what Michod thinks, and I'd like to avoid un-necessary strawman arguments. I don't think we should conflate Michod's views on this with Okasha's, and should give credit to Michod for accepting that cell and group-level fitness are inexorably correlated. But perhaps more importantly, the conclusion that in simple mathematical models cell and group-level fitnessess cannot be decoupled is not itself novel: Dr. Bourrat himself published this result as the central message of his 2015 paper on fitness decoupling . So I'd recommend modifying the language in the MS here around whether this is a novel finding .Essential revision #1 concerns the novelty of the idea that commensurable measures of fitness cannot be decoupled and our treatment of previous publications in the domain. We do agree with the reviewers that this idea has already been developed in the literature and that a better description of our specific contribution is to add a new formal argument supporting this claim through mathematical modelling and under more general conditions .Our intention is in no way to create strawman arguments. We agree that Michod and collaborators have clarified their position in several publications. In particular, we recognise that they (1) mention that particle and collective fitness must be correlated and (2) judiciously introduce counterfactual fitness to solve this problem. However, we argue that the export-of-fitness metaphor implies that the two quantities between which a fitness transfer occurs are commensurable. As a result, we think that the export-of-fitness model is incompatible with the use of counterfactual fitness (see our response to Reviewer #3\u2019s public review for more details).In light of this comment, we made the following changes to the manuscript:We edited the wording in several places to distinguish the contributions of Michod and Okasha. We note that Okasha relies extensively on Michod and colleagues\u2019 work to propose his three-stage model.In the glossary, we removed the mention of counterfactual fitness being a \u2018recent development\u201d in the export-of-fitness literature.formally its impossibility under reasonable assumptions, which is a novel contribution and not covered in Bourrat (2015).In the introduction and Section 1, we clarified that our argument about fitness decoupling is to show In the discussion, we replaced \u2018Our first main finding is the impossibility of decoupling between commensurable measures of fitness\u2019 with \u2018Our first main finding is a new formal argument cementing the position holding that decoupling between commensurable measures of fitness is impossible\u2019.2) I think the main contribution of this paper is the shift from focusing on fitnesses to traits. Specifically, the authors advocate for the concept of trade-off breaking as an indication that an ETI has occurred. I like this idea, in part because it is far easier to measure traits than fitnesses in real organisms. However, the referees have raised some important issues with this idea that should be addressed during revision. As Referees 1 and 2 pointed out, organisms evolve trade-off breaking traits all the time without affecting biological individuality. I thought Referee 1's example of the zebra herd foraging trait was quite effective here, illustrating how collective-behaviors that break trade-offs may be not meaningfully affect the level of biological individuality.Most importantly, I'd like to see this idea developed a bit more to describe the conditions under which trade-off breaking is an indication of an ETI. Specifically, I'd like the authors to clarify: (1) Is every case of trade-off breaking a case of an ETI? (2) If not, are all ETIs characterized by trade-off breaking? Or (3) Is trade-off breaking one possible way of driving an ETI, but not a universal trait of ETIs? (4) If the latter is true, and trade-off breaking is not necessary or sufficient to distinguish an ETI, then what else is needed to distinguish an ETI? The authors have made a compelling case that the fitness decoupling literature has significant problems, but it is not yet clear that trade-off breaking is the solution.To clarify my thinking on this a bit more: trade-off breaking seems like a useful tool when there are clear trade-offs that can be ameliorated by multicellular adaptation . But what about a case of simple multicellular adaptation in the absence of pre-existing trade-offs? For example, consider a simple organism like a snowflake yeast that is obligately multicellular, reproducing by group fragmentation. Over generations, cells evolve novel traits that improve the fitness of cells living in multicellular groups, but these traits reduce the growth rate of cells. It's easy apply the counter-factual approach and see how the same traits increase the fitness of groups and the cells living in these groups, while decreasing the counterfactual fitness of solitary cells (they grow slower). To me, this is a straightforward use of the counterfactual fitness approach, in line with what Michod suggests in their 2020 paper, and is indicative of an ETI in progress. This isn't hypothetical, it's precisely what we see in snowflake yeast . I don't think our model system is very unusual here, and I would think that this kind of simple group-level adaptation is common during the transition to multicellularity.Thus, the 3 referees and I agree that it is essential that the authors clarify whether trade-off breaking is a universal trait of ETIs, or is one potential diagnostic trait that an ETI has occurred. If it is not universal , then it would be useful to outline the conditions under which one might want to use a trait-based argument vs. a fitness-based argument . The referees and I generally thought that trade-off breaking could be a useful tool for determining that an ETI has occurred, even if it is not a diagnostic feature of all ETIs.Essential Revision #2 concerns the generality of the tradeoff-breaking mechanism for the study of ETIs and asks us to detail our framework in the examples of the zebra herd and the snowflake yeast. We will start by answering the first set of concerns: the example of the zebra herd is addressed in our detailed answer to Reviewer #2 and we will come back to the snowflake yeast later.As pointed out by the reviewers, we hope first and foremost that this manuscript will contribute to shifting the field from focusing on fitness to focusing on traits. We are motivated in this endeavour by our conviction that we, as a field, have reached the limits of what we can learn from fitness-based approaches to ETIs. One of the undeniable appeals of the export-of-fitness model is that it can be applied in the abstract without taking into account the likely very particular underlying constraints of the system. However, this constitutes a double-edged sword\u2014when the assumptions of the export-of-fitness model are not met, the theory cannot account for the dynamics observed. This is so because, to expect a dynamic in which counterfactual fitness decreases and whole life cycle fitness increases, one must assume three things. First, the two quantities must depend upon the same traits; second, these two quantities are maximised for different trait values; third, the ancestral trait values (as the product of previous selection on the unicellular ancestor) correspond to the maximum of counterfactual fitness within the constraints of the ancestor. In contrast, if traits and tradeoffs are the centre of the theory, not only are all previous results about counterfactual fitness and ratcheting valid but also tradeoff-breaking events\u2014which occur when the three above assumptions do not hold\u2014can be accommodated readily.However, the reviewers are correct in their assessment that we did not discuss sufficiently the generality of the fitness tradeoff-breaking approach. First, if one takes the idea of tradeoff breaking in its broadest sense\u2014that is, the emergence of rare mutations that overcome the usual constraints bearing upon a set of traits\u2014we agree with the reviewers that tradeoff-breaking events cannot be regarded as sufficient conditions for an ETI. This is so because tradeoff breaking might occur without being accompanied by a change in the level of individuality. However, we suggest that it could tentatively be proposed as a necessary condition for the evolution of individuality. Several models of ETIs involve a first step of collective formation followed by collective level changes \u2014that is, a way to assemble lower-level entities in stable collectives\u2014and secondary changes that cement the individuality of the new collectives. Tradeoff-breaking could be used as a practical way of assessing this second step, characterising what differs from collectives exhibiting low or little interaction and, thus, forming a necessary condition for a complete ETI.Pseudomonas system). From then, the observed change in cell shape fits well with the \u2018optimisation on the tradeoff\u2019, as we conceptualise it. As explored by Bozdag et al. (2021), the bigger clusters suffer from poorer oxygenation of the cells reducing the cell growth rate, which means that the increase in cluster size is accompanied by a reduction in within-collective particle growth rate (f1). A more delicate experimental question would be to establish whether the following recorded innovations constitute a proper \u2018tradeoff breaking\u2018 as we describe it in the article. One way to explore this would be to combine the existing data on cell and cluster shape , physical models of cluster volume , simple sedimentation models , and within-flake cell growth models and give the snowflake system the same transversal treatment that we give the Pseudomonas system in this article.The question of the snowflake yeast system is of particular interest to us. However, to treat the topic with the respect it deserves would require a detailed treatment that would go beyond the scope of this article. Nonetheless, we can make the following conjecture: the first snowflake mutation (ACE2) acts as the \u2018collective formation\u2018 mutation makes clearer that tradeoff breaking is the second (or third) step in an adaptive scenario that involves \u2018collective formation\u2019, \u2018optimisation on the tradeoff\u2019 and \u2018tradeoff breaking\u2019. To make this effective, we have added a new figure , (2) discussed the generality of the tradeoff approach, and (3) contextualised our approach within the broader picture of ETIs, linking it to the existing framework.We now include the snowflake yeast system in our examples (in Section 5).3) Referee 3 points out that the model developed in this paper is not itself novel, being similar in structure and analysis to her prior work. Please cite/discuss this prior work appropriately.Essential Revision #3 concerns the novelty of the modelling approach. We do agree that we are not the first to have proposed to model proto-collectives using size classes and transition between those size classes . The model can be traced back to an extension of Leslie matrices and their generalisation as class-based Matrix population models (Caswell 1989). The literature on this topic already makes use of Perron-Frobenius theory to make asymptotic predictions.Tarnita et al. (2013) present a valuable treatment of the problem using ordinary differential equations and focus on competition with the single-cell counterpart, which we ignored for the purpose of this article. Pichugin et al. (2017) uses a similar dominant eigenvalue method (as we did) to treat the problem of complex fragmentation modes .In light of these comments, we have better situated our modelling approach with respect to the existing literature by making the following changes:We added the following references to the paragraph introducing the model (Section 3): Leslie (1945), Caswell (1989), Tarnita (2013), and Pichugin (2017).We discussed in further detail the fact that we ignore competition with the unicellular ancestor as studied in Tarnita et al. (2013) (Section 5).Reviewer #2 (Recommendations for the authors):Figure 2 would be more useful if it included a comparison of fitnesses, for example between different genotypes .We have decided not to change Figure 2, as it is introduced in Section 1 where we have not yet presented the idea that fitnesses measured at the particle and collective level are necessarily equal. We believe this could potentially confuse the reader.Reviewer #3 (Recommendations for the authors):Let me start by saying the I am sympathetic to the authors' position on fitness decoupling, which I've always found to be so contrived as to be either non-sensical or meaningless, depending on how one defines fitness. I also quite like the simple proposals of sections 3 and 4 and appreciate that the authors engaged both with empirical evidence and with ways to see existing theoretical models through the lens of the tradeoff-breaking framework. This being said I have a few concerns that I would like addressed:1. the writing is fairly dense and convoluted: it took me several hours to go through the manuscript, and I'm still unsure that I'm fully grasping all details. If this is going to be engaged with by a broad audience, and especially a non-specialist one, the authors need to make more of an effort to clean up, streamline, and provide intuition. For instance,\u2013 I can't make sense of Figure 4 I don't know which ones of the dots are particles; I don't know which is the focal particle whose fitness we are tracing, I don't understand what the vertical dotted lines are, and I don't know why there are multiple horizontal lines, seemingly associated with both blue and orange dots. This lack of clarity around exactly how to measure f2 and F propagate, making it sort of hard to rigorously understand what the authors meant f2 to be in various of the examples .We have extensively reworked Figure 4 to make this clearer.\u2013 I found it tedious to go through all the math derivations and references to theorems in the main text, with, often, little to nothing by way of biological intuition that would help ground some of those results. When some biological intuition is provided, it often left me wanting some more explanation. For instance, in discussing the example of section 3.3, the authors state: \"Note importantly that the opposite directions of the dynamics of F and f2 is a consequence not a cause of the underlying tradeoff mechanism that drives the evolutionary dynamics.\" It would be great if the authors could explain why this is; and, in general, this whole example could use a bit of biology on the very bare theoretical bones. For example, it seems that all collectives of size i >= 2 have the same survival probability and the same probability to grow to the next class size; if the authors can't contextualize this with a bit more biology, at the very least it would be nice to have a sentence that simply states this assumption in words. A non-mathematical reader might have a hard time inferring that from the fact that the expressions of p_i and g_i don't depend on i.We have now provided further explanation and a biological example to address this point.\u2013 Although I understand the tradeoff in Black et al., I'm still not entirely sure I understand the tradeoff in the model in Box 2: is there supposed to be a tradeoff linking \u03b2 and q? It seems that \u03b2 is a growth rate whose increase would be good for both germ cells and soma cells. In both Boxes 2 and 3 it would be helpful to have it more clearly spelled out which modeling aspects are new to this paper and which ones are taken from the original papers, which were themselves modeling papers.g(T). This tradeoff is broken by mutations on q because somatic cells improve dispersal (if \u03c1>0) while reducing less the growth rate than mutations on \u03b2 .In the model from Black et. al, the trait \u03b2 is more akin to the underlying resource allocation trait that can be seen in other tradeoffs models, such as the trait \u03b8 from Section 3 of the main text. It corresponds to the linear abscissa on a 2D tradeoff line. The tradeoff exists in the sense that a collective cannot at the same time have a high initial growth rate \u03b2 and reach a high collective size at the moment of dispersal, We made the following edits to the boxes:We explicitly mentioned the parallel between \u03b2 . and \u03b8 (in the main text) to clarify the tradeoff involved in this example.We made more precise references to the original equations,We added a sentence: \u2018we now recast the model within our framework\u2019 to delineate the point where we start to make an original contribution.\u2013 Simultaneously, some things that are simple to state intuitively, are presented in a somewhat convoluted and tedious way: e.g., the 1st paragraph of Box 1 and associated Figure B1a make overly complicated the story that any reader would appreciate with a simple example: a bacterium that doubles every hour will make more of itself in one year than a mouse that \"doubles\" every year . I also think that some such intuitive discussions should come earlier, as the authors set up the discussion of fitness in the main text .We have now streamlined the first section of Box 1 and provided a biological intuition using an example involving bacteria.\u2013 In general, I'm not convinced that some of the mathematical detail needs to be in the main text as opposed to a detailed methods section.We have decided to keep those details in the main text. However, we provided some signposting at the beginning of Section 2 so that the reader willing to skip the mathematical details can easily do so.2. The model in Section 2 is not new. Tarnita et al. 2013 J. Theor Biol (Evolutionary construction by staying together and coming together) have used the same model to explore the origin of multicellularity. Even though that paper looks at competition of such collectives against the solitary ancestor, the ideas, setup and analyses are nearly identical to what the authors do in section 2.Thanks for bringing this to our attention. See our response to Essential Revision #3.3. This brings me to my final and broadest points:\u2013 First, if I understand correctly, in this manuscript Bourrat et al. do not seem to consider competition against an extant unicellular ancestor anymore. But, it seems to me that one of the greatest challenges would've been the persistence of the multicellular lifestyle in the earliest stages, before rare mutations can break tradeoffs and whatnot. It would be useful to at least have some discussion of this early on, so that I can situate where exactly the modeled worlds are in the origin of multicellularity and transition to individuality .Our main goal is to define a unified framework where different fitness measures that have been used experimentally can be described and their relationship studied in a mechanistic way. In particular, we want to be able to avoid making reference to the export-of-fitness metaphor, on which we share the reviewer\u2019s opinion.The paper assumes that some early \u2018collective formation\u2019 mutation, or environmental change has already happened and focuses on the early evolutionary dynamics of the proto-multicellular organisms. It also assumes that nascent multicellularity will be constrained by tradeoff inherited from the unicellular ancestors, and that underlying constraints explain in part what has been described as fitness decoupling. However, we advocate that this pattern does not hold as soon as tradeoff-breaking mutations arise .In light of this comment, we have added a sentence at the beginning of Section 2 (where we present our model) to state that we do not study the potential competition between particles and protocollectives, as done in Tarnita et al. (2013). We return to this point in Section 5, which also better situates our work with respect to the different phases of an ETI.\u2013 Second, how should we think of free riders in this framework? They don't seem to be explicitly included in any of the modeling.Free-riders, or cheaters, are not the focus of this model, but could be addressed if necessary using an additional layer of game-theory modelling. We now explicitly mention this at the beginning of Section 2.\u2013 Third, much as in the original Michod thinking, it seems to me that the current framework also sort of assumes that the collective is a sum of what the component cells do and does not consider spontaneous emergent properties that simultaneously confer benefits of group living on the group and also on the parts, such as some degree of division of labor . Such spontaneous benefits could in themselves set the stage for an ETI without requiring fundamental reversals of tradeoffs (even though these of course could follow later). Again, I'm not suggesting that the current framework should be explicitly modified to account for such dynamics, but it would be informative to have at least some discussion of whether the authors believe that tradeoff-breaking by rare mutations is the only way to get an ETI or whether it's one possible thing to look for empirically, but not necessarily the only route.We are interested in the same topic as the reviewer here: how the complexity of the repertoire of the unicellular ancestor affects the early collectives. We think that focusing on traits (and tradeoffs) instead of fitness is a necessary step in addressing this question. We do recognise that phenotypic plasticity could play a driving role in this phenomenon, leading to apparent tradeoff breaking without requiring mutations. We have included this possibility in a new paragraph within section 5 (beginning with \u2018Yet another possibility not studied in this article is\u2026\u2019).To contextualize this point further, it might be helpful to look at Ulrich et al. 2018 Nature (Fitness benefits and emergent division of labour at the onset of group living) and Brahma et al. 2017 . Both of these look at the evolution of eusociality rather than multicellularity, but the essence of the ETI can be captured by the same model . I'm not aware of similar empirical work in multicellularity, though see Gavrilets 2010 PLoS Comp Biol for theoretical work.Ulrich et al. (2018) and Tarnita et al. (2013) are now cited. We also have cited Ispolatov et al. (2012), which is relevant in this context.The three referees and I have read your revised manuscript, and really appreciate some of the new additions. In particular, there is a lot of enthusiasm for the new Section 5. Everyone found the paper to be thought-provoking and thought that this paper will make an important contribution to understanding ETIs if suitably revised.The main thing we would like to suggest is a more inclusive tone for the paper. I do not think it takes away any of the novelty of the results, and indeed, can strengthen the scholarship of the work. It is critical that you revise the introduction to more fairly reflect Michod et al.'s stated meaning (not the authors' inferred meaning) about the concepts of export of fitness, fitness decoupling, and the counterfactual account. Second, the referees and I do not see why the counterfactual account is not a valid indicator that an ETI has occurred. This does not undercut the utility of your work, showing that trade-off breaking mutations can be an indication of an ETI. But all four of us are convinced that both approaches have merit.Referee 2 has left a detailed review, highlighting five major points. The referees and I agree that it is essential to address all five points with changes to the MS, not arguments for why revision is not necessary. Please respond to their review thoroughly. I have carried forward their five summary points and provided a bit of additional context on some below, reflecting our collective post-review discussion.Thank you for your detailed report. Our response to the comments is written in purple below. We would like to note at the outset, however, that we do not see the counterfactual account, in and of itself, as a problem. Our disagreement with the literature is limited to the concepts of export, decoupling, and transfer of fitness: we believe that the counterfactual account, while fruitful, does not satisfactorily address their limits. This response letter and the changes we have made to the manuscript aim to make this point unambiguously clear.To address the comments from Reviewer 3 and the Reviewing Editor, we have pasted each of their five points together and responded to both in a single response (in purple below). Please find enclosed a new version of the manuscript, edited in light of your comments.1. Adequately address Michod and colleagues' \"counterfactual fitness\" interpretation of their own model or give a compelling reason not to in the manuscript.Despite pointing this out in the last revision, the paper still does not treat Michod et al.'s position on fitness decoupling and counterfactual fitness fairly. The paper is framed in such a way that it critiques a straw-man version of Michod et al's position on these topics , which stands in contrast to what Michod and colleagues have explicitly said they mean by these terms in their more recent papers.Thank you for these comments, which have led us to revise the way the paper is framed. It was not at all our desire to represent ideas expressed by others unfairly. Likewise, it was not our intent to attack a strawman version of the export-of-fitness account. Rather, we aim to address this concept seriously and follow through with what it implies. We sincerely regret not having been able to convince the referee of our good faith.We must stress that, in principle, we have no disagreement with the counterfactual approach proposed by Shelton and Michod (2014), which was already presented more implicitly in some earlier work by Michod and colleagues. However, our main contention is that it is not compatible with the \u2018export-of-fitness\u2019 account. This was stated in Line 715 of the second submission: \u2018The method described by Shelton and Michod (2020), once separated from the export-of-fitness model, would prove useful here\u2019. Here is our rationale why this is so.The notion of counterfactual fitness stands on its own without having to be tied to fitness decoupling/transfer/export. This point can be vindicated from the work of the proponents of the counterfactual approach. In their 2014 article, Shelton and Michod mention neither fitness transfer nor fitness decoupling, thus leaving unresolved the connection between counterfactual fitness and notions of fitness decoupling, transfer or export. The same applies to the follow-up article by Leslie et al. (2017). The connection between the \u2018fitness decoupling\u2019 account and \u2018counterfactual fitness\u2019 was only later made explicit by Shelton and Michod (2020), where the authors state (in the abstract) that \u2018during an ETI, fitnesses at the two levels, group and individual, become decoupled, in the sense that fitness in a group may be quite high, even as counterfactual fitness goes to zero.