diff --git "a/deduped/dedup_0436.jsonl" "b/deduped/dedup_0436.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0436.jsonl"
@@ -0,0 +1,39 @@
+{"text": "Recent studies suggest a high specificity of 99mTc-galactosyl neoglycoalbumin (99mTc-NGA) receptor scanning in vivo by providing both morphological and functional diagnosis of liver disease. In 22 patients with advanced breast cancer 99mTc-NGA  was exclusively trapped by the liver, the images showing 'cold spots' in areas of liver metastases formation. A two-tailed analysis was performed: the time activity curves recorded for the liver and precordial area were subjected to a kinetic receptor-calculating model allowing an estimation of the NGA-receptor concentration of the liver  as well as calculation of the residual functional liver volume (RFLV) via the S.P.E.C.T.-study. In breast cancer patients with liver metastases a significantly (P < 0.01) lower HBP-concentration was estimated (0.65 +/- 0.16 vs 0.82 +/- 0.17 mumol l-1) as evidenced by a lower 99mTc-NGA-accumulation in the liver resulting also in a significantly (P < 0.001) lower RFLV (739 +/- 348 vs 1336 +/- 184 ml). In four amonafide-treated patients (800 mg m-2 intravenous infusion over 3 h) approximately one week after one chemotherapy cycle a significant (P < 0.05) increase in HBP-concentration (0.56 +/- 0.10 vs 0.72 +/- 0.06 mumol l-1) of the liver was found corresponding with an increase in RVLF (546 +/- 297 vs 670 +/- 265 ml). These regulatory mechanisms at the HBP level measured in vivo provide further evidence that 99mTc-NGA should have promise as a clinically useful receptor radiopharmaceutical for both quantification of liver function and assessment of liver morphology."}
+{"text": "This is based upon chemical recognition and binding by the hepatic binding protein (HBP), a receptor specific for galactosylated glycoproteins. Liver tissue samples were obtained intraoperatively from patients undergoing surgery for various cancers. The concentration of specific HBP receptors in the liver  was calculated from the in vitro binding of 99mTc-NGA. One week after surgery, the in vivo HBP density was also measured in some of these patients after injection of 3.5 mg (50 nmol per patient) 99mTc-NGA (150-200 MBq) for simulation of 99mTc-NGA kinetics. Comparison of in vitro and in vivo HBP concentration in the liver showed values in the same concentration range. In patients with hepatoma or liver metastasis a significantly (P less than 0.01) decreased global HBP density was found in vivo compared to controls. The values obtained for in vivo HBP concentration in the liver amounted to 0.38 +/- 0.05 mumol l-1 liver for patients with hepatoma, to 0.4 +/- 0.1 mumol l-1 in patients with liver metastasis and to 94 +/- 0.05 mumol l-1 liver in cancer patients without liver malignancy. In vitro investigation of HBP density revealed the malignant liver tissue to have a significantly (P less than 0.0001) decreased or almost (completely) absent HBP receptor density compared to the normal tissue apart from the cancer area. It is concluded that determination of HBP density in vivo via a specific tracer is a new, simple and reliable approach for the determination of remaining hepatic function in patients with primary or secondary liver cancer."}
+{"text": "To the Editor: The genus Mycobacterium contains various obligate and opportunistic pathogens of animals, which may also be transmitted to humans and cause disease in, thus exhibiting a considerable zoonotic potential (Mycobacterium avium-intracellulare complex (MAIC) emerged as pathogens of human diseases, including lymphadenitis in children, pulmonary tuberculosis-like disease, and disseminated infections   IS1245 (901 (M. avium subsp. avium) are usually positive for IS901 and represent the main pathogen of avian tuberculosis , liver (schnauzer) and costal pleura (terrier).panel A). However, multinucleated giant cells or mineralization was not observed. In both animals, Ziehl-Neelsen stain demonstrated large numbers of acid-fast bacilli within macrophages (panel B). Samples of lymph nodes and lung were processed for mycobacterial culture by using standard procedures . Colonies emerging after 2-week incubation at 37\u00b0C were investigated by PCR targeting IS1245 and IS901 .Histologic examination showed (pyo-)granulomatous inflammation of lymph nodes, tonsils, liver, spleen, and greater omentum. Additionally, pyogranulomatous pleuropneumonia was present in the terrier, and a granulomatous enteritis and pyelitis in the schnauzer. The granulomatous lesions frequently exhibited central necrosis surrounded by macrophages, epitheloid cells, and few neutrophils , panel Arophages , panel BM. avium subsp. avium infections in humans. Thus, most of these HIV-related infections are attributed to M. avium subsp. hominissuis (M. avium was not identified in most canine cases reported in the literature (M. avium, corresponding to either M. avium subsp. avium or M. avium subsp. hominissuis, have been identified sporadically (M. avium infection for both humans and animals. However, there is also evidence of direct transmission (M. avium subsp. hominissuis infection in dogs may comprise a considerable zoonotic potential, particularly if pet dogs with close contact to the owner are affected and if prolonged nonspecific clinical signs and intestinal involvement occur, as demonstrated here.Despite improved therapeutic approaches, MAIC infection represents a frequent bacterial complication in persons with AIDS. However, several studies showed a very low incidence of"}
+{"text": "Oncorhynchus mykiss) from two seasonal spawning groups (September and December lots) within a single strain were examined when the fish were 15 months of age to assess the influence of season  and body size .Growth in fishes is regulated via many environmental and physiological factors and is shaped by the genetic background of each individual. Previous microarray studies of salmonid growth have examined fish experiencing either muscle wastage or accelerated growth patterns following refeeding, or the influence of growth hormone and transgenesis. This study determines the gene expression profiles of genetically unmanipulated large and small fish from a domesticated salmonid strain reared on a typical feeding regime. Gene expression profiles of white muscle and liver from rainbow trout (IGFBP1 gene expression was up-regulated in the livers of small fish in both seasonal lots, few expression differences were detected in the liver overall. Faster growing Dec. fish showed a greater number of differences in white muscle expression compared to Sept. fish. Significant differences in the GO Generic Level 3 categories \u2018response to external stimulus\u2019, \u2018establishment of localization\u2019, and \u2018response to stress\u2019 were detected in white muscle tissue between large and small fish. Larger fish showed up-regulation of cytoskeletal component genes while many genes related to myofibril components of muscle tissue were up-regulated in small fish. Most of the genes up-regulated in large fish within the \u2018response to stress\u2019 category are involved in immunity while in small fish most of these gene functions are related to apoptosis.Although A higher proportion of genes in white muscle compared to liver showed similar patterns of up- or down-regulation within the same size class across seasons supporting their utility as biomarkers for growth in rainbow trout. Differences between large and small Sept. fish in the \u2018response to stress\u2019 and \u2018response to external stimulus\u2019 categories for white muscle tissue, suggests that smaller fish have a greater inability to handle stress compared to the large fish. Sampling season had a significant impact on the expression of genes related to the growth process in rainbow trout. Growth in fishes is a complex trait that impacts many components of fitness. Large body size provides an advantage when competing for mates and resources and can provide energy reserves during times of famine or reproduction ,2. The iOncorhynchus mykiss) have QTL for body weight and/or condition factor with genome-wide effects, with an additional 8 linkage groups housing QTL with chromosome-wide effects ). However, with DDBP1 its associated substrate ligand is linked to the degradation of a cell cycle inhibitor [26s proteasome promotes muscle growth in rats [Small Sept. fish show up-regulation for a higher number of genes related to apoptosis compared to large fish despite the fact that as a portion of total genes differentially expressed, the \u2018response to stress\u2019 category contained a higher proportion of genes in large fish. Differences were apparent in the make-up of genes contributing to this GO category between the size classes. Genes up-regulated in large fish could be categorized as enhancers of innate and acquired immune function , transcnhibitor , and thu in rats  and therheat shock protein hsp 90-alpha (hsp90a), selanoprotein s (SelS), and der1-like domain member 2, are also up-regulated in small Sept. fish. Interestingly all three of these proteins may have regulatory interactions in that hsp90a is required for SelS and general selanoprotein synthesis, and SelS interacts with der1 in shuttling mis-folded proteins across the endoplasmic reticulum membrane towards eventual ubiquination and proteasome degradation [HIG1 domain family member 2a, glutathione peroxidase, and C1 inhibitor, which all act to prevent apoptosis and protease degradation, supports the idea that small fish are experiencing more protein turnover, possibly as a response to stress, than large fish. In this regard, however, it is also important to highlight that large fish also express much higher levels of genes related to catabolism such as cathepsins b, d, m, and l, and several genes related to chitinase, collagenase, aminopeptidase, serine protease, lysozyme, and matrix metalloproteinase activities. This indicates that catabolic activities appear to be somewhat higher in large fish and may reflect upon their increased metabolic activity. These results are similar to the up-regulation of genes related to carbohydrate, lipid, and amino acid metabolism coupled with the up-regulation of proteolysis in GH-transgenic and domestic fish compared to wildtype fish [Genes of mixed function, such as those related to protein degradation regulation such as radation . The obsype fish .The larger Dec. fish showed differential expression of more genes in white muscle and fewer genes in the liver than the Sept. fish. While underlying genetic differences between the parents used to produce the two lots could contribute to the observed differences in gene expression, it is also important to consider environmental effects. While the lots were exposed to the same water, temperature, dissolved oxygen, and nutrition, they experienced a different photoperiod regime at the time tissues were sampled for RNA analysis. The Sept. lot was sampled at a time period approaching winter solstice, a period of declining growth, while the Dec. lot was experiencing increasing daylight approaching spring equinox, a period of increasing growth. The effect of season on the different growth rates between the lots is supported by the TGC profiles Figure\u00a0. As expeGadus morhua) [Photoperiod regimes have major influences on growth rates in fishes but these are not always in predictable directions and may be species-specific. Although increased light exposure produces increased growth  in rainb morhua) ,78. Our These results indicate that sampling season can have a significant impact on the expression of genes related to the growth process in rainbow trout, with the TGC showing seasonal changes in both lots. In muscle, the large fish from the Dec. lot showed increased expression of genes related to muscle and connective tissue growth while genes related to blood production and the immune system were up-regulated in the large Sept. fish. This pattern may correspond to the differences in the growth profiles of the two lots. The Dec. fish were entering a period of increased growth while the Sept. fish were entering a phase of lower growth and possibly increased stress when they were sampled. These differences highlight the importance of sampling season in interpreting findings from gene expression studies.IGFBP1 was the only gene with highly significant and consistent up-regulation in the liver across seasons.A greater number of genes in white muscle demonstrated consistent associations with fish size regardless of sampling season, suggesting they may be better predictors of the growth response in rainbow trout compared to those in liver. Differences between large and small Sept. fish in the \u2018response to stress\u2019 category for white muscle tissue, indicate an up regulation of genes related to innate and acquired immunity in the large fish, while genes related to apoptosis have higher expression levels in the small fish. Depression of immune function genes and lower levels of oxidative stress genes in small fish suggest diminished stress handling capabilities. Genes within the \u2018response to external stimulus\u2019 and \u2018establishment of localization\u2019 categories also showed major expression differences between large and small Sept. fish. These categories show up-regulation of genes related to transcription, translation, and protein production in the small fish. However, since genes related to apoptosis were also up-regulated in the small fish, this suggests that higher cellular turnover rates may diminish the expected physiological effects of increased transcription, translation, and protein production. Genes related to blood and energy production showed up-regulation in the large fish compared to the small fish indicating an increased energy demand in the muscle of the large fish. In contrast to muscle tissue, Rainbow trout from the Lyndon commercial strain of rainbow trout  were reared at the Alma Aquaculture Research Station (AARS) using a protocol approved by the Animal Care Committee at the University of Guelph following the guidelines of the Canadian Council of Animal Care. Adults from the same strain but spawning in different seasons were selected as parents for the seasonal lots. Five paternal half-sib families were created by crossing 5 females with a single male on September 18, 2008 (referred to as Sept. lot) Figure\u00a0. A seconAll the progeny within a lot were reared together in a common environment at 8 to 10\u00b0C under a natural photoperiod from hatching to the end of the experiment. They were fed a commercial salmonid ration containing 44-55% protein and 15-22% fat depending on life stage, which corresponded to the thermal growth coefficients devised for rainbow trout (1.5-3% of body weight daily) . Tank dewhere wt2\u2009=\u2009mean weight of the fish at time t2 (days) and wt1\u2009=\u2009mean weight of the fish at time t1 (days)thermal growth co-efficient (TGC) , and tan4)2SO4, 10\u00a0mM EDTA, 25\u00a0mM Na3C6H5O7, adjusted to pH\u00a05.2) and then stored at \u221280\u00b0C until use. White muscle was excised immediately below the dorsal fin and well above the lateral line to avoid contamination with red muscle. A small square plug measuring less than a 1\u00a0cm was excised and the surface skin removed. Small fragments of tissue (~100\u00a0mg sections) from below the dermal connective tissue bundle  were placed in the RNA preserving solution.Samples of liver and white muscle were collected from 50 to 55 fish within each half-sib lot which were purposefully size-selected to represent the fastest and slowest growing fish in each lot. The tissues were placed in an RNA preserving solution . PCR conditions began with initial denaturation (95\u00b0C for 10\u00a0min), followed by 35 cycles of denaturation (95\u00b0C for 1\u00a0min), annealing (30\u00a0s), and extension (72\u00b0C for 30\u00a0s), and concluded with final extension (72\u00b0C for 5\u00a0min). An annealing temperature of 58\u00b0C was used for all primers except BX887563, which used 54\u00b0C.DNA was extracted from the liver samples using a standard phenol chloroform protocol . The pur-1, 1\u00d7 PCR buffer, 0.2\u00a0mM dNTP, 1.75\u00a0mM MgCl2, 0.2\u00a0\u03bcM of each forward or reverse marker primers, 0.01 U \u03bcL-1 Taq DNA polymerase). PCR conditions began with initial denaturation (95\u00b0C for 5\u00a0min), followed by 30 cycles of denaturation (95\u00b0C for 30\u00a0s), annealing (58\u00b0C for 1\u00a0min), and extension (72\u00b0C for 1\u00a0min), and concluded with final extension (72\u00b0C for 10\u00a0min).The sex of the progeny was determined using the rainbow trout Y-specific marker OmyY1 . PolymerTotal RNA was isolated from the liver and white muscle of the 2 largest and 2 smallest fish by weight in each half-sib family for both the Sept. and Dec. lots. TRIzol  extractions were performed according to the manufacturer\u2019s instructions and the resulting RNA concentrations were determined using a Nanodrop8000 spectrophotometer. The samples were stored at \u221280\u00b0C until future use. The presence of distinct 18S and 28S rRNA bands after agarose gel electrophoresis was used to confirm that the RNA was not degraded. The extraction process was repeated until a minimum of 40\u00a0\u03bcg of RNA was collected from each sample. Since large and small fish are growing at different rates it is possible that rRNA makes up a different proportion of the total RNA in the small and large fish. This would result in different amounts of mRNA within a unit volume of total RNA. Therefore, we avoided this potential bias by using purified mRNA. Prior to cDNA synthesis, mRNA was purified from the total RNA using \u03bcMACS mRNA Isolation Kits \u2013 Small Scale  according to the manufacturer\u2019s instructions.Gene expression levels were profiled for liver and muscle using 0.5\u00a0\u03bcg pools of mRNA composed of 0.1\u00a0\u03bcg of mRNA from each of five individuals Figure\u00a0. BecauseGH, IGF-I and II, actin, and myosin. Approximately 0.2-0.3\u00a0\u03bcg of cDNA was prepared from the mRNA using a SuperScript\u00ae Plus Direct cDNA Labeling System with Alexa Fluor\u00ae aha-dUTPs  and labeled with either Alexa Fluor 555 or Alexa Fluor 647. Briefly, the mRNA was reverse transcribed using an anchored oligod(T)20 primer and random hexamers in cDNA synthesis reactions that incorporated Alexa Fluor-labeled nucleotides following the manufacturer\u2019s specifications. The concentration and dye incorporation rates of the cDNA were calculated using a Nanodrop 8000 spectrophotometer. Samples were stored at \u221220\u00b0C until hybridization, which occurred on either the same or the following day.Gene expression was profiled using an Agilent salmonid microarray  containing cDNAs from 43,663 genes selected from Atlantic salmon expressed sequence tag databases . This chip allows quantification in expression of genes involved in muscle growth such as The cDNA was hybridized overnight to the microarrays according to the hybridization protocol described in Two-Color Microarray-Based Gene Expression Analysis (Agilent Technologies). Dye flips were included to compensate for any dye effects. Because air ozone levels could affect the Alexa Fluor 555 signal, we adopted the Agilent Ozone protection protocol following the manufacturers\u2019 specifications to prevent degradation. This was followed by washing to remove any cDNA that had not hybridized to the array. The microarrays were scanned on a GenePix 4200A scanner . Genepix Pro 6.1 software was used to process images, align the spots, integrate the Genepix Array List file with the microarray images, and quantify the spots. The arrays were subject to manual review and all unacceptable spots were flagged and excluded.http://www.genomics.agilent.com) software was used to analyze the microarray data. Data were preprocessed by removing data points where the signal intensities ratio of both channels was less than a baseline threshold value of 0.1. Lowess  normalization was applied to the data to normalize the spot intensity from various replicates. Spots that did not have a value for all of the replicates within a lot were removed from the analysis. Cross-array lowess normalizations were performed by the software and for each spot, t-statistic, p-value (probability), and the ratio between the two dyes were calculated by the Genespring software. Following