\u201dThus, it does appear that Michod and colleagues have proposed two versions of the export-of-fitness account. To our knowledge, they have not explicitly stated that one replaced the other . Regardless of whether they have been explicit, in the interest of presenting a complete picture of the problem, we have decided to present and discuss both versions.actually be decoupled, as was also appreciated by Shelton and Michod (2014). To this end, we prove mathematically that these fitnesses are equal for a wide range of models .The earlier account was not explicit about the counterfactual nature of fitness and can be (and has been) interpreted as relating two \u2018actual\u2018 fitnesses. We oppose this interpretation of fitness transfer/export/decoupling because actual cell and collective fitness cannot not exist\u2014thus, there is nothing material that can be transferred from a counterfactual to an actual quantity. Second, since fitness is not a conserved quantity in the model, there is no total fitness that could be distributed and transferred between potential and actual fitnesses, as is the case for energy in physics. Nevertheless, we recognise that counterfactual quantities are valuable for other purposes.The second account explicitly refers to \u2018counterfactual\u2018 fitness. We oppose this interpretation of fitness transfer/export/decoupling because there is no meaningful \u2018transfer\u2018 possible between counterfactual and actual quantities. We justify this point via two arguments. First, by definition, a counterfactual fitness does We think that making these points is not superfluous because the metaphor of fitness transfer is difficult to apply to experimental systems and has been used in inconsistent ways. In some of his most recent writings, Michod seems to agree that the straightforward interpretation of the term \u2018transfer\u2019 raises difficulties: \u2018speaking of fitness \u201ctransfer\u201d may suggest that fitness is a conserved quantity in the model which it is not\u2019 . As an example, when discussing fitness transfer, Shelton and Michod (2020) discuss parameters in Hamilton\u2019s rule in the following way: \u2018the evolution of altruism transfers fitness from the level of the cells to the level of the group\u2019. They reason that this is so because the cost of altruism (C \u2018units of fitness\u2019 in Hamilton\u2019s rule) is \u2018subtracted from the cell level\u2019 while its benefits (B \u2018units of fitness\u2019) is \u2018added to members of the group\u2019 . However, we would like to point out that the parameters C and B do not correspond to counterfactual cell and group fitness: they both appear in the expression of collective fitness making them de facto actual rather than counterfactual quantities.That being said, we do agree with the reviewer that our original manuscript failed to make these points clear and could be interpreted, at times, as a rejection of counterfactual fitness in its entirety. To clarify our position, we have now reworked the Introduction to:Present the idea of counterfactual fitness that we credit to Shelton and Michod .Link counterfactual fitness, following Michod and Shelton (2020), to fitness decoupling/transfer.Recognise the general value of counterfactual fitness, but highlight the difficulty when it is used to explain fitness transfer/decoupling.Further, in Section 1, we now provide several paragraphs presenting our rationale regarding why fitness decoupling/transfer, even if understood from a counterfactual perspective, is not adequate.In the Discussion, we have added a section on the usefulness of counterfactual particle fitness in the context of ETIs and how it can be integrated into the tradeoff-breaking framework.2. Make a compelling case for the lack of utility of the counterfactual fitness version of fitness decoupling in the manuscript or remove this claim from the manuscript.Given that you are using a Darwinian Individuality approach to define what is an individual , I do not see how one can dismiss the utility of the counterfactual fitness approach. Empirical measures consistent with the counterfactual fitness account can provide insight into whether groups have been acting as Darwinian Individuals. For example, consider a lineage evolving along a pre-existing trade-off between traits that improve the fitness of groups and those that improve the fitness of cells, like those identified in Figure 8's depiction of 'optimization on the trade-off'. Using the example given in this section, consider the mutations that make snowflake yeast cells elongate, which increase group size (and thus fitness) but decrease cell growth rates (and thus fitness). This would be detected in the counterfactual fitness account and would rightly be interpreted as a trait that evolved due to selection acting among groups, not among individual cells, demonstrating that groups are Darwinian Individuals. Trade-offs have not been broken, but nonetheless, groups replicate, have heritable variation in group-level traits, and selection acts on those traits, resulting in adaptations that would not have occurred among unicellular populations. I think we can all agree that in this case, measurements consistent with the counterfactual approach would indicate that groups are a Darwinian Individual according to PGS's criteria, right? On a related note, I couldn't figure out why the MS embraced the 'Ratcheting' model but rejected the counterfactual account since ratcheting is based on the same trade-offs that would be detected by the counterfactual fitness account.However, I think your paper raises very valid limitations on the scope of counterfactual fitness- not all multicellular adaptations will necessarily create a trade-off between fitness in groups and fitness in isolated cells, reverting lineages to unicellularity may often prove impossible, and measuring fitness can be difficult, etc. From my perspective, both the counterfactual fitness account and the trade-off breaking account may be useful indicators that an ETI has occurred. And importantly, I do not think either is necessary or sufficient to show an ETI has occurred without additional information about the traits themselves. They both require context to be informative.Since neither is necessary for an ETI, a lineage may evolve along either route . I could imagine cases where it is much easier to identify trade-off breaking mutations and others where it is easier to identify counterfactual fitness. Having both tools available is a net positive for our field, and I don't see the need to limit ourselves only to tradeoff-breaking mutations. All the more so if trade-off breaking mutations are not necessary or sufficient to show an ETI, making the decision to only consider them somewhat arbitrary.interpretation of fitness decoupling/transfer from the counterfactual, not that the idea of counterfactual fitness can be relevant for explanation in ETIs. Our framework is compatible with both fitness decoupling and tradeoff-breaking observations and provides them a unified interpretation in terms of a tradeoff. We have reworked our manuscript to avoid suggesting that the second kind of observation should replace the first.We wish to state again that our intention is not to dismiss the counterfactual approach. Instead, we are raising concerns regarding its interpretation in the context of the export-of-fitness (fitness transfer and decoupling) account. As mentioned in our response to the previous point, we find the idea of fitness decoupling/export/transfer inadequate in both its actual and counterfactual versions. Thus, with respect to counterfactual fitness, our main contention is with the To ensure that our position does not come across as against the idea of counterfactual fitness, in addition to the point mentioned in the previous response, we:Have made the point unambiguously that we find the notion of counterfactual fitness useful (throughout the manuscript).We changed the title of Section 2.4 and reworked the paragraph.We have also reworked the snowflake yeast example in Section 3.3, which clarifies the link between actual and counterfactual fitness.3. Consistently describe what is and is not novel about the finding of incommensurability.This is described clearly in the original review and I have no additional context to add.Our work gives a general mathematical proof of the incommensurability of individual and collective fitness. It vindicates the point made previously by Shelton and Michod (2014) ), Bourrat (2015b) and Black et al. 2020 .any population growth model where collective sizes reach a stable distribution, the long-term growth rates of particles and collectives are equal.Our results are more general than the ones previously published because they do not rely on any a priori assumption regarding the relationship between collective and individual fitness, viability, and fecundity , or on writing the model in a MLS1 or MLS2 framework. We show that in To address the reviewer\u2019s points, we have made the following changes to the manuscript:In the abstract, we have replaced the passage mentioned with \u2018Extending and unifying results from the literature, we show that fitness of particles and collectives can never decouple because calculations of particle and collective fitness performed over appropriate and equivalent time intervals are necessarily the same provided the population reach a stable collective-size distribution\u2019.\u201dLines 95\u201397 have been deleted.We have rewritten lines 109\u2013110. We added that our work is \u201c\u2019generalising and unifying results from the literature\u201d and provided references to acknowledge previous work.Lines 190\u2013191 have been deleted..\u2019In Section 2.4, we have now explicitly stated the novelty of the generality of this result: \u2018while these points have been made in the literature (see Section 1) our mathematical proof applies to a larger class of population dynamics models\u2014it is not tied to a MLS1/MLS2 framework and does not require any assumptions regarding the relationship between particle and collective life history traits, such as the usual assumption that collective viability is a linear function .Thank you for pointing out that we were not sufficiently clear about the role of tradeoff breakings in ETIs.We agree that tradeoff breaking can occur within a single level. This was stated in the sentence before the one quoted by the reviewer: \u2018\u201cconstraints breaking\u201d are already considered key events in the evolution of body plans and are expected to be a widespread mechanism for the emergence of novelties\u2019 (L 652). However, we suggest that the observation of tradeoff breaking in the context of an ETI is a relevant event that can be used as a marker of an ETI even if it is not sufficient.Our model aims to be compatible with both fitness decoupling and tradeoff-breaking observations. We have reworked our manuscript to avoid suggesting that the second kind of observation should replace the first; rather, it should come at a later stage of the transition .Regarding the operationalisation of the concept, please note that, in our framework, the tradeoff-breaking mutation is an abstract phenotypic change that could, in principle, be due to ecology, behaviour, or plasticity, in addition to genetic mutations. Developing a general method to select relevant traits and detect tradeoff breaking in any transition in individuality moves beyond the scope of the present manuscript, but is a promising avenue for future research.To clarify these points and address the comments from the Reviewer and Reviewer Editor, we have:Changed the sentence in the Abstract (line 42) from \u201ctradeoff-breaking stands as a useful marker for ETIs\u201d to \u201cThus, when observed in the context of ETIs, tradeoff-breaking events stand as a useful marker for these transitions.\u201dChanged the sentence on line 537 from \u201c\u2026 tradeoff-breaking observations are the mark of significant genetic innovation and, thus, can be used as a hallmark of ETIs,\u201d to \u201cas we advocate that such tradeoff-breaking observations in this context are the mark of significant innovation and, thus, can be used to detect ETIs.\u201dChanged the sentence in lines 417\u2013418 from \u201c\u2026one genuine marker for an ETI is the capacity for a lineage to break away from such a tradeoff,\" to \u201cthe capacity for a lineage to break away from such a tradeoff could be used to detect an ETI.\u201dLines 669\u2013670 have been rewritten and now read as: \u201cFinally, we propose that rare tradeoff-breaking events are a crucial part of ETIs and could be used experimentally for their detection.\u201dLines 687\u2013689 now read: \u201cOur second main result is a general adaptive scenario of ETIs that is compatible with both experimental fitness-decoupling observations and tradeoff-breaking deviations.\u201dGiven more context throughout the manuscript and following the response to the previous comments regarding why tradeoff breaking can be a marker for an ETI.In Section 5, we have:Italicised \u201cin and of itself\u201d in the sentence: \u201cThus, a tradeoff-breaking event in and of itself cannot be used as a marker of an ETI.\u201dContextualised our claims in the following: \u201cHowever, considering a trait in the context of an ETI, where collectives are formed and maintained (phases 1 and 2) and establishing how those collectives change and potentially overcome the constraints that historically bear on particles (phase 3) offers a good empirical handle to study ETIs. Conversely, the lack of any tradeoff-breaking observation in such an empirical system could be used as an indication that the transition is still in its early stages.\u201dTo answer specifically regarding the other information necessary for an ETI, we would need to have clearly identified discrete higher-level entities (or entities in the process of discretisation), what Clarke (2013) calls \u201cmechanisms of demarcation.\u201d We now mention this in Section 5.Finally, to clarify the scope of the tradeoff-breaking model, we have included a sentence at the beginning of Section 3 stating that we consider mutations to be abstract changes in phenotype that could potentially be induced by environmental changes (on top of genetic mutation and phenotypic plasticity).5. Clarify, in the manuscript, how the dynamics of F and f2 fail to explain the dynamics described in lines 407-417.This is described clearly in the original review and I have no additional context to add.F and f2 explain the evolutionary dynamics of the system because there is no actual causal link between the dynamic of the counterfactual fitness f2 and the actual evolutionary dynamics. The value of f2 is entirely irrelevant to the dynamics of trait values. Starting from a given initial condition , the trait would have the same dynamics whether f2 decreases, increases, or even oscillates. The only quantity that matters and explains the trait changes is the gradient of F.There seems to be disagreement between us and Reviewer 3 regarding what constitutes an explanation of dynamics. We do not think that the relative dynamics of f2 might be of interest, but not because its dynamics relative to F explain anything about the dynamics of the trait value theta \u2014rather, because it gives us a hint that there might be a tradeoff here between traits that contribute to collective fitness or would contribute to counterfactual cell fitness.Now, measuring the counterfactual fitness Once again, our point is that the counterfactual fitness approach is valuable, but only when taken for what it is: a counterfactual, and not what it is not (and what it cannot be)\u2014that is, something to be transferred to F. Using it in the context of export-of-fitness is inconsistent, as we have argued in the previous points.We have edited line 407\u2013417 to reflect this . We made a reference to this paragraph in Section 2.4 when we discuss the general usefulness of fitness-decoupling observations.Reviewer #2 (Recommendations for the authors):Recommendations for authorsThese have a 1:1 correspondence with the items above1. Adequately address Michod and colleagues' \"counterfactual fitness\" interpretation of their own model or give a compelling reason not to in the manuscript.This is now done in the Introduction, Section 1, and the Discussion.2. Make a compelling case for the lack of utility of the counterfactual fitness version of fitness decoupling in the manuscript or remove this claim from the manuscript.We have now clarified the link between counterfactual fitness and fitness decoupling/transfer, and why counterfactual fitness does not provide a satisfactory account of fitness decoupling. However, we have stressed that the concept of counterfactual fitness, in and of itself, can be useful in the context of ETIs.3. Consistently describe what is and is not novel about the finding of incommensurability.We have now clarified, from the abstract onwards, what is novel about our result.4. Consistently acknowledge that trade-off breaking is insufficient as a marker for an ETI.We have now done so.5. Clarify, in the manuscript, how the dynamics of F and f2 fail to explain the dynamics described in lines 407-417.These lines have been rewritten.Reviewer #3 (Recommendations for the authors):I really enjoyed reading the revised manuscript, especially the newly-added Section 5. I think everything is much clearer now, and although parts and ideas have been discussed elsewhere, bringing them together in a coherent conceptual and theoretical framework is novel and useful. It certainly got me thinking in directions that I hadn't previously considered.I have only one final comment. I appreciate the idea of separating the stage of collective formation from the subsequent ones and the authors' clarification that they focus on the latter (and hence that they choose not to focus on the competition with unicellular ancestors). But what if some collective property is needed precisely to overcome the competition with the unicellular ancestor? That's certainly how the experiments in Ulrich et al. 2018 came about: because in a model similar to the one in Tarnita et al. 2013 but published in the context of eusociality in Nowak et al. 2010, we predicted that a necessary condition for eusociality to evolve was for per capita fitness to get a drastic boost at very small, incipient group sizes, without any time for additional mutations. And in 2010 we had no clue what that could be. In 2018 we showed experimentally that spontaneous, rudimentary division of labor could do it.In other words, one might sometimes be in a situation where Stage 2 is altogether skipped precisely because only collectives that snap right into some form of Stage 3 already during stage 1 can outcompete the ancestor. Put differently, Stage 1 = Stage 3.Again, it seems to me that there are many ways to skin a cat and the authors are making it clear now that rare mutation doesn't have to be the only route to an ETI, but that some overarching framework based on traits and tradeoffs might offer a more productive way to look broadly at ETIs. I just wanted to alert them to the possibility that tradeoff breaking might not necessarily always occur after Stage 1 (and the associated competition with the ancestor) but sometimes might even occur during it. Unless I'm missing something?Thank you for your feedback\u2014we are glad that our revisions added clarity. We agree that Stage 2 can be skipped. This is included in Figure 8 as Route 2 and also in the section on the importance of phenotypic plasticity for a primordial form of division of labour. We have modified the relevant part of Section 5 to clarify that Stage 3 can immediately follow Stage 1.It must be noted that since the focus of this article has been predominantly on fitness-decoupling observations, we have explained Route 1 in more detail. Nevertheless, Route 2 is very interesting and might, in fact, be widespread in nature."} +{"text": "Breast cancer detection largely relies on imaging characteristics and the ability of clinicians to easily and quickly identify potential lesions. Magnetic resonance imaging (MRI) of breast tumors has recently shown great promise for enabling the automatic identification of breast tumors. Nevertheless, state-of-the-art MRI-based algorithms utilizing deep learning techniques are still limited in their ability to accurately separate tumor and healthy tissue. Therefore, in the current work, we propose an automatic and accurate two-stage U-Net-based segmentation framework for breast tumor detection using dynamic contrast-enhanced MRI (DCE-MRI). This framework was evaluated using T2-weighted MRI data from 160 breast tumor cases, and its performance was compared with that of the standard U-Net model. In the first stage of the proposed framework, a refined U-Net model was utilized to automatically delineate a breast region of interest (ROI) from the surrounding healthy tissue. Importantly, this automatic segmentation step reduced the impact of the background chest tissue on breast tumors' identification. For the second stage, we employed an improved U-Net model that combined a dense residual module based on dilated convolution with a recurrent attention module. This model was used to accurately and automatically segment the tumor tissue from healthy tissue in the breast ROI derived in the previous step. Overall, compared to the U-Net model, the proposed technique exhibited increases in the Dice similarity coefficient, Jaccard similarity, positive predictive value, sensitivity, and Hausdorff distance of 3%, 3%, 3%, 2%, and 16.2, respectively. The proposed model may in the future aid in the clinical diagnosis of breast cancer lesions and help guide individualized patient treatment. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a new functional imaging technique used to assess the physiological properties of the microvascular system in lesions and tissues. This technique is based on the acquisition of baseline images before enhancement as well as consecutive multiperiod high-temporal resolution images after intravenous contrast agent injection. DCE-MRI relies on the calculation of the MRI signal intensity over time and obtains semiquantitative or quantitative parameters that reflect the dynamic enhancement characteristics of the contrast agent in the tissue of interest. In this sense, the perfusion of a lesion is directly represented by the shape of the time-resolved signal intensity curve that represents the dynamic enhancement pattern. Semiquantitative parameters are those derived from the enhancement curve of the tissue for its visual analysis, such as maximum slope, peak value, and area under the curve (AUC). Quantitative parameters refer to contrast-related microcirculatory parameters computed using pharmacokinetic models. Compared with conventional MRI methods, DCE-MRI can not only obtain information about the morphological characteristics of a lesion but also reflect physiological changes in the lesioned tissue.Automatic tissue segmentation is paramount for the accurate computer-aided clinical diagnosis of breast cancer using MR-based imaging techniques. Historically, MRI has proven advantageous in detecting breast cancer compared to other imaging methods such as X-ray mammography, as such images effectively provide detailed tissue characteristics that include the invasive range, texture characteristics, structural status, and voxel strength . In partEffective breast tumor segmentation results have been achieved using traditional machine learning methods, such as fuzzy c-means (FCM) , 3, actiRecently, the U-Net architecture has beenThere have also been many attempts to improve the U-Net model . The resTo address the various drawbacks and limitations of current MR-based breast tumor segmentation techniques, we propose in the current work a two-stage deep learning segmentation framework. This framework incorporates a dense residual module based on dilated convolution , 29 as w2, matrix size\u2009=\u2009280\u00d7340, slice thickness\u2009=\u20091.0\u2009mm, slice gap\u2009=\u20090\u2009mm, and total scan time\u2009=\u200975\u2009s. The contrast agent used was gadopentetate, and the dose was 0.2\u2009ml.All breast DCE-MRI images were acquired using a Siemens MAGNETO MESSENZA 1.5\u2009T scanner with a 4-channel phased-array surface breast coil. All patients were in the prone position during image acquisition. T1-weighted fat suppression was utilized to quickly simulate gradient echo. All breast image volumes were acquired in the transverse position with the following parameters: repetition time\u2009=\u20094.6\u2009ms, echo time\u2009=\u20091.7\u2009ms, turning angle\u2009=\u20097\u00b0, Field-of-view\u2009=\u2009280\u2009\u00d7\u2009340\u2009mmA total of 160 breast tumors cases were included in our clinical database, with 2960 2D T2-weighted slices of size 512 \u00d7 512. The images in the axial direction of the transverse plane were utilized for this study. The average length of the DCE-MRI sequences in the database was 112 slices. The number of slices containing breast tumors ranged from 20 to 82 for individual DCE-MRI sequences. The 160 cases were randomly divided into 128 cases for the training set and 32 cases for the test set. Before training, data enhancement operations such as mirroring, scaling, and elastic deformation were performed on the training set. The training set was expanded to 14112 images.The ground truth marking of the breast ROI region was performed manually by clinicians with the help of the LabelMe software. The label marking and 3D display of breast tumors was performed using the 3D slice medical imaging software , and theIn general, the breast cancer lesion occupies only a small part of the total breast area, which places breast tumor identification into a category of difficult machine learning problems where the classification is largely unbalanced. Hence, advanced and complex segmentation frameworks, usually involving multistage processes, are generally appropriate for this application , 16, 30.The breast region ROI was first segmented from the rest of the image, which is important for the later segmentation of tumors. Sketching a rough ROI region of the breast has been shown to effectively remove the influence of other background tissues on the segmentation results , 30. TheTo accurately segment breast tumors from the breast ROI, we propose an end-to-end improved U-Net model, which combines a dense residual module based on dilated convolution with a recurrent attention module. The standard U-Net model and the proposed recurrent attention U-Net model are shown in Figures In the proposed model, we integrated various components to improve the performance of the standard U-Net model. First, all ReLU activation functions were replaced with PReLU activatikt1, of size 1 \u00d7 1 was used to halve the number of channels, N, of the input feature matrix, xt \u2208 \u211dW\u00d7H\u00d7N. Importantly, this caused the width, W, and height, H, to remain unchanged and generated a feature set, xt1 \u2208 \u211dW\u00d7H\u00d7N/2 (N/2 different 1\u2009\u00d7\u20091\u2009\u00d7\u2009N kernel), where t= is the residual unit number. The purpose of this process was to reduce the number of calculations that the model was required to perform. In addition, dilated convolution with the kernel, kt2, formed by 3 \u00d7 3 kernel size and dilation rate of dt= was used to enlarge the model's receptive field and to extract the output feature set, xt2 \u2208 \u211dW\u00d7H\u00d7N/2. Finally, the number of feature matrix channels is restored to N by a standard convolution with a kernel, kt3, of size 1 \u00d7 1, and an output feature set, xt3 \u2208 \u211dW\u00d7H\u00d7N. Except for the last (third) convolutional layer that was followed by a GN [\u2217 denotes the convolution operation, c = , and xt0=xt. For simplicity, we omitted the bias term. To obtain the final output of the current residual unit, we summed the current residual unit input, xt, and output, xt3, as well as all previous unit inputs, . After a PReLU activation function, the final feature map, xt+1 \u2208 \u211dW\u00d7H\u00d7N, was then obtained, as described byBased on the residual unit , to expa by a GN , the res by a GN . This coIn our self-built breast dataset, there were various cases where the lesion areas were very similar to the background. In such cases, automated segmentation can be very difficult. This problem can be alleviated by enhancing the receptive field of the model to obtain richer global context information. There are many ways to enhance the receptive field of a U-Net network. For example, a larger kernel size for convolution or pooling can be used to ensure that the model receives information from a larger receptive field at each pixel and extracts more discriminable features. However, a larger convolution kernel will also greatly increase the computational complexity of the model. Furthermore, the encoding and decoding paths constructed in the standard U-Net are not strictly symmetrical. Although the skip connection and concatenation operations are introduced to ensure that the encoding and decoding processes are consistent in size, the information cannot be guaranteed to be completely reversible. In complex edges and blurred border areas, image information loss is therefore unavoidable. Dilated convolution can alleviate these difficulties to a certain extent and can refine the segmentation results. Dilated convolution can obtain a larger receptive field without changing the size of the convolution kernel and keep feature size unchanged compared to traditional convolution.d and convolution kernel of k \u00d7 k was expanded to kd \u00d7 kd by inserting d \u2212 1 zeros between each parameter of the convolution kernel. Thus, kd=k+(k \u2212 1) \u00d7 (d \u2212 1).Dilated convolution with a dilation rate of kd \u00d7 kd, the effective value used for calculation is only k \u00d7 k. When the set dilation rate increases, the proportion of the effective features will decrease. Moreover, the feature information captured by the model will be sparser. To alleviate this issue, we set up three dilated convolutions with dilation rates of = within the block. Increasing the dilation rate cascade can fully cover the receptive field, avoid dilation or missing edges in the receptive field, and can solve the problem of information continuity loss.However, dilated convolution also has some disadvantages, which are elaborately described in . For a dx \u2208 \u211dW\u00d7H\u00d7N, of the convolution block, to compress its resolution from H \u00d7 W to 1 \u00d7 1, and extract global information. The output, x\u2032\u2208\u211dN1\u00d71\u00d7\u2009, of a GMP process is fed into the GRU together with the hidden state, h \u2208 \u211dN/2, which is initialized as a zero vector, and a new hidden state, h\u2032\u2208\u211dN/2, is extracted according tol = and L is the number of GRU. By raising the dimension of h\u2032 to be the same as x\u2032, we obtained the attention weight, \u03b1 \u2208 \u211dN/21\u00d71\u00d7. Then, we channel-wise multiplied x with \u03b1 and added the importance to the feature map, which is an output of the convolution block. In addition, we introduced the residual structure [yl \u2208 \u211dNN\u00d7H\u00d7 is the input of the next convolution block.In the model decoder, each transposed convolution layer is followed by a recurrent attention module, which consists of multiple alternately connected GRUs and convtructure , the resIn Python3.6 and Pytorch1.3 open-source. The initial learning rate was set to 0.001 to train the model. In addition, the standardized initialization function for PReLU presented in [This study was performed on the following platforms: computer server configuration\u2013Intel(R) Core (TM) i9-9900K CPU @ 3.60\u2009GHz\u2009\u00d7\u200916, Nvidia GeForce RTX 2080Ti, Linux OS Ubuntu18.04, Programming language\u2013ented in was usedented in as an opp\u2009 and g are the network output predictions and corresponding ground truth, respectively. The network output predictions were obtained by mapping the output pixels to probabilities using a soft-max function. Furthermore, pi0 is the probability that voxel at position i is a lesion, and pi1\u2009 is the probability that voxel at position i is a nonlesion. Similarly, gi0 indicates whether the voxel at position i is a lesion and gi1 indicates whether the voxel at position i is a nonlesion. The values of \u03b1 and \u03b2 control the punishment depth FPs and FNs, respectively. We were able to control the weight between false positives and false negatives by adjusting the hyperparameters \u03b1 and \u03b2. In the model, we set the values to \u03b1\u2009=\u20090.3 and \u03b2\u2009=\u20090.7.The number of lesion voxels in our breast dataset was significantly lower than that of nonlesion voxels, which is very common in the field of medical image segmentation. Note that the breast lesion area is generally significantly smaller than the whole breast area. Therefore, the influence of training a model with such an imbalanced dataset on segmentation accuracy is unknown. The loss function of Tversky et al. was usedX and Y represent the pixel set of the input image and ground truth.We used segmentation criteria that are standard metrics in the field of medical image segmentation, including area-based Jaccard similarity (Jaccard), dice similarity coefficient (DSC), sensitivity (SEN), specificity (SPE), and positive predictive values (PPV), as well as Hausdorff distance (HD), which can be computed according to equation \u201311):(6):(6)11):(In the first stage of the proposed model, we used the retrained U-Net model to segment the breast region ROI from the rest of the image see . The expTo evaluate the performance of the present model, we compared the segmentation results with those of the original U-Net model as well as other high-performing medical image segmentation models such as Residual U-Net , Dense UAs can be seen in To further evaluate the prediction results of the recurrent attention U-Net for single-patient sequence data, we extracted the boundary of segmentation and fused it with the ground truth image in a 3D reconstruction . The modIn addition to visual inspection, we also quantitatively evaluated the performance of all models on the test set using standard segmentation-based metrics . For 32 To compare the generalization of each model more graphically, we plotted the DSC index for each model in We also investigated the detected features at various stages of the proposed model to gain insight into how this model identified tumor tissue. The output features of each stage of the proposed model were also extracted, as depicted in With a continuous fusion of deep semantic information, the model decoder gradually refined the features of the lesion area. To explain the effectiveness of the proposed module, the weight distribution of the key convolutional layers of the proposed model is visualized and explained, see Figures Through the Grad-CAM algorithAutomatic segmentation of breast tumors is the key to the fast and early integration of this approach in clinical practice. Due to the segmentation challenges presented by the complex image background of DCE-MRI breast tumors, we proposed in this work a two-stage segmentation framework. In stage 1, FCN was deployed to conduct an automatic delineation of a breast ROI from the background image comprised of the chest and other tissues. In stage 2, we constructed a dense residual module based on the standard U-Net model with a recursive attention module and dilated convolution. For breast tumors of different sizes and shapes in images without postprocessing, a higher segmentation accuracy was achieved by the proposed model compared to other standard U-Net-based models. Therefore, our results showed that the segmentation result of our proposed model improved the DSC by 3%, the Jaccard similarity by 3%, the PPV by 3%, the SEN by 2%, and the Hausdorff distance by 16.2 compared to the standard U-Net model.Despite these enhancements, there are still some limitations of the current study that should be noted. Although the segmentation accuracy was markedly improved, the values achieved by the proposed models may still not be sufficient for clinical application. The proposed model was only evaluated on a single-source, single-sequence DCE-MRI breast dataset. For multisequence or multicenter studies, its effectiveness has yet to be verified. Furthermore, if the breast area was small or the position of the breast tumor was near the chest cavity, the segmentation may have exhibited additional errors. In addition, this framework is not strictly end-to-end, and in future studies, an in-depth study of the 3D segmentation of breast tumors is needed.Nevertheless, this study showed that the proposed model can intuitively assist clinicians with observing the position and volume of breast tumors. This work may in the future help clinicians to quantitatively analyze the segmentation results from a 3D perspective and establish a prediction model to improve the diagnosis, radiotherapy, and prognosis of breast tumors , 39."} +{"text": "N-(4-hydroxyphenyl)-3-(4-methoxyphenyl)acrylamide (ACR-2) and 2-cyano-N-(4-hydroxyphenyl)-3-phenylacrylamide (ACR-3) on copper in 1.0 M nitric acid solution using chemical and electrochemical methods, including mass loss as a chemical method and electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization (PP) as electrochemical methods. By Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1HNMR), and mass spectroscopy (MS) methods, the two compounds were verified and characterized. There is evidence that both compounds were effective corrosion inhibitors for copper in 1.0 M nitric acid (HNO3) solutions, as indicated by the PP curves, which show that these compounds may be considered mixed-type inhibitors. With the two compounds added, the value of the double-layer capacitance was reduced. In the case of 20 \u00d7 10\u22125 M, they reached maximum efficiencies of 84.5% and 86.1%, respectively. Having studied its behavior during adsorption on copper, it was concluded that it follows chemical adsorption and Langmuir isotherm. The theoretical computations and the experimental findings were compared using density functional theory (DFT) and Monte Carlo simulations (MC).The objective of this research was to explore the impact of corrosion inhibition of some synthetic acrylamide derivatives 2-cyano- The problem of corrosion annually causes losses from closing and stopping facilities, the deterioration of several industries, and accidents due to the collapse of bridges and dams ,2,3. DueInhibitors are arguably the most practical corrosion methods of protection. Inhibitors may be organic materials with some functional groups such as N, S, or O or heterogeneous rings ,6,7. TheUsing natural polymers as corrosion inhibitors is profitable, environmentally friendly, and non-harmful. Inhibitors of this type can also be synthesized easily, including grafted and modified polysaccharides. Polymeric compounds are better at adsorbing than monomers since polymers have multiple adsorption sites . It is pVarious studies yielded a good number of organic compounds used as corrosion inhibitors owing to the existence of N, S, and O atoms and double bonds in them. Heterocyclic compounds are the most important of these compounds. Recently, heterogeneous organic compounds containing N, S, and O atoms have been of interest to researchers to find copper corrosion inhibitors in various acid solutions. These compounds are of great interest due to their adsorption properties and the creation of an oxide layer on the metal surface ,20. DensN-(4-hydroxyphenyl)-3-(4-methoxyphenyl)acrylamide (ACR-2) and 2-cyano-N-(4-hydroxyphenyl)-3-phenylacrylamide (ACR-3) against the copper dissolution in an acidic medium (1.0 M nitric acid solution) by using chemical and electrochemical methods as well as characterization techniques from Monte Carlo simulations and density functional theory (DFT) to discover the adsorption type and corrosion mechanism on the metal surface.This study aims to focus on the evaluation of the potency of eco-friendly, nontoxic novelty synthetic compounds, namely a 2-cyano-3 solution which was purchased from the El-gomhouri Company in Egypt, in the absence and presence of various concentrations of ACR-2 and ACR-3. The experimental calculations were conducted in 1.0 M HNOOne drop of piperidine was added to acetamide 1 and 4-methoxy benzaldehyde in ethanol, followed by 1 hour of reflux. Using ethanol and DMF (4:1) as a mixture, the filtered solid product was crystallized.Benzaldehyde and acetamide 1 were dissolved in ethanol, and the reaction mixture was refluxed for 1 hour in the presence of catalytic piperidine. After filtration, EtOH was added for washing, and the solid product was crystallized by ethanol and DMF mixture (5:1) .For the measurements of weight loss, copper samples with a thickness of 2.0 \u00d7 2.0 \u00d7 0.2 cm were used, which were polished to different degrees of sandpaper, then washed with alcohol to remove impurities and dirt, distilled water, and then the process of good drying of the samples, followed by weight loss measurements by the usual methods at different temperatures which calculated according to following Equation (1) ,22: (1)%W and W\u00b0 are the mass loss of metal without and with investigated compounds, respectively.\u22122 to 10\u22125 Hz in the presence of alternating current signals of 5 mv; then, the inhibition efficiency was measured by the following equation [Electrochemical calculations were made at a temperature of 25 \u00b0C and using a three-electrode system of platinum, calomel, and copper. In this system, the platinum electrode was employed as a counter, and the calomel electrode as a reference. In the end, the copper electrode was used as the working electrode . To obtaequation .(2)\u03b7%=RctR and ctR,0 are charge-transfer resistances of inhibited and uninhibited copper, respectively. \u22121 and within the range of \u2212250 mV. Inhibitory efficiency values were calculated through the following equation [For measurements of the polarization value, experiments were carried out at 1 mV scorri,0, and corri mean current density values lack the existence of the protection of compounds. With a computer monitor attached to Potentiostat/Galvanostat/ZRA. A Gamry framework system based on the ESA 400 is included. Gamry applications also include DC105 polarization software and the EIS300 EIS software, Gamry Instruments, PA, USA.The DFT was computed using Gaussian 09W software, Gaussian, Inc., CT, USA The B3LYP approach optimized the acrylamide derivatives on a 6-311++G basis set . Based on highly and lightly occupied and unoccupied orbitals (HOMO and LUMO), detailed analyses were carried out on the parameters of quantum chemicals (HOMO and LUMO) ,27. DurisubsE is the total energy of Cu (1 1 1) and H2O particles, AcrylamideE signifies free inhibitor energy, and totE embodies the total energy of the total system.3, 1.0 M HNO3 + 20 \u00d7 10\u22125 M of the tested compounds.SEM was used to examine the surfaces of the original Cu sample that was mechanically polished and the Cu sample that was naturally submerged for five hours in a solution containing an inhibitor . Test wi\u22121, corresponding to hydroxyl, amino, cyano, and carbonyl groups, respectivelyas in The IR of compound (ACR-2) displayed absorption bands at 3406, 3353, 2217, and 1675 cm1H NMR showed a singlet signal at TM 3.87 ppm, corresponding to the methoxy group, in addition to multiplet signals at TM 6.75\u20138.02 ppm for aromatic protons. Moreover, singlet signals were found at TM 8.16, 9.34, and 10.03 ppm for the methine, OH, and NH protons, respectively as in The m/z (%): 295 , 294 , 279 (36.83), 242 (49.26), 202 (54.60), 197 (100.00), 159 (87.57), 145 (63.55), and 88 (57.60). The MS exhibited a molecular ion peak at m/z = 294 congruous to a molecular formula C17H14N2O3 as in MS \u22121, corresponding to hydroxyl, amino, cyano, and carbonyl groups, respectively as in The IR of the compound (ACR-3) showed absorption bands at 3401, 3338, 2220, and 1653 cm1H NMR (DMSO-d6): \u03b4 6.76\u20137.99 , 8.24 , 9.38 , and 10.17 .1H NMR showed multiple signals at TM 6.76\u20137.99 ppm for aromatic protons, in addition to singlet signals at TM 8.24, 9.38, and 10.17 ppm for the methine, OH, and NH protons, respectively as in The m/z (%): 264 , 259 (36.22), 245 (62.84), 205 (58.23), 174 (51.07), 113 (56.05), 95 (74.61), 61 (100.00), and 56 (87.29). The MS exhibited a molecular ion peak at m/z = 264 , congruous to a molecular formula C16H12N2O2 as in MS Of the main credible methods, weight loss tests are among the most important processes when assessing the inhibitor\u2019s capacity to protect the metal against corrosion, and this is evident in adsK values could be determined from the intersections of the lines on the C/\u03b8-axis and adsK was connected to the standard adsorption free energy \u2206adsG\u00b0 as follows:The inhibition pathway can be determined through the isotherm of adsorption, which provides a detailed explanation of this pathway. It is also possible to see the inhibitor and the metal interaction through isotherm diagrams. To determine the most suitable diagrams, Langmuir, Freundlich, and Temkin models were tested ,33. The adsK was calculated, and through it, the free energy \u2206adsG\u00b0 values were computed using the equation:The value of the adsorption constant adsG\u00b0 value is around \u221220 kJ mol\u22121 or lower, while a value of \u221240 kJ mol\u22121 or higher indicates a charge exchange or transfer between the copper surface and the ACR-2 and ACR-3 molecules [adsG\u00b0, the presence of physical adsorption was confirmed. The enthalpy values resulting from electrostatic interactions between charged molecules and charged metal (physisorption) range up to 41.9 kJ mol\u22121, whereas those, resulting from chemisorption, range up to about 100 kJ mol\u22121. Physisorption produces molecules with small absolute enthalpy values. When the examined chemicals are present, \u2206adsS\u00b0 values are large and negative, indicating the increase in the ordering on copper surface [olecules . From th surface ,36.corrE), anodic and cathodic slope , corri, and \u03b8; through it, a significant decrease in the values of corri can be observed with the addition of different concentrations of ACR-2 and ACR-3, the increase of which leads to a reduction in the current density to the minimum [corrE, which proves the mixed character of these compounds [corrE value shift, whereas if corrE value shifts below 85 mV, the inhibitor is considered a mixed type. This study reveals that the studied inhibitor is a mixed-type inhibitor, as measured by a maximum displacement in corrE of 85 mV [By following the cathodic and anodic curves, adding different concentrations of inhibitors will display a declining trend in the current, which indicates the tight restriction of the anodic and cathodic interactions, as shown in minimum . In addiompounds ,41. Infeof 85 mV . Y0 and maxf are the magnitude of the CPE and frequency at which the imaginary component of the impedance reaches the maximum value, respectively. n is an adjustable factor, n = \u03b1/(\u03c0/2), and \u03b1 is the phase angle. As the concentrations of the compounds increase, the values of dlC decrease because the thickness of the adsorbed layer on the copper surface increases [ctR values. The creation of a protective coating at the metal solution contact can be attributed to this. There was only one charge transfer mechanism during copper dissolution, determined by the appearance of a semicircle without any impact on the molecule being studied. A decrease in double-layer capacitance was observed with increasing inhibitor doses [The results of AC impedance were extracted, which are presented in ncreases . Based oor doses . This caQuantum chemical simulations utilizing the DFT mode were carried out on acrylamide derivatives and the components\u2019 molecular function under investigation. DFT was used to obtain the optimized molecular geometries of the corrosion inhibitor to test the effect the molecular structure has on its performance . MoleculadsE) was calculated which indicates how strongly the inhibitor binds to its substrate surface (copper). Inhibitory molecules are said to release rigid adsorption energy when they adsorb to metal surfaces, whereas on the other hand, deformation energy is released when the adsorbed component relaxes at the surface [adsE negative indicate that strong and spontaneous adsorption occurred on the copper substrate. The results of The position at which ACR-3 and ACR-2 were adsorbed onto the Cu (1 1 1) contact is shown in surface . ACR-2 a\u22125 M). 