corrections spots with a minimum of 1.2-fold change or more were considered differentially expressed (p\u2009\u2264\u20090.05). Microarray data were submitted to the NCBI Gene Expression Omnibus [Genespring GX 7.3.1 ( Omnibus  under thhttp://www.blast2go.com/b2ghome) to assign GO terms to the differentially expressed microarray sequences. This software applies an automated BlastX alignment of the sequences to the NCBI non-redundant database. The BlastX expectation value threshold was set to 1.0E-6 whereas all other parameters were set to the default values. If Blast2GO was unable to assign GO terms to a sequence, manual evaluations were considered using AmiGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi) assignments. A heterogeneity G-test (http://www.uoguelph.ca/~rdanzman with links to the software module) was used to compare the number of genes differently expressed in the GO categories at GO biological process levels 2 and 3 (GOslim generic categories) between groups using a backwards elimination procedure, using counts assigned within the Blast2GO program to the various level 2 and 3 GOslim categories. If significance was detected, the software reported the tabular category with the greatest heterogeneity. This category was removed from the analysis and the remaining categories were re-analyzed in a stepwise fashion until no significant differences remained. The GO categories removed at each step , are reported in Additional files provided. However, in several cases, the number of genes assigned to a grouping was small in number, and often with an absence of gene assignments within 1 of the 2 cells being compared. Therefore, only GO categories that differed by more than 5%  of all the genes assigned across all groupings were considered and discussed. The comparisons evaluated by the heterogeneity G-tests were: large vs. small fish within each seasonal lot; large vs. large fish from different seasonal lots, and small vs. small fish from different seasonal lots. This yielded 4 contrasts where two tested for the effects of size within a season and two tested for seasonal effects on fish of the same size category.Target gene lists obtained from the significance analysis were re-annotated using Blast2GO  from the fish in the Sept. lot used in the microarray experiment was used to make cDNA using multiscribe reverse transcriptase . Twenty fish were used for the liver analysis and 18 fish for the muscle analysis. Real-time PCR primers were designed from gene sequences  using PerfeCTa(R) SYBR(R)GreenFastMix(R)  with 15\u00a0\u03bcl reaction volumes containing 200 nM of each primer. Threshold lines were adjusted to intersect amplification lines in the linear portion of the amplification curve and cycles to threshold (Ct) were recorded. Standard curves for each gene and the reference gene were constructed using serial dilutions based on pools of mRNA from eight of the individual fish used for qPCR. PCR data were analyzed using the method described by Bookout and Mangelsdorf [Real time PCR was used to validate the expression of three genes in each of liver and muscle that were differently expressed in both the Sept. and Dec. lots. The one exception was gelsdorf . BrieflyThe authors state this research is free of conflicts of interests.This study was conceptualized by RGD and MMF. ALK conducted the microarray and PCR studies and analysis with advice from RGD and MMF. ALK drafted the manuscript with input from MMF and RGD. All authors read and approved the final manuscript.Genes up-regulated in the liver of large rainbow trout compared to small rainbow trout.Click here for fileGenes up-regulated in the liver of small rainbow trout compared to large rainbow trout.Click here for fileGenes of unknown function up-regulated in the liver of large rainbow trout compared to small rainbow trout.Click here for fileGenes of unknown function up-regulated in the liver of small rainbow trout compared to large rainbow trout.Click here for fileGh-test Results for the liver at GO level 2.Click here for fileAgilent Sequences Corresponding To Each GO ID For GO Levels 2 And 3 In The Liver Of The Sept. Fish.Click here for fileAgilent Sequences Corresponding To Each GO ID For GO Levels 2 And 3 In The Liver Of The Dec. Fish.Click here for fileGh-test Results for the liver at GO level 3.Click here for fileGenes up-regulated in the white muscle of large rainbow trout compared to small rainbow trout.Click here for fileGenes up-regulated in the white muscle of small rainbow trout compared to large rainbow trout.Click here for fileGenes of unknown function up-regulated in the white muscle of large rainbow trout compared to small rainbow trout.Click here for fileGenes of unknown function up-regulated in the white muscle of small rainbow trout compared to large rainbow trout.Click here for fileGh-test Results for white muscle at GO level 2.Click here for fileAgilent Sequences Corresponding To Each GO ID For GO Levels 2 And 3 In The White Muscle Of The Sept. Fish.Click here for fileAgilent Sequences Corresponding To Each GO ID For GO Levels 2 And 3 In The White Muscle Of The Dec. Fish.Click here for fileGh-test Results for white muscle at GO level 3.Click here for fileGenes Up-regulated in White Muscle in Large Rainbow Trout compared to Small Rainbow Trout within the GO \u2018response to external stimulus\u2019 category.Click here for fileGenes Up-regulated in White Muscle in Small Rainbow Trout compared to Large Rainbow Trout within the GO \u2018establishment of localization\u2019 category.Click here for fileGenes Up-regulated in White Muscle in Large Rainbow Trout compared to Small Rainbow Trout within GO \u2018response to stress\u2019 category.Click here for file"}
+{"text": "To develop guidelines to estimate the number of contributors to two-, three-, and four-person mixtures containing either high template DNA (HT-DNA) or low template DNA (LT-DNA) amounts.Seven hundred and twenty-eight purposeful two-, three-, and four-person mixtures composed of 85 individuals of various ethnicities with template amounts ranging from 10 to 500 pg were examined. The number of alleles labeled at each locus and the number of labeled different and repeating alleles at each locus as well over all loci for 2 HT-DNA or 3 LT-DNA replicates were determined. Guidelines based on these data were then evaluated with 117 mixtures generated from items handled by known individuals.The number of different alleles over all loci and replicates was used to initially categorize mixtures. Ranges were established based on the averages plus and minus 2 standard deviations, and to encompass all observations, the maximum and the minimum values. To differentiate samples that could be classified in more than one grouping, the number of loci with 4 or more repeating or different alleles, which were specific to three- and four-person mixtures, were verified. Misclassified samples showed an extraordinary amount of allele sharing or stutter.These guidelines proved to be useful tools to distinguish low template and high template two-, three-, and four-person mixtures. Due to the inherent higher probability of allele sharing, four-person mixtures were more challenging. Because of allelic drop-out, this was also the case for samples with very low amounts of template DNA or extreme mixture ratios. Interpretation of DNA mixtures derived from crime scene evidence samples is a major challenge in forensic DNA analysis. Evidence samples are typically deemed mixtures if they demonstrate more than 2 alleles at one or more loci, although allowances may be made for stutter and other artifacts of the short tandem repeat (STR) profiling process. Peak height imbalance at heterozygous loci may also indicate a mixture. Mixtures arise when 2 or more individuals contribute to an evidence sample. Contributors to an evidence sample may include perpetrator(s), victim(s), and/or other individuals who have come into contact with the crime scene, whether connected to the crime or not.Several different approaches can be taken to interpret DNA mixtures and to evaluate the strength of a comparison between an evidence sample and potential contributors. For some samples, the individual contributors to a mixture can be separated, or deduced -5 and, oLikelihood ratio (LR) based methods implicitly assume that the number of contributors to a mixture is known. Before computing the LR, one must specify prosecution and defense hypotheses on which to condition in the numerator and the denominator, respectively, of the ratio. Each hypothesis contains a specified number of individuals, for example, the prosecution hypothesis may be that the sample is a mixture of DNA from a suspect and an unknown, unrelated person and the defense hypothesis may be that the sample is a mixture of DNA from 2 unknown, unrelated persons. Therefore, the first steps in the calculation of the LR are determination of the number of contributors to the mixture and specification of the mixture components under each of the competing hypotheses.The most general formulation of the Probability of Exclusion does not require explicit specification of the number of contributors to a mixture, as no attempt is made to explain all of the alleles that are observed. Budowle et al  also desInterpretation guidelines from the Scientific Working Group on DNA Analysis Methods specify that the minimum number of contributors to a mixture can be determined based on the locus that exhibits the greatest number of peaks, with an allowance for tri-allelic loci and/or stutter . FollowiWhile locus-by-locus allele counting can provide an estimate of the minimum number of contributors to a mixture, it may not indicate the actual number of contributors to mixtures, particularly those with 3 or more contributors ,11. ThatUncertainty in the number of contributors to a mixture has unknown effects on the restricted RMNE. However, the effect on the LR of uncertainty in number of contributors has been explored. Bounding the LR has been suggested as a means to avoid anti-conservative bias when the number of contributors to a mixture is in dispute -15. The One could argue that a better approach than opting for the minimum number of contributors to a mixture might be