3\u2212 adsorption to create a negatively charged surface, making the adsorption of the cations in solution easier [3 groups, which act as electron donors.An experimental study and theoretical calculation can be used to demonstrate the adsorption of Cu by calculating the type of functional moieties, electron density, and the charge on the Cu surface. It has been reported that the Cu surface is positively charged in the nitric acid solution, i.e., several positive charges are present on the Cu surface . The posn easier . Organic3 solution as mixed-type inhibitors. By adsorbing these compounds onto copper surfaces, these compounds exhibited Langmuir adsorption isotherms, which corresponded to the inhibition process. The spontaneity of the reaction was inferred due to the presence of the negative value of \u0394G\u00b0ads. DFT revealed an increase in the inhibition strength of the compounds ACR-3 and pc ACR-2 due to the ability to donate and gain electrons from the highest occupied energy (HUMO) orbital to the lowest unoccupied orbital (LUMO). The corrosion process depends on the relative ability of the compounds to donate electrons to the metal. In the end, the results were found to agree with the measurements. Experimental experiments proved the effectiveness of 2-acrylamide derivatives in inhibiting copper corrosion in 1.0 M HNO"} +{"text": "Fraxinus excelsior (FEAE), Zingiber zerumbet (ZZAE), and Isatis tinctoria (ITAE), that efficiently inhibit the corrosion of mild steel in phosphoric acid. The anti-corrosion and adsorption characteristics were assessed using a combination of experimental and computational approaches. Weight loss, potentiodynamic polarization, and electrochemical impedance spectroscopy methods were used to evaluate the inhibitive performance of the inhibitors on the metal surface. Then, both DFT/DFTB calculations and molecular dynamic simulations were further adopted to investigate the interaction between organic inhibitor molecules and the metal surface. The protective layers assembled using the active constituents, such as carbonyl and hydroxyl groups, of the three plant-based products offer high electrochemical stability at high temperatures and robust protection against aggressive acidic solutions. All electrochemical measurements showed that the inhibition performance of extracts increased by increasing their concentration and improved in the following order: FEAE > ZZAE > ITAE. Further, these extracts worked as mixed-type inhibitors to block the anodic and cathodic active sites on the mild steel surface. Multi-level computational approaches revealed that FEAE is the most adsorbed inhibitor owing to its ability to provide electron lone pairs for electrophilic reactions. The experimental and theoretical results showed good agreement. These results indicate the possibility of replacing conventional compounds with natural substituted organic products in the fabrication of hybrid materials with effective anti-corrosion performance.In the present work, we present the superior corrosion inhibition properties of three plant-based products, Phosphoric acid is commonly used to remove rust from iron and steel during metal surface treatments. It reacts with rust and turns the reddish-brown color of ferric oxide into a black ferric phosphate, which can be easily removed [3PO4 is also used to remove radioactive rust from the containers and pipes of nuclear reactors, and it is a major component of most commercial rust transformers. The chemical transformation or treatment of metal and alloy surfaces is of considerable economic importance [3PO4 has become a major problem. To address this issue, various surface treatments have been developed to protect metallic surfaces in aggressive solutions. Recently, the use of organic inhibitors to protect the surfaces of metals, particularly steel, from highly corrosive acids has gained significance. The choice of inhibitor depends on the type of acid, its concentration, temperature, and velocity of flow, the presence of dissolved inorganic and organic substances, and the type of steel exposed in the acidic solution. Thus, it is necessary to consider the affinity, solubility, and adsorption capability of the synthesized inhibitors on the metal surfaces [3PO4 by reducing the corrosion rates of the equipment associated with it [Phosphoric acid , Zingiber zerumbet (ZZAE), and Isatis tinctoria L. (ITAE) were used to fabricate sustainable corrosion inhibitors, and their inhibition performance and interfacial behavior were evaluated. A brief description of each of these natural products is provided in the In this study, three plant extracts derived from The performance of these three plant extracts was investigated using electrochemical impedance spectroscopy (EIS), potentiodynamic polarization (PDP), and weight loss (WL) studies. In addition, the morphologies of the surfaces were evaluated using scanning electron microscopy (SEM) and Fourier-transform infrared (FT-IR) analysis. As mentioned above, the interfacial mechanism must be understood to elucidate the adsorption configuration of the inhibitor molecules and their interactions with the steel surface. Multilevel computational approaches based on density functional theory (DFT) and molecular dynamics (MD) simulations were performed to confirm the experimental results and verify the reactivity of the inhibitor molecules in terms of the charge-transfer ability and adsorption energy of the selected constituents.F. excelsior seeds, Z. zerumbet roots, and I. tinctoria leaves were milled, and 50 g of each powder was boiled in 500 mL of deionized water for 2 h. The resulting solutions (extracts) were cooled and filtered to eliminate impurities. Following the filtration process, the extracts were dried in an oven at 60 \u00b0C to obtain crystalline residues . Each precipitate was dissolved in 500 mL of deionized water, and the concentration of the extracts was expressed as w/v and was in the range of 0.005\u20133.00 g L\u20131. 3PO4, was prepared by diluting analytical grade H3PO4 (98% BDH) with deionized water. WL experiments were performed using a 50-mL solution, whereas electrochemical measurements with and without different concentrations of the inhibitors were conducted using a 100-mL solution.The MS specimens used for the corrosion tests had the following chemical composition (in %): 0.28 C, 0.22 Si, 0.73 Mn, 0.015 P, 0.006 Si, 0.007 N, and balance Fe. Before being placed in the test solutions for the experiments, MS rods of 5-cm in length and 1-cm in diameter were abraded using a 60\u20131200 grit size abrasive paper, cleaned thoroughly with deionized water and ethanol, and dried at laboratory temperature using an air stream. The aggressive solution used in this study, 2 M HWLCR, in g cm\u22122 min\u22121).t is the exposure duration (min) and A is the exposure area of the specimen. Using the CR values, the inhibition efficiency (WLIE%) was estimated using the following relationship:CR with and without various concentrations of the plant extracts, respectively.The pre-cleaned and weighed samples were placed in glass vessels containing the aggressive solutions with and without various concentrations of the plant extracts, which were placed in a thermostatic water bath for an 80-min immersion period. The MS specimens were removed from the test solutions at the end of each experiment, rinsed several times with deionized water and ethanol, dried in an air stream, and weighed on a digital scale with a 0.1 mg precision. For each assay, the weight loss (\u0394W) was computed by subtracting the final weight from the initial weight, and the following equation was used to calculate the MS corrosion rate as the reference electrode. EIS measurements were conducted at an open circuit potential in a frequency range of 0.1 Hz\u201330 kHz with a signal amplitude perturbation of 10 mV. The impedance responses were fitted to the appropriate equivalent circuit using the simulation program ZSimDemo (3.20 software), and corrosion parameters, such as solution resistance (sR), polarization resistance (pR), and double-layer capacitance (dlC), for various electrochemical processes occurring at the metal surface were estimated. Later, PDP scans were performed in the potential range from \u2013700 to \u2013200 mV with respect to the open-circuit potential at a scan rate of 1 mV s\u20131. The corrosion current density and high (2.5 g L\u20131) concentrations of each plant extract were examined using scanning electron microscopy (SEM) at the Center of Nanotechnology, King Abdulaziz University. The corrosion products were subjected to FT-IR analysis to confirm the adsorption of the inhibitor molecules on the MS surface, and the FT-IR spectra of the inhibitor powder and corrosion products were compared. The KBr disk technique was employed to record the FT-IR spectra using a Perkin-Elmer spectrophotometer , with a 400\u20134000 cm\u20131 range. The plant extract powder, which was mixed with KBr and compressed into a disk, was used as the initial sample for the FT-IR characterization. The second sample consisted of the corrosion products of an MS surface that had been immersed for three days in a 2 M H3PO4 solution containing 2.5 g L\u20131 of each plant extract, rinsed with deionized water, dried in air, scraped with a small quantity of KBr, and compressed into disks.The surface morphologies of MS immersed in HUnless otherwise specified, all experimental measurements were carried out in stagnant solutions at 30 \u00b0C using a thermostatic water bath (LOBT-B12) with temperature ranging from 0 \u00b0C to 99 \u00b0C and temperature stability of \u00b10.5 \u00b0C.\u20131 for energy, 0.5 kcal mol\u20131 \u00c5\u20131 for force, and 0.001 \u00c5 for maximum displacement. Broyden mixing and small thermal smearing were used to speed up the SCC convergence [adsE) between the iron surface and any given inhibitor can be defined as follows [surf+inhE is the total energy of the inhibitor/Fe(110), surfE is the energy of the clean iron slab, and Einh is the energy of the isolated inhibitor.The quantum chemical (QC) properties and interfacial behavior of the major compounds present in the plant extracts on metal surfaces were investigated using multi-level computational model. Density functional theory (DFT), density functional-based tight-binding (DFTB), and molecular dynamics (MD) simulations were used to highlight and correlate the molecular features of the extract molecules with their adsorption mechanisms. First, the charge transfer behavior of plant extracts were evaluated using DFT method at B3LYP of Gaussian 16 as described elsewhere . All expvergence . The Fe follows ,36:(5)Ea2O, H3O+, PO43\u2212, and the inhibitor molecule), and a vacuum layer was created. MD calculations were preceded by geometrical optimization; then, four inhibitor molecules and the corresponding molecular ions were allowed to interact with the iron surface. Subsequently, all MD simulations were performed using a COMPASS II force field with an NVT ensemble at 298 K with a fine calculation accuracy and a simulation time of 500 ps with a step time of 1 fs [totalE, surf+solE, inh+solE, and solE are the energies of the simulation system, iron slab and solution, inhibitor molecule and solution, and solution.To simulate the effect of temperature on the synergistic adsorption mechanisms of the inhibitor molecules in aqueous solutions, the Forcite module in Materials Studio was used to perform the MD calculations . As an i of 1 fs ,39,40. TWL%) was significantly enhanced by the addition of plant extracts to the electrolyte as a function of the inhibitor concentration. \u03b3 was calculated using Equation (7) [tion (7) .(7)\u03b3=CR\u03b3 increases gradually with concentration and reaches a maximum value of 20.8 at 3.0 g L\u20131. However, for ZZAE and ITAE, the \u03b3 values increase very slightly until 0.25 g L\u20131, after which they increase sharply and reach the maximum values of 13.9 and 12.8, respectively, at 3.0 g L\u20131. Based on these results, the inhibition efficiencies of the plant extracts can be arranged as follows: FEAE > ZZAE > ITAE.As shown in the figure, 3PO4 solutions. The semicircular Nyquist plots reveal that the charge-transfer process occurs during the dissolution of the metal. The semicircle appears to deviate from the ideal capacitive behavior, corresponding to the frequency dispersion of the interfacial impedance resulting from the surface heterogeneities and roughness factors [3PO4 increases with the inhibitor concentration, indicating that the inhibitor molecules in the solution hindered the surface transfer of the aggressive species. Therefore, the corrosion inhibition can be attributed to the adsorption of inhibitor molecules on the metal surface, which can block the corrosion sites. At high concentrations of the investigated plant extracts, an induction loop emerges at low frequencies, which could be attributed to the relaxation of the adsorbed inhibitor molecules or the redissolution of the passivated surface [The data on the resistive and capacitive behaviors of the plant-based extracts at the interface were obtained using EIS ,43. Figu factors . However surface .\u20131 or no inhibitors. A more complex electrical circuit, as shown in \u20131 of the inhibitors. The two equivalent circuits consist of the following main elements: sR represents the solution resistance, dlCPE is a constant phase element, instead of pure capacitors, that represents numerous non-homogeneities characteristic of corroding electrodes, and pR is the polarization resistance with barrier-type characteristics, including the charge transfer (ctR), diffuse layer(dR), accumulation (aR), inductance (LR), and film (fR) resistances [Pure electric models can explain impedance behavior and can be used to decouple electrochemical processes at the metal-solution interface in order to determine the physical and chemical parameters of the electrochemical system under study. By fitting the proposed electrical circuit to the experimental data, as shown in istances ,50.pR can be calculated using the following formula [Using the equivalent circuit model shown in formula :(8)Rp=RcdlC) is defined as follows:dlQ and n are the major components of CPE. n is a CPE exponent which provides information about the surface inhomogeneity. For a real electrode processes, its value is often between 0 and 1. dlQ is the non-ideal double-layer capacitance separating the metal\u2013electrolyte interface. RIE%) values with and without various concentrations of the inhibitors. Chi-square (\u03c72) values of the order 10\u22123 supported the validity of the proposed circuits, which was also demonstrated by the consistency of the experimental data ZCPEnditions .pR increase, whereas those of dlC decrease significantly, with increasing inhibitor concentration. Larger impedances result in lower interface activities, i.e., fewer electrons or ions passing through the interface, thereby lowering the corrosion rates. The decrease in dlC can be attributed to a decrease in the value of the double-layer dielectric constant or electrode surface area, or an increase in the double-layer thickness [dlC can be attributed to the replacement of the pre-adsorbed water molecules on the metal surface with the inhibitor species, which have a lower dielectric constant, and/or an increase in the thickness of the electrical double layer, indicating that the inhibitor molecules act via adsorption at the metal/solution interface and reduce the MS corrosion rates. The pR values increase with increasing inhibitor concentration, leading to an enhanced R%IE. According to the R%IE, the plant extracts can be arranged as follows: FEAE > ZZAE > ITAE. This arrangement is consistent with the results obtained from the WL measurements.hickness . As the 3PO4 with and without the three plant extracts (corrE), corrosion current density (corri), slopes of the anodic and cathodic branches (a\u03b2 and c\u03b2), and inhibition efficiency (iIE%). A general comparison is presented in the form of the polarization resistance (pR) by employing the Stern\u2013Geary equation [Potentiodynamic scans were performed to determine the effects of the plant extracts on the anodic dissolution of the metal and cathodic hydrogen evolution. The PDP curves of MS in 2 M Hextracts reveal tequation :(11)Rp=BcorrE with the presence of the plant extracts were shifted to more positive values in comparison to that in the absence of inhibitors, which indicates that the inhibition for anodic iron dissolution is stronger than the cathodic hydrogen evolution reaction. This indicates that the formed protective layers can cause a significant reduction in the amount of corrosive species reaching the metallic substrate. Thus, the steel surface treated by the plant extracts exhibited higher corrosion resistance than the untreated samples. In addition, based on the general criterion in the corrosion field [corrE less than 85 mV after adding inhibitors with respect to the corrosion potential of the blank reflects the mixed-type behavior of inhibitors. As seen in corrE change is significantly less than 85 mV and the maximum displacement obtained was 42, 27, and 25 mV for FEAE, ZZAE, and ITAE, respectively, with respect to 2 M H3PO4 without inhibitors, suggesting a mixed-control behavior of the plant extracts. a\u03b2 and c\u03b2 changed slightly with increasing extract concentration, confirming that the plant extracts functioned via a mixed adsorption mechanism. Moreover, the presence of plant extracts in the corrosive medium does not change the anodic and cathodic mechanisms, indicating that the inhibitors act by simply blocking the active sites on the metallic surface [corri and subsequently increases the iIE%. \u20131 increases the iIE%, which may have significant implications. At high concentrations, more inhibitor species are present, and a greater surface coverage is achieved. Polarization resistance, pR, recorded in pR with an increasing extract concentration imply lower corrosion metal dissolution rates. From the inhibition efficiency (iIE%) data, the studied inhibitors can be arranged as follows: FEAE > ZZAE > ITAE.Obviously, the values of on field ,19, a po surface . IncreasThis arrangement is consistent with the results obtained from the EIS and WL measurements.\u20131) and high (2.5 g L\u20131) concentrations of the plant extracts are shown in \u20131 of each plant extract, the corrosion was significantly diminished, along with the number and depth of the pits (\u20131) of the plant extracts (\u20131).SEM images of the polished and corroded MS surface without and with low , \u03bd (C=C), and \u03bd (C-H) vibrations with their shifts compared with plant extract powder supports the interaction of the plant extracts with the MS surface. The weakness of band intensities of the film formed on the MS surface revealed a strong interaction between MS and plant extracts species, mainly through electrostatic interaction, as well as the formation of a chemical bond between Fe and plant extracts species.The application of FT-IR spectroscopy to identify and determine the type of organic functional groups contained in the plant extract that can adsorb by establishing bonds with the steel surface is well recognized . Figure r2 \u2248 1) and slopes ranging from 1.02 to 1.10, confirming that the inhibitor molecules were adsorbed on the steel surface in a single layer according to the Langmuir adsorption isotherm. The deviation of the slopes from unity (adsK) and is related to the Gibbs free energy change of adsorption (\u0394adsG) by the following equation [\u20131, R is the universal gas constant (8.314 J mol\u20131 K\u20131), and T is the absolute temperature in K. adsK values revealed that FEAE has the highest adsorption ability for the steel surface. The greater the adsK, the stronger and more efficient the adsorbed layer on the steel surface and the better the surface coverage [adsG imply favorable adsorption of the extract species on the steel surface and the practicability and spontaneous nature of the adsorption process, especially the FEAE. The calculated values of \u0394adsG lie between \u201322.65 and \u201329.77 kJ mol\u20131, confirming that the extract species are adsorbed on the MS surface through both physisorption and chemisorption (mixed mode) mechanisms [During the adsorption process, a higher degree of surface coverage by the inhibitor is critical for effective corrosion protection. Furthermore, the interfacial mechanism of the corrosion inhibitors depends on the direct or indirect adsorption of the inhibitor particles onto the metal surface, which reduces the direct contact between the aggressive species and the metal surface. The interfacial behavior was evaluated using the Langmuir ,55, Temkom unity indicateequation :(12)Kadscoverage . The negchanisms .3PO4 with and without 2.5 g L\u20131 of plant extracts is shown in The effect of temperature on the inhibited acid/metal systems is complex. It has a significant influence on the electrochemical corrosion reactions due to multiple changes on the metal surface, such as inhibitor molecule desorption, rapid etching, and breakdown and/or rearrangement of the inhibitor molecules ,65. ConsIE% with increasing temperature from 30 \u00b0C to 60 \u00b0C and may provide a good criterion for the stability and the performance of the inhibitors with temperature. The decrease in the inhibition efficiency was between 2.3% (FEAE) and 7.8% (ITAE), demonstrating the excellent corrosion protection of the plant extracts, particularly FEAE. These results indicate the formation of an organic protective film on the MS surface in which strong donor\u2013acceptor interactions become important.To assess the strength of the plant extracts as inhibitors over the studied temperature range, A is the pre-exponential factor, and R is the universal gas constant (8.314 J mol\u22121 K\u22121). h is Plank\u2019s constant, and \u20131 of the plant extracts. The rate of metal dissolution is determined based on corrosion rate obtained from WL method. Straight lines are observed in both cases, having slopes in H3PO4 without inhibitors are consistent with the values reported in literature for steel in the same acidic medium [The c medium . However process ,68.A, in the Arrhenius-type relationship for the corrosion behavior and heterogeneous reactions is related to the number of active centers. Assuming energetic surface homogeneity, these active regions had different energies. The tendencies of dissimilarity in A and The pre-exponential factor, rrelated .\u20131 for the chemisorption process and less than 40 kJ mol\u20131 for the physisorption process. It can be seen that the RT as per Equation (16), suggesting that MS corrosion is a unimolecular reaction [The a values , indicatreaction .(16)Ea\u2212The uninhibited solution has negative extracts . GrigoryTo shed light on the electronic structures of the main compounds present in the plant extracts, we first modeled the isolated molecules, and their density distribution was described by frontier molecular orbital (FMO) theory. The optimized geometries and FMO distributions of FEAE, ZZAE, and ITAE are shown in HOMO and lower ELUMO exhibit potential reactivities for donor\u2013acceptor interactions with metal surfaces. Calculations revealed that the electron donor\u2013acceptor capabilities of the inhibitors followed the order ITAE > ZZAE > FEAE. The same order was obtained while describing the molecular reactivity based on the energy gap. Although the HOMO and LUMO were distributed on a small part of the FEAE inhibitor, its ability to donate electrons did not vary as much as other inhibitors , indicating that ITAE exhibits low reactivity under the present electrolyte conditions. Therefore, the presence of a lone pair of electrons on the O and N atoms is not sufficient to form strong coordinate bonds in the absence of the hydroxyl group. In addition, as there are several hydroxyl groups in a FEAE molecule, the FEAE inhibitor are expected to have higher chemical reactivity and adsorption potential than the ZZAE inhibitor, in addition to higher stability and polarity. This is in agreement with the experimental findings. Therefore, information on electronic properties and intermolecular interactions obtained using ELF and ESP is highly reliable for predicting the adsorption performance of the inhibitors.O\u2013Fe) ranges between 1.64 and 2.12 \u00c5, whereas the Fe\u2013C bond distance in the case of parallel adsorption of the ITAE molecule is 2.19 \u00c5. All measured distances are close to the sum of Fe\u2013O (rO + rFe = 1.98 \u00c5) and Fe-C (rC + rFe = 2.08 \u00c5) covalent radii [We used first-principles DFT calculations, which can accurately describe the ability to form chemical bonds, to study the possible interactions between the inhibitors and the metal surface. At the molecular level, parallel adsorption configurations have been systematically investigated for their potential to increase the surface coverage of large organic inhibitors . The mosnt radii . This suAdsorption energy calculations indicated that the adsorption of FEAE on the Fe surface was energetically more favorable (\u22128.33 eV) than that of ZZAE (\u22125.14 eV) and ITAE (\u22124.07 eV). ITAE did not form covalent bonds with the Fe surface via N atoms, showing less charge transfer between the N sites and the surface. Therefore, the adsorption of the inhibitor molecules is consistent with the high electron localization in the bonding regions, as discussed above, by the ESP-mapped molecular vdW surfaces of the molecules. Likewise, the formation of O\u2013Fe and intermolecular hydrogen bonds are the main driving factors, besides the \u03c0-current of the aromatic rings, for the robust interaction between the FEAE and ZZAE inhibitors and the Fe surface. From these results and the structure of the inhibitor molecules, we concluded that a high degree of charge transfer and electron localization enhances the adsorption performance of large inhibitors owing to the sufficient number of lone-pair regions favorable to interactions with iron d-orbitals.2O, H3O+, and PO43\u2212) was explored. The complexity of the systems, the size of the inhibitors, and the diversity of the influencing factors made first-principles simulations difficult. Therefore, MD studies were performed to determine the role of the adsorbed molecules in the solutions, deepening our theoretical understanding of the possible arrangements of inhibitors on the organic-metal interface. The most stable configurations of the adsorbed inhibitors on pure iron in aqueous systems are shown in The dynamic behavior of the inhibitor molecules on the iron surface in the presence of all corrosive species (HintE of FEAE (\u2013948.62 kJ mol\u20131) is more negative than those of ZZAE (\u2013589.64 kJ mol\u20131) and ITAE (\u2013514.67 kJ mol\u20131), indicating that FEAE has the strongest adsorption ability and, consequently, covers the largest surface area of Fe. The difference in the interaction energies of the studied systems can also be explained based on the intense interaction between the active sites, especially the hydroxyl groups, and the surrounding medium. The intermolecular interactions and orientation of the inhibitor molecules facilitate charge transfer between the active regions and the Fe surface, which further enhances the corrosion inhibition effect. Therefore, the presence of more active sites in large molecular chains increases the adsorption stability of the inhibitors by forming a dense molecular layer over the metal surface. These results are consistent with those obtained from the first-principles DFT calculations and the experiments.Moreover, the three inhibitor molecules formed hydrogen bonds through the O\u2013H and N\u2013H groups. The number of hydrogen bonds between the aqueous FEAE molecules was higher than those of ZZAE and ITAE, confirming that the dense molecular layer on the iron surface originates from the coordinate bonds between the molecules, which eventually interact with the Fe surface. After the final equilibrium, the interaction energy is also an important factor for evaluating the adsorption behavior of the inhibitor molecules. The interaction energies were calculated for four adsorbed inhibitors. The 3PO4 solutions. In order to give a fair comparison between the studied plant extracts in the present work and those reported in the literature as corrosion inhibitors for steels in H3PO4 solutions, Analyzing various results in the field of corrosion inhibition studies, there is a growing body of literature that recognizes the importance of using plant extracts as corrosion inhibitors for metals and their alloys in different aggressive environments. However, a search of the literature revealed a few studies which report the corrosion inhibition behavior of plant extracts on steel surface in H3PO4, and the following conclusions can be drawn:3PO4 increased with the inhibitor concentration.Regardless of the technique used, the inhibition efficiency for mild steel corrosion in 2 M H\u22121, the inhibition ability of the plant extracts followed the order: FEAE (95.1 %) > ZZAE (91.7 %) > ITAE (89.4 %), which was confirmed by surface analyses based on SEM and electrochemical measurements.At 2.5 g LThe plant extracts act as mixed-type inhibitors by simply blocking the anodic and cathodic active sites without changing the corrosion mechanism.dlC value decreases significantly with the addition of the extracts, indicating that the thickness of the film adsorbed on the steel surface depends on the concentration of the extracts.The The SEM and FT-IR techniques confirmed the formation of the adsorbed film. The adsorbed layers exhibited effective anti-corrosion behavior.The adsorption of the extracts obeyed the Langmuir adsorption isotherm, and the free energy of adsorption indicated a mixed type of adsorption for the inhibitor species on the metal surface.The adsorption of the extracts increased considerably in the parallel mode with the increase in the number of lone-pair electrons (reactive sites) in the molecular structures.Direct chemisorptive interactions through the \u03c0-current of the aromatic rings and functional groups played a leading role in the stability of the parallel arrangement of the studied inhibitors.Corrosion inhibitors should be chosen not only based on their inhibition efficiency, but also their environmental impact. Despite their high efficiency, many synthetic compounds are not suitable for corrosion inhibition because of the environmental hazards they pose. In this work experimental, DFT, DFTB, and MD studies were conducted on the heterocyclic compounds derived from FEAE, ZZAE, and ITAE to evaluate their action as corrosion inhibitors for mild steel in 2 M H"} +{"text": "In efforts to contain the COVID-19 pandemic, health colleges at Qatar University shifted their clinical training to virtual internships (VI) and project-based learning (PBL). The shift was new to students and faculty alike, and a major change that posed many challenges. This study aimed to explore the experience of changing to VIs during the pandemic from both the clinical instructors' and health sciences students' perspectives. A qualitative study was conducted based on the framework of readiness to change. It involved focus group discussions with students from the departments of Public Health and Human Nutrition and in-depth interviews with clinical instructors using appropriate online platforms. A total of 4 focus groups with 20 students and 4 interviews with instructors were conducted. Transcripts were analyzed following the inductive-deductive approach. The major themes that emerged from the analysis described students' and clinical instructors' perceptions of the necessity and efficiency of the switch to VI; the design of the VI and the extent of the clinical/field experience and skills that it offered; confidence in the ability to succeed in this type of internship and confidence about reaching expected goals; academic and moral support from clinical faculty and coordinators and the communication process with faculty and preceptors; and finally, the benefits gained and how employers would view this type of internship. Health sciences students' readiness for VI was generally low. Several student and faculty needs have to be addressed, specifically regarding the design of the program and the level of preceptors' communication with students. The findings would direct health programs, clinical instructors, and preceptors to better understand students' needs and efficiently plan for virtual internships during not only emergencies but also whenever there is a need to deliver online experiential learning courses. Worldwide, many educational institutions have experienced an urgent shift of teaching and learning activities to full-scale online modes in response to the COVID-19 pandemic. Although there are benefits to online education, including learning flexibility and inclusiveness, there are several challenges: increased workload for instructors, difficulty motivating students, lack of opportunities for interaction, technical issues, and lack of appropriate teaching materials health programs, like many of their counterparts around the world, had been challenged by the COVID-19 pandemic. At the College of Health Sciences (CHS), the Department of Public Health (PH) and Department of Human Nutrition (HN) faculty and clinical preceptors struggled to create safe and appropriate alternatives to practice-based learning due to the suspension of routine services in the health care system. Similarly, students struggled to cope with the abrupt change in their mode of learning. These two programs are only offered for female students at QU.When the university started the virtual clinical practice, the PH department shifted its health education practicum to a project-based internship (PBI) replacing their clinical training in Primary Healthcare Centers in which they would finalize their internship after having their community-level training at the Ministry of Public Health and other public health organizations in Qatar. In the PBI, students constructed different instruments after reviewing the literature to assess the needs of specific audiences in regard to different public health topics. They collected online data and conducted the analysis. Then, they tailored the findings toward specific activities to promote the health of the chosen audiences or to enhance the services provided for them in various settings. Students also built creative models to plan for implementing these activities and services and suggested a variety of methods to monitor the implementation process to enhance their outputs. In addition, they planned for effect evaluation by suggesting specific outcome indicators and different research designs.The HN internship is a structured program including rotation in community sites such as Qatar Diabetes Association (QDA) and clinical sites such as cardiac, oncology, renal, food service, and other medical departments of Hamad Medical Corporation (HMC) hospitals. During the pandemic, most clinical rotations were immediately shifted to virtual rotations, and different student tasks were suspended such as patient education, counseling, and working in interdisciplinary teams. These were replaced by an extended number of hours of professional development classes where students attended some workshops by dietitians abroad and got more involved in case simulations using the telehealth concept, case studies, case presentations, preparation of awareness campaigns, and writing articles.The decision to shift to full-scale online education was taken by the national authority overseeing the pandemic response. Under such emergency circumstances, students and instructors were not prepared for the transition, which may be critical for their readiness for change. In turn, readiness for change is important for the success of targeted outcomes , 14. OriHolt et al. suggest In line with the framework of readiness for change to facilitate effective VIs, it is essential to support students with initial readiness for emergency change, addressing the following five aspects. First, discrepancy refers to understanding the need to use VIs in such an emergency in order to reach the aspired goals, and have the knowledge/skills about VIs. Second, appropriateness refers to students' belief that the current use of the online learning platform and the design of VIs is appropriate for the given purpose. When students understand how the change is implemented and what strategies are being used to support VIs, they have a better chance to develop a strong belief in the appropriateness of the change and get motivated in and engaged with the changed mode of learning \u201320. ThirA fourth aspect of readiness for change is principal support. Leadership support is significant to improving motivation and self-efficacy to ensure the success of change efforts . LiteratWhile current literature highlights the critical role of readiness for change, little is known about how change recipients report their readiness in the context of an urgent shift, when the decision to make the change is enforced externally. Understanding CHS students' experiences while shifting to virtual internships, and the factors shaping this process would direct health programs, clinical instructors, and preceptors to better understand students' needs and efficiently plan for virtual internships during a not only emergency but also whenever there is a need to deliver on-line experiential learning courses. This study aimed to explore health sciences students' and instructors' experience of an emergent shift to virtual internship and their perceptions of VI replacing on-site experiential learning.A qualitative design was employed in this study, which took place at the time when the COVID-19 pandemic was at its peak in Qatar (Spring semester 2020). Purposive sampling was followed to recruit participants who are considered rich cases and would help answer the overarching research question. Our study ran in two parallel streams, one involving focus group discussions with senior female students registered for clinical practice from the departments of Public Health (PH) and Human Nutrition (HN), and the second involved conducting in-depth interviews with clinical instructors . Online platforms (WebEx and Microsoft teams) were used for data collection, given regulations surrounding social distancing and restrictions on in-person gatherings. An interview guide and a focus group guide were developed considering the main constructs of the readiness framework and based on the discussions of the research team and a review of the relevant literature. Interviews and focus groups were recorded with participants' permission, and a member of the research group took written notes simultaneously.To invite participants, emails and reminders were sent out through Blackboard to all students registered for clinical training in spring 2020, providing research information and a link to an electronic consent form. We conducted four focus groups until data saturation was reached, where no new themes emerged . A trainSimilarly, clinical instructors, including clinical coordinators and faculty responsible for bedside teaching in clinical sites were contacted by email with electronic consent. Four semi-structured interviews were conducted on online, each lasting 45 min.All interviews and focus groups were audio-recorded and transcribed into texts for analysis. An inductive-deductive approach was employed in the analysis. Inductive qualitative analyses were used to discover emerging themes, and the transcripts were analyzed for predetermined themes mapped to the readiness framework as well . Coding N = 20), two with senior health education students from PH (N = 11), and two with senior students from HN (N = 9). We also conducted four individual interviews with 2 clinical instructors from PH and another 2 clinical instructors from HN. The major themes that emerged from the analysis described students' and clinical instructors' perceptions of the necessity and efficiency of the switch to VI; the design of the VI and the extent of the clinical/field experience and skills that it offered; confidence in the ability to succeed in this type of internship and confidence about reaching expected goals; academic and moral support from clinical faculty and coordinators and the communication process with faculty and preceptors; and finally, the benefits gained and how employers would view this type of internship.In total, we conducted four focus groups with students from CHS , collaboration and teamwork, online skills, planning skills, critical thinking, and creativity. A nutrition preceptor mentioned:\u201cAs Food service is an inevitable part of the Dietetics department, the rotation will help the students to apply their knowledge of food systems management and understand the functions of the dietitian in food service and administration. As throughout the rotation they are participating in the supervision of food production, sanitation inspections, menu planning, sensory evaluation and kitchen design.\u201dMost of the public health students agreed that the PBI would not enrich their resume, nor enhance employability when they apply for jobs in the near future. They were concerned that employers would prefer having an internship in a clinical setting during the pandemic instead of having an online public health project or a project-based internship.\u201cNot sure how the employers will view this type of internship. Like. I know that it's a new thing, but I don't know how they would accept and view such an internship as they would ask about any previous experience in the field or things like that\u2026 I'm not sure how they would see it since it was like 4 credit hours\u2026\u201d [PH student 8]Students from HN felt a need to compensate for the lost practical experience and suggested:\u201cAfter the situation gets better, we could revisit the sites we weren't able to. For the sake of practicality, we could maybe have a choice of choosing 2 or 3 rotations out of all the rotations we have missed, or we could even shorten the time spent in each practical rotation when visiting on-site\u2026.\u201dThe PH clinical instructors were also concerned about how employers will view this type of internship and suggested that the department needs to support students and issue certificates explaining this type of internship.\u201cAs I mentioned, the project was realistic they built on their experience from the previous site, this is something to highlight. In addition, they had research skills, writing, literature review, and online presenting skills. Yet, I have a concern, yeah, maybe it won't be taken as a field internship. Maybe we need to support these students with a certificate, saying that this online training was a real training when applying for a job.\u201d [PH teaching assistant]This study aimed to explore health sciences students' and instructors' experience of an emergent shift to virtual internship and their perceptions of VI replacing on-site experiential learning. Being sensitized to the main constructs of the readiness framework, major themes arose from the analysis reflected on the participants' understanding of the shift to VI, the appropriateness of the overall design of the VI, confidence in one's own abilities to succeed through the VI, efficient communication and instruction, leadership support and technical support, and personal benefits from the VI in the long term.In general, students had a sufficient understanding of the need to shift to VI and described the decision by the university to move to VI to be prompt and efficient. When exploring student safety during the pandemic, many students perceived that the switch to VI was necessary to maintain their safety, the safety of their families, and that of patients. Such a finding is understandable because our study was done at a time when the pandemic was at its peak, a time when the psychological effects of the pandemic, such as fear and anxiety, were highly seen . On the The majority of students in our study had negative perceptions about the overall appropriateness of the VI program. A theme from our study indicated that students were not satisfied because the program did not enable them to gain the needed skills and competencies that are usually gained from on-site practice. The students' views about the importance of gaining such competencies are consistent with what was reported in the literature \u20138. StudeOur study results reflect that students' efficacy depended on certain factors, such as their previous exposures to clinical settings, how much they would be earning essential skills from VIs, and the extent of communication and feedback they were being provided with. Since most of them had the needed online skills and were familiar with clinical settings from their previous rotations, CHS students were confident that they would succeed in this type of internship. Nevertheless, students were not confident that they would reach the expected goals by taking this VI. Such a negative perception was driven by the lack of gaining some of the skills that are essential for clinical practice, such as effective communication with patients and doctors , becauseAs for their perceptions of support, the majority of students said they received academic and moral support from QU clinical faculty and coordinators. Faculty members provided good quality and frequency of communication with students as well as providing them with timely and constructive feedback. However, there were a few negative student perceptions about the communication process with clinical preceptors assigned from training sites. Only a small number of students were not satisfied with both the quality and the frequency of such communication. It is evident from the literature that such an issue with communication is a major obstacle that usually creates a negative impact on the students' confidence and capability to undertake new challenges and changes \u201336. Our Nevertheless, students perceived gaining several benefits from this unique VI. They improved their online skills in data collection and implementation of health education sessions and enhanced their online communication skills. In addition, some of them got exposed to online counseling and telehealth, which according to literature, comprises a valuable asset for the future careers of medical students . FinallyFindings from this study provide students' and clinical instructors' insights into the VIs that were being offered as an alternative to the standard onsite clinical training. If students are not ready, they will tend to reject change and face it with negative reactions such as sabotage and absenteeism . Just liThe findings of our study set a base for future research that can suggest what improvements should be implemented in the design of the VI program, and evaluate the impact of such improvements. In that sense, it would be important to consider the introduction of new interactive learning techniques that can enhance students' clinical skills and hence provide more benefits from the VI. Moreover, this study shed the light on several student and faculty needs that should be addressed to optimize the students' experience of VI. There will be an urgent need for training of preceptors, specifically in relation to improving their skills of communication and providing feedback during VIs.Applying qualitative research methods helped us recruit rich cases and understand the experience in depth. In order to enhance the credibility of our findings, we applied triangulation is different ways: data collection through interviews and field notes, and independent data analysis by three members of the research team. On the other hand, one of the principal limitations of the study is the inability to have the themes validated by the study participants due to time constraints. The study assessed students' readiness from the perspective of members and students from only one health college of QU. However, to enrich the understanding and provide broader views of the VI it would be important to assess the perspectives of students and members of other health colleges in QU and those in other universities in Qatar.Results from this study highlighted health sciences students' experience in switching to VI during the COVID-19 pandemic in Qatar. Our study revealed that students held several negative perceptions about the different aspects of the change, and hence their readiness for shifting to VI was generally low. However, the study suggested other positive views including the prompt and efficient decision by the university to move to VI, good moral support from faculty and preceptors as well as several perceived benefits of this type of training. The finding would support planning for efficient VI for health sciences students.