to determine the number of contributors best supported by the data. Several recent publications have explored this idea using maximum likelihood to estimate the most likely number of contributors to a mixture. Egeland et al  proposedTo complement the maximum likelihood and maximum allele count approaches, we examined 728 two-, three-, and four-person purposeful mixtures and identified characteristics that can assist in estimating the number of contributors to the mixtures. To date, only conceptual mixtures have been examined and this is the first empirically based study of this nature. To account for allelic drop-out and drop-in, template amounts ranged from 10 pg to 500 pg. Considering empirical observations unique to mixtures in different quantitative ranges and the total number of different alleles seen across all loci, a set of guidelines was developed. The guidelines were then applied to a set of 117 items handled by 2, 3, and 4 individuals.Protocols for preparation and quality control of personnel, workspace, equipment, and consumable preparation were performed as previously described in Caragine et al .Buccal swabs and blood samples from known donors, and items handled by individuals whose DNA profiles were known, were used. Eighty-five different donors were sampled representing the diverse population of the City of New York. The race was known for 61 (72%) of the donors as they were laboratory employees. They could be categorized generally as follows: 20% Asian, 16% Black, 54% Caucasian, and 10% Hispanic. The remaining 24 or 28% of the donors represented an anonymous sampling of the City of New York, which is composed of 9.8% Asians, 26.6% Blacks, 44.7% Caucasians, and 27% Hispanics  . Touched items were sampled with a patent pending swab from the Office of the Chief Medical Examiner of the City of New York pre-moistened with 0.01% sodium dodecyl sulfate . The swaMA, USA)  pretreat\u00ae (Qiagen) using an Alu-based real time polymerase chain reaction (PCR) assay based on the method described by Nicklas and Buel  following the manufacturer\u2019s recommendations with the exception of a two-minute annealing time, a half-reaction volume, and 2.5 U of AmpliTaq Gold\u00ae for either 28 (High Template DNA [HT-DNA]) or 31 (Low Template DNA [LT-DNA]) cycles. HT-DNA samples were amplified in duplicate with at least 100 pg of DNA in each replicate. LT-DNA samples were amplified in triplicate with 100 pg or less of DNA in each replicate , 2:1 (n\u2009=\u200934), 4:1 (n\u2009=\u2009100), 1:1:1 (n\u2009=\u200918), 2:2:1 (n\u2009=\u20091), 3:1:1 (n\u2009=\u200920), 4:1:1 (n\u2009=\u20096), 5:1:1 (n\u2009=\u200963), 5:5:1 (n\u2009=\u200913), 1:1:1:1 (n\u2009=\u200923), 5:1:1:1 (n\u2009=\u20092), 4:1:1:1; (n\u2009=\u20091), 3:1:1:1 (n\u2009=\u20091), 2:1:1:1 (n\u2009=\u20091), 4:3:2:1 (n\u2009=\u20096), 3:2:1:1 (n\u2009=\u20095), 3:3:2:2 (n\u2009=\u20095), and 2:2:1:1 (n\u2009=\u20096). The ratios for purposeful mixtures amplified for 31 cycles included 1:1 (n\u2009=\u200952), 2:1 (n\u2009=\u200943), 3:1 (n\u2009=\u20098), 4:1 (n\u2009=\u200994), 5:1 (n\u2009=\u20092), 1:1:1 (n\u2009=\u200920), 2:2:1 (n\u2009=\u20092), 3:1:1 (n\u2009=\u200921), 4:1:1 (n\u2009=\u20093), 5:1:1 (n\u2009=\u200961), 5:5:1 (n\u2009=\u200917), 1:1:1:1 (n\u2009=\u200914), 2:1:1:1 (n\u2009=\u20092), 3:1:1:1 (n\u2009=\u20093), 4:1:1:1 (n\u2009=\u20093), 5:1:1:1 (n\u2009=\u20093), 4:3:2:1 (n\u2009=\u20096), 3:2:1:1 (n\u2009=\u20096), 3:3:2:2 (n\u2009=\u20097), and 2:2:1:1 (n\u2009=\u20096).Regarding samples generated from touched items, 53 samples were amplified with HT-DNA amounts and 64 samples with LT-DNA amounts.xl Genetic Analyzer using 0.375 \u00b5L per of GeneScan\u00ae 500 LIZ\u00ae Size Standard (Applied Biosystems) per injection and HIDI formamide (Applied Biosystems). Samples amplified for 31 cycles (LT-DNA) were prepared with 5 \u00b5L of PCR product in a total volume of 50 \u00b5L, and were injected with 1kV for 22 seconds, 3 kV for 20 seconds or 6kV for 30 seconds as needed .Studies have shown that replication serves to identify allele drop-out and drop-in -26. TherThe distribution of the number of different alleles expected from the purposeful mixtures was determined by creating conceptual mixtures using the same donors. Without allowing drop-out or drop-in, the number of different alleles observed in each of the conceptual mixtures was determined. Because some combinations of donors were used for more than one purposeful mixture, there were fewer conceptual mixtures than actual mixtures.To investigate the effect of varying mixture ratios on the number of different alleles detected, samples were divided into mixtures with similar contributions from all donors and samples with more extreme mixture ratios. For two-person samples, 1:1 and 2:1 mixtures were grouped together  and compared to samples with more extreme mixture ratios . For three- or four-person samples, mixtures with the smallest contributor having at least a 20% contribution were grouped together. For three-person samples, these ratios were 1:1:1, 2:2:1, and 3:1:1 . More extreme ratios included 4:1:1, 5:1:1, and 5:5:1 . For four-person samples, the similar ratios were 1:1:1:1, 3:3:2:2, and 2:1:1:1 , while the more extreme ratios were 3:1:1:1, 4:1:1:1, 5:1:1:1, 4:3:2:1, 3:2:1:1, and 2:2:1:1 .Although there was some overlap between the distribution of the total number of different alleles expected for two-person and three-person mixtures and for three-person and four-person mixtures, there were some distinctions . These rIn order to explore additional characteristics of two-, three-, and four-person mixtures, 728 mixtures of varying template amounts, and numbers and ratios of contributors were examined. In this data set, 9 characteristics distinguished three-person from two-person mixtures and 8 characteristics distinguished four-person from three-person mixtures . There wTo determine whether the empirical data supported theoretical predictions for the number of different alleles, the same 728 mixtures were examined. If a low amount of template DNA is amplified, an expected allele consistent with one from a contributor may not be detected, or may drop out. Alternatively, an allele not consistent with the DNA of the contributors may be seen or may drop in. As replication can identify these phenomena, the number of labeled repeating alleles observed was first measured for both HT-DNA and LT-DNA mixtures (data not shown). However, both sample sets displayed significant overlap among the mixture categories such that it was not feasible to delineate any distinct separations.The number of labeled different alleles was examined for HT-DNA  and LT-DResults are enumerated for HT-DNA  and LT-DThe maximum, minimum, and the average plus or minus 2 standard deviations of the number of different alleles observed in all HT-DNA samples can help distinguish among two-, three-, and four-person samples . Two 5:1Although in this data set no more than 50 different alleles were labeled for any of the two-person mixtures tested, theoretically it was possible to generate 51 alleles from one combination of 2 of the donors. Using all data sources, the presence of 52 or more different alleles in HT-DNA mixtures indicated that the sample consisted of more than 2 contributors. Forty-six or fewer different alleles suggested that the sample could best be described as originating from 2 individuals. These groupings signify that samples containing 47, 48, 49, 50, or 51 alleles should be examined for the additional criteria in For HT-DNA three-person mixtures, at most 64 alleles were seen, although 1 combination of 3 donors consisted of 66 different alleles. Therefore, 67 or more different alleles signified a four-person HT-DNA mixture. Using the cutoffs from Only 1 HT-DNA purposeful four-person mixed sample showed 51 different alleles. Two of the 4 donors to this 400 pg 2:2:1:1 experienced significant drop-out, with 7 and 11 missing alleles. Thus, this sample may be better described as a three-person than a four-person mixture. Although in this study all other four-person mixtures amplified had 58 or more different alleles, 1 combination of 4 donors was composed of 57 different alleles. Therefore, the presence of 52-56 different alleles suggested a three-person mixture while 57-66 different alleles warranted additional scrutiny.Following these guidelines, 7 or 14% of the 50 HT-DNA four-person mixtures assessed better resembled three-person mixtures than four-person mixtures. Each of these 7 samples had 61 or fewer different alleles and would fall within the zone encompassing both three-and four-person mixtures. Furthermore, these samples did not demonstrate the characteristics listed in Considering the estimated ranges, as well as the maximum and the minimum numbers of different alleles observed, LT-DNA samples had less distinct divisions, likely due to allelic drop-out. For example, the lowest expected number of different alleles from the combinations of 3 donors tested was 47 although some three-person samples were observed to have fewer than 47 different alleles . NeverthIn fact, the 25pg 5:1:1 mixture, which had the minimum number of alleles, 42, did not meet the On the other hand, another 25 pg 5:1:1 mixture had 48 different alleles. Unlike the previous samples, this mixture had 7 loci with 4 different alleles and 1 locus with 5 different alleles, both of which are qualities of three-person mixtures. As none of the contributors were missing more than 2 alleles, this mixture is probably best described as three-person in spite of the low total allele count. Accounting for all data, samples with 52 or more alleles could have more than 2 contributors, whereas samples containing 47-51 different alleles should be further evaluated.Regarding LT-DNA two-person mixtures with 50 pg or more, samples with 55 or more alleles contained more than 2 contributors and values from 47-54 should be assessed for other characteristics. Using the cutoffs in To distinguish between three-person and four-person mixtures, the upper end of the distribution of the number of different alleles seen in the three-person samples and the lower end of the distribution of the number of different alleles seen in