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Qatar University Institutional Review Board (QUERG-CHS-2020-1). The patients/participants provided their written informed consent to participate in this study.HB, HA, XD, AE-A, and AA-M: conceptualization of the study, designing the interview guides, and critical review of the manuscript. JM: data collection and write-up of the manuscript. RS and MD: write-up of the manuscript. GA-J: conceptualization of the study, designing the interview guides, data collection, data analyses, write-up of the manuscript, critical review of the manuscript, journal submission, and response to reviewer comments. All authors contributed to the article and approved the submitted version.This study was funded by a Qatar University Emergency Response Grant (QUERG-CHS-2020-1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "To summarize the available evidence from systematic reviews with meta-analysis on the effects of music-based interventions in adults diagnosed with cancer.An overview of systematic reviews was conducted. CINHAL, Embase, PEDro, PubMed, Scopus, the Cochrane Library and Web of Science were searched from inception until November 2022. Systematic reviews with meta-analysis in individuals with cancer (any type), any comparator, and outcomes of cancer-related pain, fatigue, and psychosocial symptoms were eligible. The methodological quality of systematic reviews and the amount of spin of information in the abstract were assessed. The Graphical Representation of Overlap for OVErviews tool (GROOVE) was used to explore the overlap of primary studies among systematic reviews.Thirteen systematic reviews, with over 9000 participants, containing 119 randomized trials and 34 meta-analyses of interest, were included. Music-based interventions involved passive music listening or patients\u2019 active engagement. Most systematic reviews lacked a comprehensive search strategy, did not assess the certainty in the evidence and discussed their findings without considering the risk of bias of primary studies. The degree of overlap was moderate (5.81%). Overall, combining music-based interventions and standard care seems to be more effective than standard care to reduce cancer-related pain, fatigue, and distress. Mixed findings were found for other psychosocial measures.Music-based interventions could be an interesting approach to modulate cancer-related pain, fatigue, and distress in adults with cancer. The variability among interventions, together with important methodological biases, detract from the clinical relevance of these findings.The online version contains supplementary material available at 10.1007/s00520-023-07938-6. Nearly 18 million individuals are diagnosed with cancer every year . PatientNon-pharmacological therapies are of interest for the clinical management of long-term diseases, as considered to be safe, low-cost, and with minor side effects . Among thttps://doi.org/10.17605/OSF.IO/Y67BU). This overview has followed the preferred reporting items for overviews of reviews (PRIO) statement and the PRISMA for abstracts [The overview protocol was prospectively registered at the Open Science Framework carried out an electronic search from inception to November 2022 in the following databases: CINHAL, Embase, PEDro, PubMed, Scopus, the Cochrane Library, and Web of Science. Medical Subject Headings (MeSH) terms associated with the intervention (music) and the medical condition were combined. A comprehensive search strategy was first constructed for PubMed and then adapted for other databases. The lists of references of previous overviews were manually checked. The detailed search strategies are listed as Supplementary file TM carrieP: Adults diagnosed with cancer without restrictions in body location/system or the cancer stage.I: music-based interventions, used alone or as adjuvant to usual or standard careC: no restrictions regarding the control intervention.O: physical , and psychosocial measures .S: systematic reviews with meta-analysis .The eligibility criteria were established following the PICOs framework :Systematic reviews were not included when: a) the publication was written in a language other than Spanish or English; b) there were not meta-analyses for the condition of interest; c) music-based interventions were meta-analyzed together with other experimental treatments; and d) meta-analyses included non-adult participants, population without cancer, or non-randomized controlled trials. Possible outcomes of interest that were not analyzed in at least two systematic reviews were not considered. Congress proceedings, thesis dissertations, and network meta-analyses were also excluded.Duplicate records were removed using the Mendeley desktop software, v2.72.0. and manually checked. One researcher (ATM) screened the remaining records based on the title and the abstract. The full text of eligible studies and those lacking an abstract were then revised. A consensus was achieved for three studies with a second researcher (AMHR) who independently double-checked the entire selection process.Data were extracted with a standardized form that included: a) first author plus et al., the year of publication, and the number of clinical trials of interest; b) sample size ; c) the characteristics of participants ; d) description of the experimental and control interventions; e) music style used; f) outcome measures; and g) main results from meta-analysis. We aimed to extract the overall effect size from each meta-analysis. When this was not reported, results from sub analyses were included. Two corresponding authors were contacted by e-mail to clarify some information , 40. A rTwo independent reviewers (ATM and MJCH) evaluated the methodological quality of systematic reviews using the AMSTAR-2 tool . As recoThe abstracts of the systematic reviews were assessed in isolation to quantify the occurrence of spin of information. Two independent reviewers (ATM and PGG) utilized a 7-item checklist , where eFindings have been narratively described based on the outcomes of interest. To identify the most relevant key terms across systematic reviews, the VOSViewer software, v. 1.6.18 was used to conduct a co-occurrence analysis and bibliometric mapping. The degree of overlap of primary studies among included systematic reviews was evaluated with the Graphical Representation of Overlap for OVErviews (GROOVE). The GROn\u2009=\u200929) is described in the Supplementary file Search strategies retrieved a total of 926 eligible records. After removing duplicates, 466 records were screened. We eventually included 13 systematic reviews and 34 meta-analyses in the qualitative synthesis , and \u2018selective reporting or overemphasis on the beneficial effect of music-based intervention\u2019 (n\u2009=\u20095). No spin of information was found in three abstracts [The overall spin-abstract score was 21, with a mean value of 1.6\u2009\u00b1\u20091.3 points . The most common forms of spin were \u2018concluding a positive effect despite high risk of bias of primary trials\u2019 and this remained moderate even after adjusting for chronological structural missingness. The citation matrix and the CCA calculation can be found in Supplementary file n\u2009=\u20096) concluded that music-based interventions plus usual or standard care were more effective than control interventions to reduce cancer-related pain [All systematic reviews measuring pain as an outcome Supplementary file2 (DOCX 36 KB)Supplementary file3 (DOCX 15 KB)Supplementary file4 (DOCX 52 KB)Supplementary file5 (DOCX 18 KB)Supplementary file6 (DOCX 25 KB)Below is the link to the electronic supplementary material."} +{"text": "On September 28, 2022, the first case of Omicron subvariant BF.7 was discovered among coronavirus disease 2019 (COVID-19) infections in Hohhot, China, and then the epidemic broke out on a large scale during the National Day holiday. It is imminently necessary to construct a mathematical model to investigate the transmission dynamics of COVID-19 in Hohhot.Re). Finally, we explored the effects of higher stringency measures on the development of the epidemic through scenario analysis.In this study, we first investigated the epidemiological characteristics of COVID-19 cases in Hohhot, including the spatiotemporal distribution and sociodemographic distribution. Then, we proposed a time-varying Susceptible-Quarantined Susceptible-Exposed-Quarantined Exposed-Infected-Asymptomatic-Hospitalized-Removed (SQEIAHR) model to derive the epidemic curves. The next-generation matrix method was used to calculate the effective reproduction number (R0) was approximately 7.01 (95%CI: 6.93\u2009~\u20097.09), and then Re declined sharply to below 1.0 on October 6, 2022. Scenario analysis of higher stringency measures showed the importance of decreasing the transmission rate and increasing the quarantine rate to shorten the time to peak, dynamic zero-COVID and an Re below 1.0, as well as to reduce the number of peak cases and final affected population.Of the 4,889 positive infected cases, the vast majority were asymptomatic and mild, mainly concentrated in central areas such as Xincheng District. People in the 30\u201359 age group primarily were affected by the current outbreak, accounting for 53.74%, but females and males were almost equally affected (1.03:1). Community screening (35.70%) and centralized isolation screening (26.28%) were the main ways to identify positive infected cases. Our model predicted the peak of the epidemic on October 6, 2022, the dynamic zero-COVID date on October 15, 2022, a number of peak cases of 629, and a cumulative number of infections of 4,963 : 4,692\u2009~\u20095,267), all four of which were highly consistent with the actual situation in Hohhot. Early in the outbreak, the basic reproduction number (Our model was effective in predicting the epidemic trends of COVID-19, and the implementation of a more stringent combination of measures was indispensable in containing the spread of the virus. As the most widespread and severe public health crisis in the last 100 years, coronavirus disease 2019 (COVID-19) has affected more than 200 countries to varying degrees . From MaRecent outbreaks of COVID-19 in mainland China were distributed in multiple locations, with wide coverage and frequent occurrences, involving many provinces, cities, counties and districts nationwide, so the epidemic prevention and control situation remains severe and complex. On September 28, 2022, one new indigenous confirmed case of COVID-19 was reported in Hohhot, Inner Mongolia Autonomous Region. This was the first time that the Omicron subvariant BF.7 had appeared in China. The variant, called BA.5.2.1.7 or BF.7 for short, is an offshoot of the Omicron subvariant BA.5, which has the characteristics of extremely strong transmissibility, pathogenicity and immune escape capacity. As of October 3, 2022, the cumulative number of COVID-19 cases reported in Hohhot has exceeded 500, suggesting that the anti-epidemic situation is not optimistic. A serious concern at present is that there is no clear description or consensus on the epidemiological characteristics and future course of the current outbreak. There is an urgent need to explore the epidemiological characteristics of COVID-19 in Hohhot and to develop a prediction model to estimate the incidence trend and determine the priorities of prevention and control measures to provide a scientific basis for an effective response to subsequent outbreaks.R0) for the epidemic in Japan based on the SEIR compartmental model using a least-square-based method with Poisson noise and Susceptible-Exposed-Infected-Removed (SEIR) compartmental models are particularly popular . Huarachon noise . A Bayeson noise . Some ston noise , 16.Although the above models have achieved rather good prediction results, the SEIR model and its parameters still need to be further modified in due course because the transmission characteristics of the mutant strains differ considerably from those of past strains, and the speed and intensity of implementation of prevention and control measures are inconsistent across regions. Our objectives were to (1) establish a dynamic model based on the current epidemiological situation in Hohhot, (2) predict the development trends of COVID-19, including the peak, size, and reproduction number, and (3) evaluate the effectiveness and priority of different non-pharmaceutical interventions in halting the spread of COVID-19.Daily COVID-19 report data were obtained from the Inner Mongolia Autonomous Region Health and Health Commission,In the study of dynamic models of infectious diseases, individuals are abstracted into several compartments: Susceptible (S), Exposed (E), Infected (I) and Removed (R), and transitions between individuals constitute different transmission models. The classical transmission models include SI, SIR, SIS and SEIR, of which SIR and SEIR occupy a central place in epidemiology . Given tS, Q_s, E, Q_e, I, A, H, R) and 14 parameters method has become very popular for Bayesian computation in complex statistical models. The basic idea is to first construct a Markov Chain whose desired distribution is close to its equilibrium distribution, and then generate samples of the posterior distribution through this Markov Chain, and finally perform Monte Carlo integration based on the valid samples when the Markov Chain reaches its equilibrium. To compensate for the low acceptance probability of MCMC sampling, we used the Metropolis-Hastings (M-H) algorithm to sample the posterior distribution of \u03b3I,\u03b3A,\u03b3H, 21. We R0) is defined as the number of second-generation cases caused by an individual infected in a fully susceptible population without any intervention. It is a threshold indicator for measuring the transmission capacity of infectious diseases and determining the point of disease-free equilibrium. Notably, the effective reproduction number (Re) is more appropriate than R0 for assessing the effectiveness of vaccines or other non-pharmaceutical interventions. It can be used to track changes in the reproduction number, and whether the epidemic is controlled depends on whether Re is consistently less than 1.The basic reproduction number was used to evaluate the goodness-of-fit of the model.The calculations of the SQEIAHR compartmental model were performed using Python software . The figures were produced by GraphPad Prism software . The coefficient of determination on October 6, 2022, and then rapidly decreasing after arriving at the second peak (n\u2009=\u2009645) on October 10, 2022 and males (n\u2009=\u2009957) were almost equally affected, and the sex ratio was approximately 1.03:1 (n\u2009=\u2009629), with the sum of the two dropping below 100 cases on October 15, 2022, after which the epidemic gradually died out . The numdied out ,C. Our mdied out . The aboR0 of approximately 7.01 (95%CI: 6.93\u2009~\u20097.09). The second stage occurred after October 4, 2022. Following the implementation of a series of measures, such as multiple rounds of nucleic acid testing and resident travel control, Re began to decline rapidly until it dropped below 1.0 on October 6, 2022, and has since maintained a steady trend increasing the exponential decline rate of the transmission rate (2.000 vs. 1.001), (2) increasing the maximum quarantine rate (0.700 vs. 0.499) and the exponential increase rate of the quarantine rate (2.000 vs. 0.999), and (3) increasing the exponential decline rate of the transmission rate (2.000 vs. 1.001), the maximum quarantine rate (0.700 vs. 0.499), and the exponential increase rate of the quarantine rate (2.000 vs. 0.999). Our results showed that scenario design 3 was the optimal strategy: the time to reach the peak, the time to achieve dynamic zero-COVID, and the time for the ectively .Overall, the peak of the epidemic in Hohhot has passed, and the number of new cases is steadily declining, indicating that the epidemic has been effectively controlled. The time-varying SQEIAHR compartmental model we proposed in this study captured some important qualitative features and hence could provide guidance in policy-making. To the best of our knowledge, this is the most comprehensive report investigating the latest epidemiological characteristics and development trends of COVID-19 in Hohhot.According to epidemiological investigations, the majority of cases in the current outbreak were asymptomatic and mild, which may be related to the fact that COVID-19 vaccination reduces the threat of serious illness and death . People Dynamic model is an important method for the theoretical and quantitative study of infectious diseases. It can be used to describe the pattern of disease transmission, predict disease status and assess the effectiveness of prevention and control measures . The timR0 was a constant greater than 7. After the implementation of a series of control measures, Re showed a rapid downward trend, and it took only nine days from the discovery of the first case to achieve an Re below 1.0. After the outbreak of COVID-19 in Shanghai on March 1, 2022, Re did not drop below 1.0 until day 45 (The four key indicators predicted by our model were (1) the epidemic peak on October 6, 2022, (2) dynamic zero-COVID on October 15, 2022, (3) the number of peak cases of 629, and (4) the cumulative number of infections of 4,963, all of which were in fairly good agreement with the actual situation in Hohhot. Compared to the outbreak also caused by Omicron BF.7, the number of infections in Beijing far exceeded that in Hohhot . The real day 45 . This reRe below 1.0, while substantially reducing the number of peak cases and accumulated cases. These results emphasized the importance of decreasing the transmission rate by travel restrictions and the necessity of increasing the quarantine rate by close-contact tracing. Liu W et al. also recommended that isolation measures be implemented in communities and outbreak sites in a timely manner to ensure social distancing between people, thereby reducing the level of human contact , the Major Science and Technology Project of Shanxi Province (202102130501003 and 202005D121008), and the Special Foundation on COVID-19 of Shanxi Health Commission (16).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The avian spleen is an important immune organ in birds and its size can be used as an index of immune system responses in different conditions. Based on the lack of knowledge in computed tomography of the spleen in chickens, the present study was conducted to assess the inter-and intraobserver reliability in the measurement of the spleen dimensions and attenuation, as well as the feasibility of utilization of these measurements as a predictor of different diseases. For these purposes, the spleens of 47 chickens were included in the study. Two observers measured the dimensions and attenuations of the spleen, which were finally compared with the clinical diagnosis. The results showed an excellent interobserver reliability in the length, width, and height of the spleen , and a good interobserver reliability was observed during the evaluation of the average Hounsfield units of the spleen (ICC: 0.818). The intraobserver reliability was excellent in all the measurements (ICC\u2009>\u20090.940). Additionally, no statistical differences were detected in the spleen size and attenuation between the normal and diseased groups. Based on the present results, the computed tomographic measurements of the spleen could not predict the clinical diseases of the chickens; however, the low rates of the inter- and intraobserver variability suggest the reliable utilization of these computed tomographic measurements in routine clinical application and follow-up examinations. The It is common for clinicians and researchers to correlate the size of the spleen with the clinical diseases but there is no published evidence supporting this fact. Also, recently, the spleen size is used as an index of immune system response in infectious diseases or as a biomarker in different immuno-toxicological, ecological, and evolutionary studies, in which the results are not properly translated for clinical use , 4\u20136.Although the spleen can be evaluated easily through necropsy, diagnostic imaging offers a detailed antemortem evaluation of the spleen\u2019s size, shape, and parenchyma. However, radiography was the pioneer in avian diagnostic imaging , nowadayTo the best of the authors\u2019 knowledge, the sparsely available knowledge of the spleen in avian CT is mainly based on case reports. There is a lack of research that describes the CT features of chicken spleens in normal and diseased patients. Due to this lack of knowledge, this study was conducted to investigate the following aims:To evaluate the size, shape, and attenuation of the parenchyma (HU) of the spleen in normal and diseased chickens;To assess the intra-and interobserver agreement on the objective evaluation of the spleen in plain CT;To present reliable indices for evaluation of the chicken spleen size.We hypothesized that, first, the spleen size, attenuation (HU), and suggested indices differ in normal and diseased chickens; second, more specific differences may exist between different pathological conditions; and, finally, there is an excellent interobserver agreement in the objective evaluation of this organ.2.The study was a retrospective design, so no institutional animal care and use approvals were requested officially and written informed consent was obtained from the owners for the participation of their animals potentially in retrospective studies at the time of admission in the hospital.The archives of the diagnostic imaging section were investigated retrospectively between 2014 and 2020 for all chickens that underwent CT in the Department of Small Animal and Horses, University of Veterinary Medicine of Vienna. The CT scans were performed using a SOMATOM\u00ae Emotion 16-slice helical CT scanner . Consistent scan parameters used included 130\u2009kV tube potential, 80\u2013100 mAs, 0.6\u2009mm slice width, and helical scan mode with a collimator pitch of 0.8. For reconstruction, a soft tissue kernel as well as a sharp bony kernel were used. Chickens were included in the study if the entire coelomic cavity was included in the field of view (FOV) and the quality of the images was recorded \u201cdiagnostic\u201d by a third-year resident of ECVDI (YV). The included chickens were categorized based on the final diagnosis and laboratory results recorded in their medical history into five main groups: 1. normal or trauma or orthopedic patients, 2. infection, 3. inflammatory disorders with unknown origin, 4. neoplasia, 5. egg binding, and 6. egg peritonitis. The \u201cinfection category\u201d was categorized into bacterial, viral, fungal, and parasitic infection subcategories.The gender of the included cases was extracted from the medical record for further statistical analysis. All the images were reviewed using the soft tissue kernel reconstruction with the assessed window width (WW) and window level (WL), depending on the preference of the observers using the Osirix_Lite\u00ae software . The cases were excluded during the evaluation if the spleen was displaced or effaced due to the crowded celomic cavity . The observers were not aware of signalments and the final diagnosis of the cases.3.In total, two veterinary radiologists evaluated the included cases: a third-year resident of ECVDI with experience in avian diagnostic imaging (YV) and an assistant professor specialized in avian and reptile diagnostic imaging (MG). The same guidelines were used by these two radiologists for the objective evaluation \u20133. The mThe attenuation of the spleen was measured by drawing a circle in the center of the spleen in the transverse plane. The ROI was placed a minimum of 1\u20132\u2009mm far from the borders. Then, the minimum of HU (MinHU), the maximum of HU (MaxHU), the average of HU (AvHU), and the standard deviation of HU (SDHU) were recorded.The thickness of the pectoral muscle (MTH) was measured on both sides using the transverse plane just caudal to the cranial curvature of the keel. The caliper was placed from the junction of the sternal keel to the surface bisecting the angle . The attThe pectoral muscle\u2019s thickness was used as an indicator of body condition. Furthermore, it served as a rating tool together with the spleen size to create quantitative indices and ratios.Consequently, three ratios were suggested by dividing the spleen width, length, and height by the pectoral muscle\u2019s thickness and included in the measurements for further statistical analysis.4.As the body condition score (BCS) was not recorded in the medical records of the cases, thus CT images were used for subjective scoring. A poultry specialist, with a doctorate in avian and poultry medicine and PhD candidate in poultry medicine (SB), recorded the body condition score of each patient by subjective evaluation of the pectoral muscle in the transverse plane based on the keel scoring guideline .For the subjective evaluation of the spleen\u2019s shape, the dorsal plane was used, while the MPR plane was adjusted on the length of the spleen. Then, the area of the spleen was measured using the Adobe Photoshop\u00ae CS5 . Each spleen was colored with 10% opacity of black and layered on another to present the morphological variation, the common area, and the shape of the spleens.5.P-values below 0.05 were considered statistically significant. The normality of the data was assessed by the Shapiro\u2013Wilk Test. The results of measurements (spleen dimensions and attenuation) and suggested indices were compared with the recorded final diagnosis by the Kruskal\u2013Wallis test. The correlation of the spleen dimensions and HU in relation to muscles\u2019 measurements was assessed by the Spearman rank-order correlation coefficient. Principal component analysis (PCA) was used to illustrate the relationship among the three dimensions of the spleen, the abovementioned six different disease categories, two different groups , gender, and BCS. PCA was performed by R software (R Core Team (2019), version 3.6.1. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/).The data were collected and analyzed using SPSS 10.0 for Windows by a third-year resident of ECVDI under the supervision and guidance of a statistician, PhD in biostatistics (EL). An agreement between intra-and interobserver measurements was assessed by the intraclass correlation coefficients (ICCs) using a two-way mixed-effects model. ICC values less than 0.5, between 0.5 and 0.75, between 0.75 and 0.9, and greater than 0.90 were interpreted as poor, moderate, good, and excellent reliability, respectively . P-value6.Overall, 50 chickens were included during the first archive screening and three of them were excluded during the evaluation due to crowded coelomic cavities. Finally, objective and subjective evaluations were carried out in 47 chickens. The number of chickens in different groups was unequal . Means \u00b1p-value<0.001), width, and height of the spleen . The average measure ICC was calculated as 0.940 and 0.958 with a 95% confidence interval from 0.861 to 0.971 and 0.921 to 0.977 for the left and the right pectoral muscles, respectively, for the abovementioned measurements (p\u2009<\u20090.001). Additionally, a good interobserver reliability was observed during the evaluation of the AvHU of the spleen. The average ICC was 0.818 with a 95% confidence interval of 0.673\u20130.898 (p\u2009<\u20090.001). The intraobserver reliability was excellent in all the measurements , for the spleen\u2019s height in the normal/traumatic group , and for the length of the spleen in the egg peritonitis group . The highest MinHU of the spleen was calculated in the parasitic infection group , the highest MaxHU and SDHU were detected in the viral infection group , the highest AvHU was calculated in the egg peritonitis group . Moreovep\u2009=\u20090.013, with a mean rank thickness of muscle of 21.93 for females and 35.86 for males. Thus, subsequently the suggested ratios in the present study containing the thickness of the muscles showed a significant difference between males and females. No statistically significant differences were detected in other parameters between females and males.The Kruskal\u2013Wallis H-test showed a statistically significant difference in the thickness of muscles between males and females, \u03c72(2)\u2009=\u20096.151, H-test showed that there was a statistically significant difference in the suggested ratios . WS/ MTH was different between different BCSs, \u03c72(2)\u2009=\u200911.131, p\u2009=\u20090.011, with a mean rank ratio WS/MTH of 30.32 for BCS 2, 16.31 for BCS 3, 18.33 for BCS 4, and 19.60 for BCS 5. HS/MTH was also different between different BCSs, \u03c72(2)\u2009=\u20098.952, p\u2009=\u20090.030, with a mean rank ratio HS/MTH of 29.68 for BCS 2, 18 for BCS 3, 17,67 for BCS 4, and 17.40 for BCS 5. Additionally, a significant difference was detected in ratio LS /MTH between different BCSs, \u03c72(2)\u2009=\u200913.784, p\u2009=\u20090.003, with a mean rank ratio LS/MTH of 30.86 for BCS 2, 18.69 for BCS 3, 11 for BCS 4 and 14 for BCS 5. No statistically significant differences were detected in other parameters of different BCSs.The Kruskal\u2013Wallis r\u2009=\u20090.339, n\u2009=\u200914, value of p\u2009=\u20090.02).A positive correlation exists between the width, height, and length of the spleen . FurtherThe PCA showed a large overlap of the spleen dimensions in different groups of diseases, BCS, and genders . UnfortuFinally, 7.The present study revealed no statistical differences in the spleen size, attenuation, and suggested indices between the normal and diseased groups. The results showed an excellent interobserver reliability in measuring the thickness of the pectoral muscles as well as the length, width, and height of the spleen, while a good interobserver reliability was observed during the evaluation of the AvHU of the spleen. The intraobserver reliability was excellent in all the other measurements.The role of the spleen is fully understood in mammals, and the probable causes of splenomegaly are well described ; howeverSimilar to other species, the assessment of the avian splenic size is mainly subjective . BecauseFurthermore, the spleen size has a high variability among individuals, it can be also affected by several factors and conditions . The splBasically, there are many controversial theories to explain the size of the spleen. As the factors that reduce the spleen size have a direct effect on the suppression of the immune response , it mighHowever, CT is a diagnostic imaging modality with an excellent reproducibility for objective evaluation of nodules and small structures especially larger than 10\u2009mm, while measurement of small irregular structures stays challenging , 21. TheSmith (2004) emphasized on the necessity of presenting valid indices to evaluate the spleen size . In the In the latest part of the subjective evaluation of the spleen by overlying the shapes of the spleens in all the birds, the common shape was almost the shape of a pistachio nut, which also was presented previously by John in 1994 . AdditioThe lack of research that described the CT features of the chicken spleens in normal and diseased patients limits a proper discussion and comparison of the findings. An additional limitation of this study is the absence of any pathological examination (as the chicken survived), aside from the abovementioned intrinsic limitation due to the retrospective inclusion of the referral patients and therefore unequal sample size in the different diseased groups. Accordingly, the spleens were not removed and evaluated grossly for true size, nor were the spleen tested histopathologically. Thus, it should be considered that more than one disease process may apply to each bird.In conclusion, measuring the spleen dimensions and attenuation has high reproducibility using the same guideline, thus the authors suggest their guideline for routine clinical application. Based on the present results, the computed tomographic measurements of the spleen could not predict the clinical disease of the chickens; however, this result may be affected by the limitations of the retrospective nature of this study. Hence, further evaluations are recommended to evaluate the effect of the different diseases on spleen presentation.The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because the study was a retrospective design, so no institutional animal care and use approvals were requested officially and a written informed consent was obtained from the owners for the participation of their animals potentially in retrospective studies at the time of admission in the hospital. Written informed consent was obtained from the owners for the participation of their animals in this study.YV and SB: conception and design. YV, MG, CK, and SB: acquisition of data, revising article for intellectual content, and agreement to be accountable for all aspects of the study in ensuring that questions related to the accuracy or integrity of any part of the study are appropriately investigated and resolved. YV and SB: analysis and interpretation of data. YV and SB: drafting the article. All authors contributed to the manuscript and approved the submitted version.The Article Processing Charges (APC) for the current manuscript was paid from the resources of the Open Access Fund of the University of Veterinary Medicine Vienna (Vetmeduni).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Moreover, whole-blood glycated haemoglobin (HbA1c), as a marker of average glycaemia, was also measured and a correlative analysis of this parameter with metal concentrations performed. Significant alterations in the plasma concentrations of magnesium, zinc (both increased by 13.2% and 25.2% respectively) and copper (decreased by 7.9%) were observed over this period (plasma selenium concentration was unchanged), with BMI values correlating with plasma magnesium (p = 0.004) and zinc (p = 0.022) concentrations. At 9 months post-surgery, an increase in mean zinc/copper ratio was observed (0.86\u00b10.29 compared to 0.63\u00b10.14 pre-surgery). Comparison of whole-blood HbA1c concentrations pre- and post-surgery revealed a reduction from 6.50\u00b11.28% pre-surgery to 5.51\u00b10.49% post-surgery. Differences in plasma HbA1c and magnesium at either pre- and post-surgery correlated significantly, as did HbA1c and magnesium levels when pre- and post-surgery values were analysed together. Collectively, this work reveals that bariatric surgery, in conjunction with lifestyle/dietary changes, lead to improvements in the nutritional status of magnesium, zinc and copper. Furthermore, the observed improvements in magnesium and zinc were associated with weight loss and in the case of magnesium, to better glycaemic control.Obesity enhances the risk of type-2 diabetes, cardiovascular disease and inflammatory conditions and often leads to metal dyshomeostasis, which contributes to the negative health aspects associated with the disease. In severe cases, bariatric surgery can be recommended to achieve sustained weight loss and improvement in health. Here, magnesium, zinc, copper and selenium concentrations were examined in 24 obese patients before and 9 months after undergoing Roux-en-Y gastric bypass surgery. All patients lost weight over this period, with the mean BMI reducing from 51.2\u00b17.1\u00a0kg/m The worldwide prevalence of obesity has increased over the past 50 years, to reach what are described to be pandemic levels is 30\u00a0kg/ms Bl\u00fcher , and is s Bl\u00fcher . Obese is Bl\u00fcher . Althougs Bl\u00fcher . These c2), or those with milder obesity (BMI of 35\u201340\u00a0kg/m2) in addition to other significant disease, surgical procedures often represent the best strategies for intervention. These can be recommended when non-surgical measures have been attempted but the person has either not achieved, or been able to maintain clinically beneficial weight loss. An individual must also be assessed as fit for anaesthesia/surgery and commit to long-term follow-up and lifestyle changes are also prescribed. However, these generally have a high cost and are often associated with unwanted side\u00a0effects and only relatively modest weight loss , and associated dietary recommendations influence total plasma concentrations of magnesium, copper, zinc and selenium in patients before and 9 months after surgery. We also explore whether potential relationships exist between the plasma concentrations of these nutrients and both BMI and glycaemic status.A total of 24 Roux-en-Y surgical patients were recruited from York Hospital, York, United Kingdom. Male or female patients over the age of 18 referred to the hospital for gastric bypass surgery for obesity were included. Patients were excluded if they were under 18 years old, were diagnosed with type 1 diabetes or endocrine disorders other than type 2 diabetes, had a history of alcohol or drug abuse, had a significant psychological history, had history of deep vein thrombosis or pulmonary embolism, were taking warfarin, were pregnant, developed post-operative complications or had a history of active malignancy. With the exception of one participant (who was of Asian-British decent), all participants were Caucasians. Plasma samples were collected following approval by the National Research Ethics Service Committee Yorkshire & The Humber - Sheffield (REC reference: 11/H1308/16) after obtaining written informed consent. Two\u00a0weeks before surgery patients were placed on a low-calorie/low-fat diet, containing 800\u20131000\u00a0kcal/day. At this point, they commenced taking an over the counter multi-vitamin pill until 4 weeks post-surgery. After surgery patients were given a puree diet for the first 4 weeks . They were then either prescribed Forceval multivitamin , magnesium (30\u00a0mg), selenium (50\u00a0\u00b5g) and zinc (15\u00a0mg) or advised to take another complete multi-vitamin and mineral supplement of their choosing. Other supplements given post-operatively after 4 weeks were ferrous fumarate (210\u00a0mg twice a day for menstruating women or once daily for non-menstruating women and men), ferrous gluconate (300\u00a0mg once daily), Adcal D3 , lansoprazole FASTAB and vitamin B12 injection (1\u00a0mg intramuscular injection every 3 months). For all subjects, blood samples were collected in both lithium heparin and EDTA tubes before surgery (no more than 48\u00a0h before the operation) and not before nutritional intervention and at a 9-month (\u00b11 week) follow-up appointment after surgery. EDTA-treated whole\u00a0blood was used to measure HbA1c by the Laboratory Medicine service at York Hospital using a boronate affinity chromatography-based method traceable to the National Institute of Standards and Technology, USA (NIST) were used as calibration and internal standards. All standards were prepared in 2% v/v butan-1-ol , 0.05% w/v EDTA (Sigma Aldrich), 0.05% v/v Triton-X-100 (Sigma Aldrich), 1% v/v ammonia in 18.2 M\u2126 deionised water . Plasma samples (collected in lithium heparin tubes) were diluted 1 in 10 with the same solution used to prepare standards with 25 ppb germanium . ClinChek\u00ae 1 & 2 and Seronorm\u2122 1 human serum certified reference materials were used to demonstrate accuracy. ClinChek\u00ae1 and 2 are materials with consensus values while Seronorm\u2122 is traceable to a NIST-certified reference material.3 volume) was prepared in-house using 0.8 mm internal diameter and polytetrafluoroethylene sample tubing (Agilent Technologies). A quartz torch with a 2.5 mm internal diameter was used (Agilent Technologies). Nickel sampling and skimmer cones were always used (Agilent Technologies). Instrument parameters were optimised each day by performing an auto-tune while aspirating a tuning solution containing 10 ppb of lithium, yttrium, cobalt, cerium and thallium. Typical instrument parameters were as follows: isotopes monitored were 24Mg, 63Cu, 66Zn, and 78Se; radio frequency (RF) power = 1550\u00a0W; RF matching = 1.70; sampling depth = 10 mm; carrier gas = 1.05\u00a0L/min; make up gas = 0\u00a0L/min; spray chamber temperature = 2\u00a0\u00b0C; helium octopole reaction system flow = 5.0 mL/min; nebuliser pump = 0.1 rps. Three certified reference materials (Seronorm 1 and ClinCheck 1 and 2) with assigned values and ranges were used to demonstrate the accuracy of the method used to determine the concentrations of Mg, Cu, Zn and Se in human plasma , controlled using Mass Hunter software . Argon was used to form the plasma . Polyatomic interferences for magnesium, copper, zinc and selenium were removed through collision-induced dissociation and kinetic energy discrimination using helium gas . The concentration of all elements was measured three times within a single run using the central 0.05\u00a0m/z of the peak. The ICP-MS was equipped with a CETAC ASX-500 series autosampler , an Integrated Sample Introduction System and Discrete Sampler-3 and a G32992A re-circulating chiller . The ISIS-DS was fitted with a Quartz Scott-type, double-pass spray chamber (Agilent Technologies), a glass micro-mist nebuliser (Agilent Technologies) and a sample loop. The sample loop (0.63 cmData are presented as mean \u00b1 standard deviation (SD). Graphs were generated and statistical analysis was performed using Prism 9.0 . Data were distributed normally and differences between groups were analysed using multiple Student\u2019s t-tests (paired or non-paired as appropriate), while correlations between linear variants were analysed with Pearson\u2019s correlation. Significance threshold in all cases was set at p\u22640.05.Anthropometric values and whole-blood concentrations of HbA1c and plasma trace elements were measured and compared in the 24 patients before and 9 months after Roux-en-Y gastric bypass surgery. Table\u00a02 pre-surgery to 37.2\u00b15.5\u00a0kg/m2 (p < 0.0001). Significant differences were also observed in the waist, hip and neck circumference, with the waist-to-hip ratio decreasing from 1.07\u00b10.08 to 1.01\u00b10.08 (p < 0.0001). These measurements indicate the treatment was successful in promoting substantive weight loss by 9 months post-surgery. The mean whole-blood HbA1c concentration reduced from 6.50\u00b11.28% to 5.51\u00b10.49% (p < 0.0001), suggesting that the intervention led to improved glycaemic control in the cohort. Indeed, 11 of the patients (54.2%) were dysglycaemic (defined as an HbA1c concentration \u22656.0%) before surgery, while only 2 (8.3%) remained dysglycaemic 9 months after surgery. Unsurprisingly, a greater reduction in HbA1c concentration was observed in those with dysglycaemia before surgery. Prior to surgery, 7 of the participants were prescribed metformin and 6 were prescribed a statin. At 9-months post-surgery, 2 were still prescribed metformin and 5 were still taking a statin.Over the period, the mean weight of the cohort reduced by 29.5% from 150.9\u00b134.9\u00a0kg pre-surgery to 106.4\u00b124.5\u00a0kg post-surgery (p < 0.0001) and the BMI by 28.3% from 51.2\u00b17.1\u00a0kg/mSignificant differences in the mean plasma concentrations of zinc , copper and magnesium were observed 9 months after surgery. Of note, a decrease in copper levels was evident even given supplement therapy with this metal. No difference was observed in mean plasma selenium concentration post-surgery over this period. Also, no significant differences were observed in the plasma concentrations of zinc, magnesium, copper or selenium between dysglycaemic and normoglycaemic individuals either before surgery or 9 months after surgery.The normal plasma reference intervals for the four analysed trace elements are shown in Table\u00a0A comparison of mean anthropometric values and mean whole-blood HbA1c and plasma trace element concentrations pre- and post-surgery between males and females was carried out Table\u00a0; Fig.\u00a02.Plasma concentrations of magnesium, copper, zinc and selenium from individual patients were compared to respective BMI values measured either before or 9 months after surgery. No statistically significant correlation between pre-surgery values or between the post-surgery values were observed (where p\u22640.05 is considered significant). Similarly, no correlation was observed when differences in plasma metal concentrations between pre- and post-surgery values were compared to respective differences in BMI values in individual patients . However significant correlations were observed between BMI values and both plasma magnesium (p = 0.004) and zinc p = 0.031) concentrations when both pre-and post-surgery values of these parameters across individuals were compared together in all patients . A correlation between changes in HbA1c and magnesium concentration pre- and post-surgery was observed lead to substantive weight loss and reduction in BMI and other anthropometric measures over a 9-month period. In addition, favourable outcomes relating to glycaemic status (as indicated by mean whole-blood HbA1c concentration) were apparent in this study. Modest improvement in plasma concentrations of magnesium and copper (and zinc/copper ratio) was observed 9 months after surgery. Although changes were generally within the normal range, improvement in the status of these nutrients would potentially impact positively on health aspects including appetite regulation through control of leptin levels, improvement in glucose metabolism and insulin action and a reduction in thrombotic risk. Whilst individual metals exhibited modest improvement, the mean zinc/copper ratio was much improved across the cohort and would be expected to lead to an enhancement in the activity of enzymes utilising these metals for catalysis. In a few individuals, surgery and dietary/lifestyle changes led to abnormally high values of magnesium, copper or zinc, which if unchecked, have the potential to lead to additional health issues. These observations suggest that, while bariatric surgery and the subsequent nutritional treatments offered to the patients lead to improved health outcomes, treatment may be further optimised by monitoring plasma metal levels and the specific nutritional supplements taken by patients after bariatric surgery."} +{"text": "Whole-exome sequencing (WES) significantly improves the diagnosis of the etiology of fetal structural anomalies. This study aims to evaluate the diagnostic value of prenatal WES and to investigate the pathogenic variants in structurally abnormal fetuses.We recruited 144 fetuses with structural anomalies between 14 and 2020 and 15 December 2021 in the study. Genetic screening was performed by WES combined with karyotyping and chromosomal microarray analysis. The molecular diagnostic yield of prenatal WES for each type of fetal structural anomaly and the identified pathogenic genes and mutations were reported.EP300, MYH3, TSC2, MMP9, CPLANE1, INVS, COL1A1, EYA1, TTC21B, MKS1, COL11A2, PDHA1 and L1CAM). Five additional pathogenic variants were classified as incidental findings. Our study showed that the overall diagnosis rate of WES was 28.1% (27/96) in the parent-fetus trio cases and 16.3% (8/49) in the proband-only cases. Fetuses with musculoskeletal anomalies had the highest diagnostic yield . In addition, FGFR3 and COL1A1 were the most common pathogenic genes.In this study, we retrospectively analyzed the clinical and genetic data of 145 structurally anomalous fetuses. These cases were classified into 9 phenotypic classes based on antenatal ultrasound findings. Thirty-eight pathogenic variants in 24 genes were identified in 35 of the 145 cases, including 14 novel variants in 13 genes (Our work expands the mutation spectrum of the genes associated with fetal structural anomalies and provides valuable information for future parental genetic counselling and pregnancy management of the structurally anomalous fetuses.The online version contains supplementary material available at 10.1186/s12920-023-01697-3. Prenatal ultrasound is a routine screening method for fetal malformations, identifying fetal structural anomalies in approximately 3% of fetuses . The anoFetal structural anomalies are usually caused by pathogenic sequence variants and chromosomal aberrations in development-related genes . Soft ulLBR, SLC26A2, FLNB, COL1A2, COL2A1, and FGFR3, have been found by WES to be frequently associated with skeletal dysplasia in fetuses. Nevertheless, incomplete phenotypic identification due to the limitations of ultrasound examination and insufficient data on the functions of many candidate genes make it very difficult to determine the clinical significance of variants identified by WES [WES has become a powerful and cost-effective diagnostic approach for genetic disorders , 5. Seved by WES \u201322. The In this study, we retrospectively analyzed the clinical and genetic data of 145 structurally anomalous fetuses. These cases were classified into 9 phenotypic classes based on antenatal ultrasound findings. Genetic screening was performed by WES combined with karyotyping and CMA. The molecular diagnostic yield of prenatal WES for each type of fetal structural anomaly and the identified pathogenic genes and mutations were reported. These data provide valuable information for advancing the clinical application of prenatal WES and for establishing systemic genotype-phenotype associations in prenatal diagnosis.From 14 to 2020 to 15 December 2021, we recruited 145 pregnancies in which the fetuses showed structural anomalies during 11\u201332 weeks by prenatal ultrasonography at the medical genetics centre of the Maternal and Child Health Hospital . The fetuses had all undergone common tests such as intrauterine infection and autoimmune screening at the hospital by obstetricians. Only those fetuses with no clear cause underwent further genetic screening. Clinical information including maternal age, gestational age, family history, and obstetric history were recorded in detail. This study was approved by the institutional ethics committee. All parents of the fetuses agreed to participate in the study and provided signed informed consent. Clinical geneticists were mainly responsible for interviewing participants, collecting data/samples and providing pre- and post-test genetic counselling to participants. Human phenotype ontology (HPO) terms were used to standardize clinical features in the study.Sample extraction was performed at different stages depending on the situation. For most pregnant women, amniocentesis was performed at 18\u201332 weeks of gestation to collect amniotic fluid. A few pregnant women