the four-person mixtures can be used. Sixty-seven or more different alleles signified a four-person LT-DNA mixture. For samples with less than 50 pg, all values from 52-56, and for samples with 50 pg or more, values of 55 or 56, suggested 3 contributors. Samples with 57-66 different alleles should also be evaluated with the Overall, 25 of the 50 LT-DNA four-person samples looked like four-person samples by the total number of different alleles labeled and/or by the patterns listed in Sixteen four-person samples were amplified with 20 to 40 pg of template DNA. None of these samples showed more than 66 different alleles and only 2 would be called four-person mixtures by the criteria in Four-person mixtures with at least 50 pg of template DNA presented fewer challenges. None of the 4 contributors to 24 of the 30 four-person mixtures with 50 pg or more of template DNA were missing more than 2 alleles. Of these 24 samples, 18 (75%) met the four-person mixture criteria of 67 or more different alleles and/or the characteristics shown in The purposeful mixtures in this study included mixture ratios of varying extremes; therefore, samples showed different degrees of allelic drop-out, as measured by the number of different alleles detected compared to the number of different alleles expected. In order to determine whether varying mixture ratios were masking any important trends, samples were divided into two categories: those with similar contributions from all donors and those with more extreme mixture ratios.The distribution of number of different alleles observed in LT-DNA and HT-DNA two-, three-, and four-person mixtures was not substantially different for similar ratios and for more extreme ratios . The verSamples generated from items handled by 2, 3, or 4 contributors were evaluated with the derived guidelines. These non-purposeful mixtures included another variable, the shedder status of the individuals who touched the items. Therefore, the distribution of the observed number of different alleles in four-person purposeful mixtures more closely approximated the expected distribution for four-person mixtures  than forThe observed results did not precisely reflect the expected number of different alleles without drop-out or drop-in. Due to the nature of a sample, degradation, the mixture ratio or the number of contributors to a mixture, allele(s) may not be amplified or may drop out, reducing the total number of different alleles observed. Alternatively, an allele that is not consistent with those of the known contributors may \u201cdrop in.\u201d This would increase the number of different alleles seen -26.All loci were evaluated in this study, but the total number of alleles observed or the power of discrimination at any particular locus was not considered. However, it was noted that 5 alleles were labeled in three-person mixtures more frequently at D2S1338, D19S433, D18S51, FGA, and vWA than at the other 10 loci (data not shown). Three of these loci also had a high propensity for allelic dropout (Overall, the samples with numbers of different alleles within the two-, three-, or four-person mixture zones resembled two-, three-, or four- person mixtures by the Extensive allele sharing could also cause a three-person mixture to appear as a two-person mixture ,11. For There was one instance wherein a two-person mixture appeared to be a three-person mixture. This 50 pg mixture had 54 different alleles, a number well beyond what is expected for a two-person mixture. Further investigation of this sample revealed that it contained 4 loci with 5 different alleles and 4 loci with 4 different alleles; thus, this sample met the Using the total number of different alleles and the characteristics listed in In summary, including only samples for which none of the true contributors were missing more than 2 alleles, 99%, 96%, and 82% of two-, three-, and four- person purposeful mixtures, respectively, met the appropriate criteria for the number of contributors. Samples that did not, exhibited extreme phenomena, such as excessive allele sharing or stutter. The success rates stated here are data driven estimates and may not be representative of all forensic samples. Although a variety of template amounts and mixture ratios were included in this sample set, forensic samples may exhibit qualities not observed in these mixtures.Mixtures generated from items handled by two-, three-, or four-persons more closely mimicked evidentiary samples. Results mirrored the purposeful mixtures except that more alleles that were consistent with the individuals who touched the items were not amplified and more alleles that were foreign to these individuals were detected. The absence of alleles could be attributed to the fact that individuals shed to varying degrees and thus if a strong shedder is the last person who handled an item, DNA from the first person may not be recovered . AllelesThus, it is more challenging to accurately estimate the number of contributors to samples from touched items than to samples from body fluids. Nevertheless, general ranges for the number of different alleles are still indicative of the number of contributors. When these values are within the intermediate ranges for categories, empirically defined characteristics have proven useful. In a few instances, extreme allele sharing, the allele sharing inherent to four-person mixtures, or the lack of sharing coupled with increased stutter could mask the true number of contributors.A probabilistic model involving allele frequencies and the probability of drop-out and drop-in could address these issues -30. For"}
+{"text": "In Reply: The purpose of Perez et al  hypothesis. Although treating a true four-person mixture as a three-person mixture may be conservative , tools tAnother concern was the level of detail provided regarding the test samples. In order to reflect the range of results encountered in forensic casework, over 728 samples covering a variety of DNA template amounts from a diverse set of donors mixed in different ratios were examined. The number of samples amplified with a specific mixture ratio and number of contributors, as well as whether the sample contained high-template or low-template amounts of DNA was noted in the methods section. The ethnic composition of the pool of contributors used was also specified. To list the exact amount of template DNA amplified for each mixture ratio and combination of donors and their individual ethnicity for each of the 728 samples as suggested by the authors of this letter  would haAlternatively, it is helpful to look for general characteristics in mixtures. To illustrate whether different mixture ratios significantly affect the determination of the number of contributors to a mixture, samples were divided into six separate graphs  of two-, three-, and four-person mixtures composed of high-template or low-template amounts of DNA. These data sets were then further subdivided into mixtures with ratios of contributors that were similar or dissimilar, and the numbers of different alleles observed, a key parameter in assessing the number of contributors to mixtures, were shown. For each of the six groupings, the number of different alleles labeled was not substantially different for mixtures with similar ratios as compared to those with more extreme ratios. Contrary to what the authors of the letter state, this and other findings may be replicated by studying similar general groupings.The letter further critiques the experimental design using defined DNA dilutions. Although there is some uncertainty in the exact amount of DNA amplified, the DNA concentration measured using our in-house assay is a good estimate . To accoThere was no subjective editing of data and allele calls in this study. All filters and analysis thresholds employed are programmed into the software and were not altered from sample to sample. Also, at the NYC OCME evidence extracts are routinely tested using multiple amplifications and thus multiple runs are available for analysis.The authors of the letter speculate that data may have been selected to fit a preconceived hypothesis. This was not the case. Each purposeful mixture was evaluated as if it was a forensic unknown using the proposed guidelines, a strength of this study. The described characteristics of three- and four-person mixtures were never observed in the set of two- and three-person mixtures studied, respectively. Some samples, however, did not meet the specified criteria and had to be classified as two-person mixtures even though they were created with DNA from three individuals, for example. The frequency that this occurred was clearly stated.The reality of forensic casework mixtures, especially those containing low amounts of DNA, is that allelic drop-out may occur for one or more components, which may mask the true number of contributors. In our study, we categorized these types of mixtures more conservatively , and theLastly, we recognize that although the study with touched items represents a blind data set, one shortcoming is that the actual number of contributors amplified is not known since one may not recover DNA from all persons who handle an item. Another large data set of purposeful mixtures would be required. Nevertheless, we maintain that our study provides useful tools to determine the number of contributors to mixtures processed in our laboratory. Based on these studies, work by our colleagues in their own laboratories may customize these guidelines for their use and/or develop more enhanced mechanisms to address the issue."}