underwent chorionic villus sampling at 12\u201314 weeks of gestation. Genomic DNA was extracted from amniotic fluid/ chorionic villi, and peripheral blood samples from both biological parents using the Qiagen DNA Blood Midi/ Mini Kit according to the manufacturer\u2019s protocol.WES of parent-fetus trios (fetuses and parents) and proband-only (fetuses) was performed on DNA samples at Berry Genomics Corporation. WES samples are also subjected to short tandem repeats (STR) to exclude maternal contamination for parentage analysis. Massively parallel sequencing was performed using NanoWES and the Novaseq6000 platform . The following metrics were achieved for all samples: mean coverage depth was ~\u2009100\u00d7, and ~\u200995% of target bases were interrogated at >\u200920\u00d7 read depth.http://picard.sourceforge.net/). Single nucleotide variants (SNV) and insertion/deletion (InDel) variants were detected using the Verita Trekker\u00ae Variants Detection System and GATK (https://software.broadinstitute.org/gatk/). Detected variants were annotated using ANNOVAR (http://annovar.openbioinformatics.org/en/latest/) and Enliven\u00ae Variants Annotation Interpretation System, authorized by Berry Genomics. Nonsynonymous, splicing, and InDel variants were filtered by the 1000 Genome Project (http://browser.1000genomes.org), gnomAD (http://gnomad.broadinstitute.org/), Berrybig data population database, dbSNP (http://www.ncbi.nlm.nih.gov/snp) etc. Variants with a minor allele frequency (MAF)\u2009>\u20090.01 were discarded. The whole exome was analyzed and late onset disorders were filtered out for each case in this study.Raw image files were processed using CASAVA v1.82 for base calling to generate raw data. The raw sequence data were aligned to the human reference genome (hg38) using the Burrows-Wheeler Aligner tool. The PCR duplicates were removed using Picard v1.57 and scientific reports. Sanger sequencing was used to confirm the inheritance of the likely pathogenic/ pathogenic variants for parent-fetus trios and proband-only cases. CNV-calling was performed using Sprinkle, a comprehensive tool developed by Berry Genetics. More specifically, XHMM PCA was used to remove sequencing background. CNVKit fixed module was used for GC and bias correction, copy number calculation in exons and long fragment regions, and CNV identification. The window size was 200\u00a0bp and the normal reference included 30 samples. Interpretation was based on CNV type, size, frequency, number of key genes and recurrence region according to ACMG guidelines. DECIPHER (https://www.deciphergenomics.org/), DGV (http://dgv.tcag.ca/dgv/app/home) and ClinGen were used to assess CNV pathogenicity. Copy number variants (CNVs) identified by WES were further investigated by quantitative PCR.Relevant inheritance patterns, family history and clinical manifestations were considered during variant interpretation. Variants relevant to the corresponding phenotypes were evaluated for pathogenicity according to the adapted American College of Medical Genetics and Genomics (ACMG) guidelines . Genetic variants were screened under the combined consideration of pathogenicity prediction using multiple tools , relative hits from different databases were occasionally included when there was a strong indication for reporting . The highly penetrant pathogenic variants that were unrelated to the fetal phenotypes were classified as incidental findings. These variants were predicted to cause moderate to severe childhood onset disorders, such as neurodevelopmental disorders and metabolic disorders. Additional relevant tests were usually carried out on the fetuses before or after birth. The results are explained in detail to the parents by clinical geneticists. Statistical comparisons were made using the Chi-square test.n\u2009=\u200937), multisystem (n\u2009=\u200936), genitourinary system (n\u2009=\u200924), and nervous system (n\u2009=\u200913) (Table\u00a0n\u2009=\u200912). The 145 cases include 96 fetus-parental trios and 49 proband-only cases. In all cases, the chromosomal aberrations were excluded by means of karyotyping and CMA.The 145 cases were classified into 9 phenotypic groups based on the ultrasound results Table . Most can\u2009=\u200996), the diagnostic rate of WES was 28.1% (27/96). In contrast, the diagnostic rate of WES in the proband-only cases (n\u2009=\u200949) was 16.3% (8/49) (Table\u00a0p\u2009<\u20090.05). In addition, variants were classified as VUSs in 13 cases . Four cases carrying 5 likely pathogenic/pathogenic variants that were not considered phenotypically correlated were classified as incidental findings. In the parent-fetus trio cases were autosomal dominant (AD), 22.8% (n\u2009=\u20098) were autosomal recessive (AR), and 8.6% (n\u2009=\u20093) were X-linked (XL) and PDHA1 (c.766G\u2009>\u2009A) were detected in two cases with central nervous system abnormalities. A pathogenic variant, c.5527dup in EP300, was identified in the case with craniofacial abnormalities.Importantly, 14 novel pathogenic variants were identified in the study Table\u00a0. Five noSMPD4 in trans with a deletion of 59.72\u00a0kb spanning SMPD4 was found in a fetus with arthrogryposis . The clinical significance of the BBS2 variant was further determined by the family history.carried a homozygous stopgain variant in FGFR3 are known to cause autosomal dominant achondroplasia and hypochondroplasia. However, a homozygous missense variant in FGFR3 was identified in one case (case 3084) with shortened long bones was inherited from the proband\u2019s parents affected by dwarfism. Two pathogenic gene variants were detected by WES in a fetus (case 4714) with shortened long bones, multiple kidneys and polyhydramnios by WES. These two diagnoses could clearly explain the structural abnormalities of the fetus.Pathogenic variants in es Table\u00a0. FurtherTTN gene were identified. TTN is usually associated with cardiomyopathy and muscular dystrophy, which was not relevant to the ultrasound findings in this case. Another example is a de novo heterozygous variant in FGFR1, which was identified in case 8212 with abnormalities of the cerebral ventricles and septum pellucidum and the agenesis of the corpus callosum. However, FGFR1 mutations are often associated with Hartsfield syndrome, which is characterized by holoprosencephaly, ectrodactyly and cleft lip/palate [Incidental genetic variations were detected in four cases Table\u00a0. These vp/palate .de novo heterozygous variant in the TRIP12 gene was detected in the case 2907 with abnormal feet. Pathogenic variants in TRIP12 are often associated with neurodevelopmental disorders, such as developmental delay and intellectual disability. The clinical significance of TRIP12 variants should be confirmed by adequate postnatal follow-up. ARID1B pathogenic variants are associated with several clinical features, including intellectual disability, developmental delay, severe speech delay, corpus callosum abnormalities, etc. The ARID1B variant was classified as an incidental finding in case 6428, because most of the clinical features associated with neurodevelopmental disorders are difficult to observe on prenatal ultrasound.A FGFR3 (n\u2009=\u20099) and COL1A1 (n\u2009=\u20094) were the most common diagnostic findings in this study with skeletal dysplasia. The variants c.1138G\u2009>\u2009A in FGFR3 were detected six times. Four missense variants in COL1A1 were detected in four different cases and 16.3% (8/49), respectively. These findings provide valuable information to promote the clinical application of prenatal WES and to establish genotype-phenotype associations in prenatal diagnosis.de novo and compound heterozygous variants. Among the 35 cases with a specific molecular diagnosis in this study, the pathogenic variants in 20 cases were de novo, accounting for 57.1%. This result suggests that de novo variants may be the predominant mutation in fetuses with structural anomalies. In addition, 15 fetuses had pathogenic variants inherited from their parents, suggesting the importance of genetic counselling and carrier screening for pregnant women.Several recent studies have shown that prenatal WES achieves diagnostic rates of 20\u201324% in fetuses with structural anomalies with normal karyotype and CMA , 26, 27.The diagnostic yield of 51.4% gene variants in cases with musculoskeletal anomalies was significantly higher than in other cases with multisystem anomalies (22.2%), nervous system anomalies (15.1%), and genitourinary system anomalies (12.5%). Furthermore, we found a higher diagnostic yield in fetuses with musculoskeletal anomalies than in those with an anomaly excluding the skeletal anomalies . This may be because the fetuses with abnormal skeletal system development have more distinct phenotypic features, just as other researchers have claimed that a specific organ system anomaly may have a higher diagnostic yield than a single structural anomaly .FGFR3 is a pathogenic gene that causes for achondroplasia with shortened long bones and over 80% of cases have been found with spontaneous gene mutations [FGFR3 were identified in 9 cases of skeletal dysplasia. Among these, a homozygous FGFR3 variant (G380R) in case 3084 was inherited from the parents of the proband affected by dwarfism, which is a rare finding. Six cases carried the same pathogenic variant G380R in FGFR3. Of these, two cases carrying the G380R variant presented with shortened long bones, and four other FGFR3 cases presented with shortened long bones and polyhydramnios. Case 6411, carrying the N540K variant, presented with short femur length only. However, the remaining two cases (3031 and 2919) with thanatophoric dysplasia had short limbs and a small chest. In addition, the FGFR3 G380R variant was found in the father with dwarfism, but not in the fetus with skeletal dysplasia, indicating the added value of trio WES.utations . Four hede novo heterozygous variant in the TRIP12 gene. Haploinsufficiency of TRIP12 has been reported to cause childhood-onset neurodevelopmental disorder including intellectual disability [TRIP12 gene variant was uncertain.On the other hand, WES can detect incidental findings that cannot be detected by prenatal imaging. For example, neurodevelopmental disorders such as developmental delay and intellectual disability cannot be detected by routine prenatal testing. Case 2907 with abnormal feet was found to carry a sability , as impoSMPD4 gene was identified with the performance of the CNV calling algorithm in WES data analysis . This suggested that the WES-based CNV calling tool could be applied as an effective way to improve the diagnostic quality and develop WES as a more powerful method in prenatal diagnosis.Remarkably, a compound heterozygous mutation of pathogenic CNV and SNV in the This study reported 14 novel variants with a clinical significance of pathogenic or likely pathogenic among the diagnostic cases. Our study suggests that WES can broaden the mutation spectrum of genes associated with fetal structural anomalies, which would further benefit the prenatal diagnosis and genetic counselling in the future. WES also has the potential to detect new causative genes for specific developmental disorders and to establish new fetal phenotype-genotype correlations in the remaining cases without genetic diagnosis .FGFR3 and COL1A1 are the most common pathogenic genes in musculoskeletal disorders, suggesting a strong genotype-phenotype correlation. Although some pathogenic genes recurred in the test results of different cohorts, the most pathogenic genes are only reported in a single study. The results indicate that the pathogenic molecular mechanism is complicated with a high degree of genetic heterogeneity and that a complete gene network needs to be discovered. WES gene variation should to be further investigated by combining of prenatal and postnatal phenotyping to provide reliable genetic diagnosis [Comparing our results with eight other recent studies on fetal structural abnormalities showed that iagnosis , 33.In this study, WES was used to identify pathogenic gene mutations in fetuses with a wide range of structural anomalies. Our study will not only widen the mutation spectrum of pathogenic gene variations causing fetal structural anomalies, but also provide valuable information for prenatal counselling.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4"} +{"text": "Whole exome sequencing (WES) has become part of the postnatal diagnostic work-up of both pediatric and adult patients with a range of disorders. In the last years, WES is slowly being implemented in the prenatal setting as well, although some hurdles remain, such as quantity and quality of input material, minimizing turn-around times, and ensuring consistent interpretation and reporting of variants. We present the results of 1 year of prenatal WES in a single genetic center. Twenty-eight fetus-parent trios were analyzed, of which seven (25%) showed a pathogenic or likely pathogenic variant that explained the fetal phenotype. Autosomal recessive (4), de novo (2) and dominantly inherited (1) mutations were detected. Prenatal rapid WES allows for a timely decision-making in the current pregnancy, adequate counseling with the possibility of preimplantation or prenatal genetic testing in future pregnancies and screening of the extended family. With a diagnostic yield in selected cases of 25% and a turn-around time under 4 weeks, rapid WES shows promise for becoming part of pregnancy care in fetuses with ultrasound anomalies in whom chromosomal microarray did not uncover the cause. Major congenital anomalies (MCA) have a prevalence of 2\u20133% and are responsible for a significant percentage of pre- and perinatal demise and neonatal morbidity ,2. The eChromosomal microarray analysis (CMA) has been widely implemented in the analysis of invasively obtained prenatal samples (amniotic fluid or chorion villi) for the genome-wide detection of both aneuploidies and microdeletions/microduplications (copy number variants or CNVs). In up to 40% of pregnancies with a fetal structural anomaly, CMA is able to diagnose an aneuploidy or CNV , still lSeveral recent metaanalyses have demonstrated an added diagnostic yield of 1.8\u201368% for prenatal whole exome sequencing (WES), with the yield largely depending on the inclusion criteria and organ system affected ,5,6,7,8.This paper describes the experiences of a single Belgian genetic center with the implementation of WES in the prenatal diagnostic workflow. In Belgium, the molecular analysis of publicly funded invasive prenatal diagnosis can only be executed at one of the eight Centers for Medical Genetics. For all indications, a genome-wide microarray is performed with national consensus guidelines in place steering the interpretation and reporting of the results . RecentlHere we discuss the opportunities and challenges for the use of WES in the diagnosis of fetuses with ultrasound abnormalities and provide suggestions for implementation of this valuable technique in other labs.Genomic DNA was extracted from either amniotic fluid, chorion villi or cultured amniocytes using the Maxwell RSC Blood DNA kit on a Maxwell RSC 48 Instrument . Library prep on 50ng of genomic DNA was performed using the Twist Human Core Exome kit according to the manufacturer\u2019s instructions on a Hamilton STAR robot . Twenty-four libraries were pooled equimolarly for sequencing on a NextSeq500 or NextSeq550 instrument with a 2 \u00d7 75 bp or 2 \u00d7 150 bp flow cell . WES data were analyzed using an in-house developed pipeline which considers only de novo, X-linked and recessive variants, either in a predefined panel or exome-wide . Additiowww.college-genetics.be (accessed on 1 December 2022)). The following criteria must be met: (1) The fetus shows ultrasound anomalies, but CMA is negative; a diagnosis is essential to guide the pregnancy/neonatal management; (2) All cases should be reviewed in a multidisciplinary team including a clinical geneticist; (3) Expert fetal ultrasound examinations are required to provide the best possible phenotypic evaluation. When beneficial, fetal MRI may be performed; (4) Pretest counseling by a clinical geneticist is mandatory, with signed informed consent by both parents; (5) Trio analysis is strongly recommended to speed up the process. Variant classification is performed based on the ACMG guidelines [in trans with a pathogenic or likely pathogenic variant and for which further clinical exams are recommended to refine variant classification, possibly leading to a genetic diagnosis (upgrade of the variant to class IV/V). By national agreement, no systematic search for secondary findings, unrelated to the fetal phenotype, is performed, in line with the framework proposed by Vears et al. [The guidelines for prenatal WES were developed at a national level and can be found at the website of the Belgian College of Genetics for exclusion of the common aneuploidies and triploidy as well as for determination of maternal cell contamination and fetal identity through comparison to the maternal profile. Next, CNV detection by CMA is performed, more precisely a SNP (single nucleotide polymorphism) array with a 400 kb resolution. However, this combined approach yields a diagnosis in less than 25% of cases: on 3453 analyses that were performed over the past nine years, QF-PCR and SNP array were positive in 786 cases (22.8%), among which 557 with a trisomy , 30 with a triploidy (respectively 3.8% and 0.87% of cases), 62 with monosomy X (respectively 7.9% and 1.8% of cases) and 134 with a subchromosomal pathogenic anomaly (respectively 17% and 3.9% of cases) .COL2A1 variant , thumb hypoplasia on the left hand and absent thumb on the right hand. The fetus with the KMT2D variant had talipes equinovarus as well as abnormal placing of the ears.Since January 2021, our center offers the subsequent option of whole exome sequencing (WES). In 2021, WES was performed in our center on 28 prenatal cases showing structural anomalies on ultrasound see . Twenty iant see , a homozMUSK variant . In all but one case, the parents opted for a termination of the pregnancy involved , the hiwww.college-genetics.be (accessed on 1 December 2022)). However, prenatal WES has not been implemented widely, as many hurdles still remain. Issues involve (1) the quality and quantity of the starting material; (2) the short TAT; (3) the interpretation of variants; (4) the ethical perspective.In Belgium, prenatal WES in a diagnostic setting is publicly funded and fully reimbursed. National guidelines describing the inclusion criteria, pre- and posttest counseling and the filtering and reporting strategy have been developed by a committee of laboratory and clinical geneticists and are publicly available on the website of the Belgian College for Human Genetics and Rare Diseases , associated with Noonan type 8 (OMIM# 615355): hydrops, ascites and hydrothorax, which were all present in this fetus, are frequent ultrasound markers in RASopathies.Correlating the genotype with the prenatal phenotype was challenging. In general, the fetal phenotype of many conditions has not been well described and may deviate quite substantially from the known postnatal phenotype. Cataloguing the phenotype of prenatal-onset syndromes is of utmost importance to guide healthcare providers in recognizing these syndromes at an early stage and know their evolution throughout the pregnancy ,17. The STXBP1, that was reported as incidental finding because of its association with developmental and epileptic encephalopathy 4 (OMIM# 612164). Given the severe phenotype of seizures, profoundly impaired psychomotor development, limited or absent ability to walk, spastic quadriplegia and poor or absent speech, prenatal testing in a future pregancy is warranted as parental gonadal mosaicism cannot be ruled out.Given the genotypic heterogeneity of Noonan syndrome and by extension non-immune hydrops fetalis , WES or COL2A1 missense mutation (case 10) displayed rhizomelic shortening and bowing of the long bones as well as microretrognathia and clenched hands on ultrasound (KMT2D (OMIM# 147920). Kabuki syndrome shows prenatal phenotypic heterogeneity, with ultrasound abnormalities that are non-specific. The most frequent ultrasound features include cardiac anomalies (49.4%), followed by polyhydramnios (28.9%), genitourinary anomalies (26.5%), single umbilical artery (15.7%), intrauterine growth restriction (14.5%) and hydrops fetalis/pleural effusion/ascites (12.0%) [FANCG nonsense variant (OMIM# 614082). FA is an autosomal recessive disorder with both phenotypic and genotypic heterogeneity, but major birth defects such as skeletal malformations , microcephaly, genitourinary malformations and intrauterine growth restriction are present in 75% of the cases. Consequently, these findings in the prenatal setting are suggestive of FA, although absence of skeletal anomalies does not exclude FA [In case of a suspicion of a fetal skeletal dysplasia, the value of adding WES to the prenatal diagnostic tools has been demonstrated before ,21,22; ttrasound a,b. The trasound c\u2013e. Anal (12.0%) . The fetclude FA . Radial clude FA ,26. RapiTHOC6, causing Beaulieu-Boycott-Innes syndrome (OMIM# 613680). There are seven reports of prenatally diagnosed Beaulieu-Boycott-Innes syndrome with variable clinical findings, such as IUGR, cerebral malformations, genito-renal abnormalities, cystic hygroma, retrognathia and suspicion of ventricular septal defect [MUSK). For fetal akinesia deformation sequence 1 (FADS1), caused by homozygous MUSK mutations (OMIM# 208150), prenatal diagnosis is based on multiple contractures, reduced motility, flattening of facial profile and\u2014with increasing gestational age\u2014IUGR, reduced cardiothoracic ratio and polyhydramnios [CHRNA1 missense variant. Recessive mutations in the CHRNA1 gene result in lethal multiple pterygium syndrome . LMPS displays a heterogeneous range of prenatal manifestations that generally include cystic hygroma, pulmonary hypoplasia, cleft palate, cryptorchidism, joint contractures, fetal akinesia, heart defects, growth restriction and intestinal malrotation [Of eight fetuses with multisystem aberrations in our series, three (37.5%) were positive. The first (case 26) was compound heterozygous for four known missense variants in l defect . Our casdramnios . The ultIn total, WES was able to pinpoint the cause of the fetal anomalies in 25% of cases (7 out of 28). Among the seven positive cases, two were de novo, four recessive and one paternally inherited (from an affected parent). Multidisciplinary genetic counseling of the prenatal results was performed and except for the parents with the fetus diagnosed with paternally inherited Stickler syndrome, all chose to terminate the pregnancy after approval by the ethical committee of the University Hospital Antwerp. For all cases, the decision to terminate was based on the WES result, effectively demonstrating its use in the prenatal setting. For five out of seven families (71.4%), the recurrence risk is high and preimplantation or prenatal invasive genetic testing can be offered in future pregnancies. For the family with the dominantly inherited variant, testing in first-degree relatives of the father can be considered as well. In the families with a de novo variant, genetic testing in future pregnancies should be discussed because of the possibility of parental gonadal mosaicism.STXBP1, associated with developmental and epileptic encephalopathy type 4, in the fetus carrying the RIT1 mutation. In case of future prenatal invasive testing, presence of both the RIT1 and the STXBP1 mutation can be evaluated.Ethically, the most demanding issue is the possibility of incidental findings in both fetus and parents. Although rigorous filtering can reduce the number of incidental findings, they can never be fully excluded as this would jeopardize the identification of the primary variant(s) explaining the phenotype. Therefore, a genetic pretest counseling as well as informed consent by both parents are mandatory, so that they are well aware of the possible outcomes. The only incidental finding we encountered was a de novo mutation in The recent update of the ISPD position statement on prenatal WES states that although the available data is insufficient to recommend which categories of abnormalities warrant sequencing, there are \u2018sufficient data to begin differentiating diagnostic yields by specific organ system or number of organ systems affected\u2019 . Our resOur data set, although limited, clearly shows the added value of WES in the prenatal setting in case of MCA. The diagnostic yield of 25% demonstrates that the rigorous selection of prenatal cases according to our national guidelines is effective; yield is highest in cases with skeletal or multisystem anomalies. Furthermore, our findings demonstrate that WES can be implemented in a medium-throughput diagnostic lab with little failures and an acceptable TAT, effectively expanding the diagnostic portfolio that can be offered to future parents." \ No newline at end of file