+{"text": "A 6-year-old boy presented with the recent development of a right sided limp. He had no known medical conditions. On examination, the patient had limited internal/external rotation and abduction of the right hip. He walked with a non-antalgic gait and was noted to have a positive Trendelenberg sign. Lower extremity radiographs (A) revealed flattening of the right proximal femoral epiphysis with greater than 50% collapse. Mild acetabular changes of the right hip were present; however, there was still concentric reduction of the right femoral head within the acetabulum. After a thorough discussion of management options, the patient received botulinum toxin injections along the adductor longus and gracilis muscles to resolve an adduction contracture. An arthrogram (B) demonstrated mild-moderate flattening of the femoral head with lateral femoral head extrusion, and moderate femoral-acetabular congruency. The patient was subsequently given a hip abduction orthosis for intermittent daytime and full nighttime bracing, and was instructed to continue stretching exercises daily. The parents were educated regarding the cyclical nature of the pathology, and that the prognosis is relatively improved in younger children with the potential to still remodel. Legg-Calv\u00e9-Perthes disease describesan idiopathic avascular necrosis of the proximal femoral epiphysis. Initial therapy includes maintenance of the femoral head within the acetabulum by an abduction splint. Additionally, daily abduction stretching exercises and physical therapy are recommended. Surgical containment can be achieved by a femoral osteotomy to redirect the involved portion within the acetabulum."}
+{"text": "Hepatoblastoma is a rare malignancy. Approximately 100 cases are diagnosed yearly in the United States. The highest incidence occurs in infants and in children younger than 5 years. Cases involving patients older than 5 years are very rare. We describe the case of a patient who was diagnosed with hepatoblastoma at an atypical age of presentation for this type of malignancy. We also performed Ovid MEDLINE search for hepatoblastoma and epidemiology reports occurring in children between the ages of 5 and 18 years. In this article we review the epidemiology and summarize case reports published between 1997 and 2012 of patients with hepatoblastoma, who were older than 5 years.Our patient is an 11 year old boy with stage IV hepatoblastoma with lung and omental metastases at diagnosis.The patient received 7 cycles of chemotherapy following the treatment plan of COG protocol AHEP 0731, off study. He also had tumor resection and omentectomy and achieved complete remission.He later had disease recurrence and after undergoing treatment with different modalities, ultimately died of his disease. Review of SEER program data shows that the incidence of hepatoblastoma in children above the age of 5 years is too infrequent to be calculated. Literature review revealed 13 cases of patients diagnosed at age older than 5 years. Most cases were published due to unusual associations and/or complications. There are no obvious unifying characteristics for these cases, although there may be a slight male preponderance and many patients in this selected series presented with elevated Alpha-fetoprotein.The reported case is rare, given the very low incidence of hepatoblastoma outside of infancy. A systematic review of characteristics and outcomes for patients older than 5 years who are enrolled in cooperative group hepatoblastoma trials may reveal important information about the epidemiology and tumor biology in this rare patient population. In patients older than 5 years, cases of hepatoblastoma are very rare. The estimated incidence is 0.1 cases per million children in the age group of 5 to 9 years and sometimes reported as \u201ctoo infrequent to be calculated.\u201dHepatoblastoma (HB) is a rare malignancy, approximately 100 cases are diagnosed yearly in the USA.per se have been mainly suggested by case reports. Some of the well-known associations of HB are with Beckwith\u2013Wiedemann, parental exposure to metals and very low birth weight (VLBW). HB has been reported in single instances in association with fetal alcohol syndrome, oral contraceptive use during pregnancy, and maternal liver transplantation with immunosuppressive therapy.The epidemiology of HB has not been studied extensively. Although its incidence over the last several decades has been well described, as well as certain associations with genetic syndromes and parental environmental exposures, risk factors  and Reis et al evaluated HB incidence and trends specifically among children 0 to 4 years in the United States from 1975 through 1999 in the 9 reporting areas of SEER.,6 The overall incidence rate of HB in this age group rose from 2.59 per million in 1975\u20131979 to 5.27 in 1995\u20131999, which represented a statistically significant 3.9% annual rise in incidence. The rate of HB was slightly higher in males compared to females and in blacks compared to whites. There was a significant annual rise in incidence for males, females, and whites. The average annual percent change (AAPC) for blacks suggested a rise in incidence but was not significant. The incidence of HB is vanishingly small, 0.3 cases per million or less, at ages older than 4 years for the period 1975\u20131999.Spector et al The study results have not been published yet and we believe that these will reveal new findings and insights into the risk factors and biology of HB in infants and young children. However, the epidemiology and biology of HB in the older patient population still remain to be explored.Given the noted increase in incidence of HB between 1975 and 1999 and paucity of epidemiologic studies, a cooperative group study\u2014COG AEPI04C1\u2014was designed for patients with HB in the 0 to 4 age group. The aims of the study were to investigate exogenous and endogenous risk factors for HB with special emphasis given to risk factors for the development of HB among children with low birth weight.In this article, we report a case of an 11-year-old boy diagnosed with stage IV hepatoblastoma with lung metastases and extensive omental studding, which is an infrequent location for metastatic disease. We review briefly the epidemiology of hepatoblastoma and summarize 13 cases of patients between the ages of 5 and 18 years, reported in the English literature over a period of 16 years (1997\u20132012).The University Hospitals Cleveland Medical Center Institutional Review Board reviewed this case report and determined that it did not qualify as human subjects research according to Federal regulations; therefore, a formal approval was not necessary. A HIPPA waiver was granted for the individual patient's information to be reviewed.2An 11\u200ay/o Hispanic male, who was previously healthy, presented to his primary care physician with persistent abdominal pain of several months. He was found to have a right-sided abdominal mass on physical examination. An abdominal ultrasound was performed and revealed the presence of a large mass, extending inferiorly from the right lobe of the liver and measuring approximately 11.6 \u00d7 11.4 \u00d7 9.6\u200acm. An abdominal CT confirmed the presence of a large hepatic mass involving segments 5 and 6 of the liver and peritoneal metastases. Alpha-fetoprotein (AFP) was elevated; it was 35,084\u200ang/mL (reference range 0\u201315\u200ang/mL). Biopsy of omental metastases revealed epithelial hepatoblastoma, embryonal type.Treatment was started with 1 cycle of vincristine/irinotecan (VI) followed by 4 cycles of cisplatin and 5 fluorouracil, vincristine, doxorubicin (C5VD). Our patient's tumor was classified as PRE-TEXT II at diagnosis and was POST-TEXT I after 5 cycles of chemotherapy. The patient had surgical resection after the initial 5 cycles of chemotherapy. His AFP decreased from 35,084\u200ang/mL at diagnosis to 2612\u200ang/mL after 2 cycles of chemotherapy and to 162\u200ang/mL prior to surgery. The AFP came down to 28\u200ang/mL post-surgery and it normalized completely 1 month later. He received 2 more cycles of C5VD postoperatively. He did not require surgical resection for his lung lesions; they completely resolved with chemotherapy. The patient was followed with CT scans of chest, abdomen, and pelvis for disease recurrence after the end of therapy. His AFP started increasing approximately 2 months post end of therapy . At first, there was no radiographic evidence of disease despite a 4-month upward trend of his AFP. At that time, a very small lung lesion was found on CT of chest. Three months later, a repeat chest CT and an MRI of his abdomen detected enlarging lung lesions and a hepatic lesion. At that time, his AFP had increased to 145\u200ang/mL. Biopsy of liver lesion proved recurrent hepatoblastoma.Treatment postrelapse was initiated with vincristine, irinotecan, and temozolomide (VIT) for 2 cycles and the patient had stable disease. After 2 more cycles, disease progression was noted. In light of these findings, 2 cycles of ifosfamide, carboplatin, and etoposide (ICE) were also given; however, the patient had disease progression with an increase in size of a hepatic lesion and new subdiaphragmatic lesions found on MRI. Of note, for our patient, the MRI was found to be a more useful imaging modality for identifying the liver and subdiaphragmatic lesions at relapse and following their progression. He underwent radiofrequency ablation (RFA) of the liver lesion and the diaphragmatic tumor implants. After the RFA, his AFP normalized and remained normal for 3 months. He continued with chemotherapy\u2014irinotecan, temozolomide, and temsirolimus. Approximately 9 months after the RFA, an increase in size of the subdiaphragmatic, liver, and lung lesions was detected, together with rising AFP. The patient underwent transarterial chemoembolization (TACE) with doxorubicin for his liver lesions and had a significant decrease in the AFP post-procedure. Because he still had lung lesions, systemic chemotherapy continued. He was given a PARP inhibitor and temozolomide on a phase 1 clinical trial followed by axitinib. His disease progressed with increase in size of the hepatic lesion and IVC involvement with intracardiac/right atrial extension. He received palliative radiation to the areas of disease involvement of his liver, IVC, and right atrium. Subsequently, he received hospice care and died of his tumor 44 months from his initial diagnosis.3Here, we review the epidemiology of hepatoblastoma (HB), the most common associations, and summarize case reports for patients with HB above the age of 5 years.Fifteen percent of all abdominal tumors in childhood are primary liver tumors, 66% of these are malignant, the most common being hepatoblastoma (HB).About 100 cases of hepatoblastoma are diagnosed annually in the USA. In comparison, in the 15 to 19 years age group, hepatocellular carcinoma accounts for 87% of cases.Review of SEER database for the 2002\u20132008 period demonstrates that among patients younger than 5 years, hepatoblastoma accounts for 91% of primary hepatic malignancy cases. Age-specific incidence was reported to be highest in the 0- to 4-year-old age group. The occurrence of hepatoblastoma is too infrequent in children older than 5 years and the actual incidence could not be calculated.,9,10International data from different regions in the world report no significant variations in the incidence of HB between different nations.Hepatoblastoma is slightly more frequent in males and its incidence is higher in patients with several genetic and cancer predisposition syndromes: Beckwith\u2013Wiedeman, Familial Adenomatous Polyposis, and Trisomy 18.Another factor which is strongly linked with higher incidence of HB is very low birth weight .,11Pre-eclampsia, poly- or oligohydramnios, high maternal prepregnancy weight, and treatment for infertility in women were also reported to correlate with higher hepatoblastoma occurrence in their children. Other associations have been observed between tobacco smoking of both parents pre- and postconception and HB and parental occupational exposure to metals. reported that among those factors, correlating significantly with reduced event-free survival is low or very high AFP at diagnosis\u2014when it is lower than 100\u200ang/mL or if it is higher than 1.2 \u00d7 106\u200ang/mL. Elevated AFP is a known hallmark of active disease in patients with HB. It is generally significantly elevated with large tumor burden and its level decreases with therapy. It is frequently utilized as a marker of disease activity and response to therapy. Patients who are found to have a quick drop in AFP and normalization early in their therapeutic course tend to have better outcomes.Several clinico-pathological factors relevant to long-term outcome in newly diagnosed patients with HB have been identified. Maibach et al De Ioris et al also identified patients with HB and low serum AFP (< 100\u200ang/mL) as high-risk subgroup with extensive disease at diagnosis, poor response to therapy, and poor survival.Metastatic disease at presentation, PRETEXT IV, age > 5 years and some other clinical and histopathological characteristics were also found to correlate with poor outcomes.In contrast to previously described clinico-pathological indicators known to correlate with the poor outcomes, molecular profiling of HB had not been performed until recently. Identification of biomarkers with a goal to improve risk stratification, recognize prognostic implications, and potentially influence treatment decisions has been sought of late. Sumazin et al analyzed 88 HBs and revealed 3 risk-stratifying molecular subtypes that are characterized by differential activations of hepatic progenitor cell markers and metabolic pathways. For example, high-risk tumors were characterized by up-regulation of certain genes and proteins, one of which was high AFP expression and high coordinated expression of oncofetal proteins and stem cell markers. Low-risk tumors had low level of expression and activity of these genes and proteins. Further analyses of targeting these genes in a prospective study of 35 HBs suggested that these candidate biomarkers have the potential to improve risk stratification and guide treatment decisions for HB patients at diagnosis.Our patient did not have any of the above-mentioned clinico-pathological risk factors associated with higher incidence of HB. Upon review of his past medical history, we found out that he was born prematurely at 34 weeks. Nevertheless, with no history of VLBW, he had a brief and uncomplicated NICU course. He did not have other symptoms, signs or physical exam findings to suggest a genetic syndrome or a cancer predisposition condition. In terms of risk factors associated with poor outcomes, his AFP level at diagnosis did not place him in the unfavorable category; however, he had metastatic disease and was older than 5 years. His case is rare because of his age at diagnosis and extensive omental metastases, not commonly found as a site of disease in patients with HB.Here, we summarize 13 cases of patients with HB older than 5 years (Table There were no unifying characteristics of these patients, other than male preponderance and elevated AFP at diagnosis. It is worth noting that they were reported from all over the world, with no particular geographic predilection of certain pathological subtypes or comorbidities. Three of the cases were reported as associations with genetic syndromes, not previously described: Down syndrome, Kabuki, and partial trisomy 18. One of the patients who presented at the same age as our patient had an underlying rare metabolic disorder\u2014metylmalonic aciduria. Two of the patients were born prematurely, although only 1 had a VLBW.Different pathological subtypes of the tumors were noted. One of the most common histological types of tumors was epithelial type, which is the same as in our patient. Of note, in 1 of those 3 tumors, cells which resembled hepatocellular carcinoma were seen. To our knowledge, our patient did not have cells that are typical of, or resemble, hepatocellular carcinoma. However, based on the biological behavior of his tumor, with short-lived response to first-line therapy for HB, there was a question of whether he had a transitional cell liver tumor, which is a relatively new entity that has been described in the recent years. This speculation was based on the aggressiveness of his disease and was not proven by histological or other analyses. At least 3 of the cases had well-differentiated tumor types and, interestingly, in one of the tumors renin secreting cells were found. One of the well-differentiated tumors had osteoid formations. There a few cases that did not have pathology of tumors reported; thus, we cannot make conclusions about overall correlation of pathological subtypes and patient outcomes.The case reports have described the different therapeutic modalities which were used. Most of the patients had surgical resection, 1 patient had liver transplant, and several patients received different chemotherapy agents. Two patients had high-dose chemotherapy with stem cell rescue. One patient had chemoembolization. Going back to our patient, his treatment consisted of a multitude of different agents and modalities. He received several different chemotherapeutic regimens, had surgery, radiofrequency ablation, trans-arterial chemoembolization, and palliative radiation. From the reported cases, we cannot make a conclusion about certain types of treatments and outcomes. This is due to this small case series and also the focus when reporting these patients had not been to review in detail the treatments utilized, but rather complications that occurred or new associations.Outcomes in these case series were variable with no clear evidence of favorable or unfavorable characteristics.4In summary, the reported case is rare given the very low incidence of hepatoblastoma outside of infancy. Our literature search revealed 13 other cases of patients with HB above the age of 5 years published over a period of 16 years. These cases were reported due to complications of treatment or associations not being described previously. We suspect that there are other cases of older patients with HB, who were participants of cooperative group clinical trials, and have not been reported separately.Although epidemiologic studies have been initiated to investigate exogenous and endogenous risk factors for HB in infants and young children in the age 0 to 4 years, the influence of older age at diagnosis on disease course and survival of patients with HB has not been well studied. Our case highlights the importance of considering a systematic approach to study characteristics and outcomes of patients older than 5 years enrolled in cooperative group hepatoblastoma trials. This will provide insights into tumor biology in this rare population and rationale for future risk stratification schemas and new risk-adapted therapeutic strategies."}
+{"text": "Binge drinking (BD) is defined as a pattern of high alcohol intake in a short time followed by periods of abstinence. This behavior is very common in adolescence, a developmental stage characterized by the maturation of the prefrontal and striatal networks, important circuits underlying the capacity to control and regulate the behavior. In this study, we conducted a voxel-based morphometry (VBM) analysis, using a region of interest (ROI) analysis of brain regions associated with inhibitory control and self-regulatory processes, in a group of 36 young college students, 20 binge drinkers (BDs) and 16 alcohol abstinent controls (AAC). Results showed increased gray matter (GM) densities in the left middle frontal gyrus in BDs, when compared with alcohol abstinent controls. Additionally, a ROI-based Pearson analysis documented positive correlations between the left middle frontal gyrus GM densities and the self-control subscale of the Barratt Impulsiveness Scale (BIS), in the BD group. These findings highlight abnormalities in core brain regions associated with self-regulatory processes in the BD group. Binge drinking (BD) is a common pattern of consumption among college students and is characterized by repeated episodes of large amounts of alcohol intake. In order to be considered BD, an alcohol ingestion episode requires a minimum consumption of four drinks for women and five for men, in a brief period of time (\u00b12 h) at least once per month, being followed by periods of abstinence see .According to the National Institute on Alcohol Abuse and Alcoholism (NIAAA), nearly 60% of US college students (age range 18\u201322 years) reported alcohol consumption and 40% exhibited a BD pattern in the past month. This behavior is seriously harmful affecting several domains of the individuals\u2019 life such as social, academic and health, being associated with the death of approximately 1,825 US college students each year . In EuroThe adolescence is a critical period for the beginning of abusive alcohol consumption . In factResearch on the putative neuropsychological abnormalities in BD documented a multiplicity of deficits underlying these executive function and self-regulatory abilities, as attention, cognitive flexibility, working memory, planning, decision-making and inhibitory control. These functional impairments have been associated with atypical functioning of several brain regions, including the dorsolateral prefrontal cortex (DLPFC), the inferior, middle and superior frontal gyri, the anterior cingulate cortex and the parietal and the temporal lobes, revealed by electrophysiological, structural and functional neuroimaging studies see , the DLPOverall, while some of the authors related their results to a neurotoxic effect of alcohol e.g., , others Taking this developmental, neurofunctional and neurocognitive findings into consideration, we hypothesized that BDs would show morphological alterations within core brain regions associated with self-regulatory processes , when compared with AAD. In order to test this hypothesis, we performed a voxel-based morphometry (VBM) study in a group of young college students that met the criteria for binge alcohol consumption and a group of AACs employing a region of interest (ROI) analysis of brain regions associated with inhibitory control and self-regulatory processes .Table 1.Participants were recruited through an online survey with college students, which included items regarding the use of alcohol  and other drugs . Then, participants with BD or AAC criteria were selected to participate and invited to a clinical structured interview. The interview covered several aspects related to alcohol and drug consumption, personal and family history of alcoholism, medical or psychopathological disorders, as well as the assessment of their laterality, impulsivity and psychopathological symptomatology. The sample included 36-college students ranging in age between 18 and 23 years old, with 20 participants with BDs (10 women) and 16 AACs (10 women). Participants were classified as BDs if they consumed a minimum of four drinks or five for men in a brief period of time (\u223c2 h), at least once per month, for the last 10 months (minimum). Participants assigned to the AAC group were completely alcohol abstinent, i.e., do not drink alcohol at all, neither now nor in the past. The demographic and drinking characteristics of both groups are shown in Exclusion criteria for both groups were defined as the following: be left-handed; scores \u2265 20 in the AUDIT; GSI \u2265 90 or scoring in at least 2 symptomatic dimensions of the SCL-90-R; uncorrected sensory deficits; personal history of traumatic brain injury or neurological disorder; regular  consumption of cannabis, personal history of regular or occasional use of other drugs ; Alcohol Use Disorder (AUD), i.e., alcohol abuse/dependence, based on DSM-IV-R criteria; personal and/or family history of any neurological or DSM-IV axis I disorder in first-degree relatives, family history of alcoholism in first-degree relatives; and magnetic resonance imaging (MRI) contraindications.Personal and family history of alcoholism plus medical or psychopathological disorders information was collected through a semi-structured interview including: a Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA), Individual Assessment Module (IAM) and Family History Assessment Module (FHAM), designed by the Collaborative Study on the Genetics of Alcoholism . In addiSociodemographic and substance use data were collected through a questionnaire that, besides sociodemographic information, included items 10, 11 and 12 from the Alcohol Use Questionnaire (AUQ) , assessiAdditionally, a diary of alcohol ingestion, questions about consumption of alcohol and other psychoactive substances  and the Portuguese version of the Alcohol Use Disorder Identification Test (AUDIT)  were admImpulsivity was assessed through the Barratt Impulsiveness Scale 11 (BIS11) . BIS is All the participants (regardless of whether they had been pre-classified as BDs or AACs) underwent the same clinical, neuropsychological and neuroimaging assessment protocol. Prior to the MRI assessment, participants were asked to abstain from BD during the three preceding days, consuming drugs and alcohol 12 h before the scanning and to avoid smoking and drinking tea or coffee for at least 3 h in advance. All participants gave written informed consent after the procedure had been carefully explained and received a financial stipend for their participation. The research was conducted in accordance to the ethical principles for medical research involving human subjects of the World Medical Association present in the Declaration of Helsinki . The Bio2, and 256 mm field of view (FoV).The neuroimaging assessment was conducted with clinically approved Siemens Magneton TrioTim 3T MRI scanner  equipped with a 32-channel receive-only head coil. Sagittal high-resolution 3D T1 weighted anatomical images were acquired using a magnetization prepared rapid acquisition gradient echo (MPRAGE) sequence with the following parameters: repetition time (TR) = 2700 ms, echo time (TE) = 2.33 ms, flip angle (FA) = 7\u00b0, 192 slices with 0.8 mm thickness, in-plane resolution = 1 \u00d7 1 mm1) executed in Matlab R2015a  with the VBM module. VBM is an automated processing technique applied to the entire brain allowing the characterization of shape and neuroanatomical configuration of different brains. Local composition of brain tissues is compared based in a voxelwise approach  space. Data was processed using SPM12 pipeline and statistical tools  that included both cortical and subcortical areas of these brain regions, known to be involved in the inhibitory circuitry .For this purpose, a review on the prefrontal-striatal network underlying self-regulatory mechanisms involved in the addiction circuitry  was perfp < 0.05. The correction was determined over 1000 Monte Carlo simulations using AlphaSim tool, distributed with REST toolkit , , .In addition, Pearson correlations  were performed to analyze the relationship between GM densities and both alcohol-related measures only in the BD group: number of times of binge drinking per month, number of months with BD pattern, grams of alcohol consumed per week, speed of drinking (grams/h during BD episodes), AUDIT scores, and BIS scores: total score, and subscales: attention, cognitive instability, motor control, perseverance, cognitive complexity and self-control. Additionally Pearson correlations between GM densities and the BIS scale and subscales scores were also calculated for both groups.K = 315, z = 3.98, p < 0.0001 uncorrected; AlphaSim correction, p < 0.05, cluster size > 29), when compared to the AACs. Figure 2 illustrates the regions where significant differences in peak-level densities were observed between BDs and AACs.Increased GM densities in the left middle frontal gyrus were observed in the BDs . Post hoc tests revealed that BDs males displayed higher GM densities than AACs males in the left middle frontal gyrus ; and BDs females displayed higher GM densities in the left middle frontal gyrus  when compared to AACs females.A group-by-gender interaction effect was observed in the left middle frontal gyrus . No significant correlations between GM densities and the BIS sub-factor self-control were observed in the AAC group. Finally, no significant correlations between GM densities and alcohol-related measures were observed in the BD group  is an additional constraint. VBM is a fully automated method, as manual segmentation methods are considered the gold standard for structural neuroimaging studies. Finally, we could have had an additional control group including light or regular drinkers, in order to compare them with BDs and with the AACs. Future studies should take the advantage of longitudinal designs with more than two follow up assessments and the combination of morphometric, genetics and behavioral measures in order to disentangle whether structural abnormalities reflect vulnerability factors or consequences of high alcohol consumption.This study suggests frontal GM abnormalities in BDs college students, which is likely to impact self-regulatory processes. The pattern of increased regional GM density suggests that developmental factors may contribute to brain alterations in BDs.SS wrote the manuscript, collected data, preprocessed the data, carried out the statistical analysis, and helped with subject\u2019s recruitment and assessment. AS coordinated data acquisition, preprocessing and statistical analysis and collaborated in manuscript writing. PM helped with data preprocessing. OG collaborated in manuscript writing and AC designed the study, coordinated subject\u2019s recruitment and assessment and data acquisition, and collaborated in manuscript writing. All authors 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": "We previously showed that autologous dendritic cells (DCs) loaded with an allogeneic heat shock (HS)-conditioned melanoma cell-derived lysate, called TRIMEL, induce T-cell-mediated immune responses in stage IV melanoma patients. Importantly, a positive delayed-type hypersensitivity (DTH) reaction against TRIMEL after vaccination, correlated with patients prolonged survival. Furthermore, we observed that DTH reaction was associated with a differential response pattern reflected in the presence of distinct cell subpopulations in peripheral blood. Detected variations in patient responses encouraged molecular studies aimed to identify gene expression profiles induced after vaccination in treated patients, allowing the identification of new molecular predictive markers.Gene expression patterns were analyzed by microarrays during vaccination, and some of them confirmed by quantitative real-time reverse transcriptase PCR (qRT-PCR) in the total leukocyte population of a representative group of responder and non-responder patients. New candidates for biomarkers with predictive value were identified using bioinformatics, molecular analysis, and flow cytometry.Seventeen genes overexpressed in responder patients after vaccination respect to non-responders were identified after a mathematical analysis, from which ten were linked to immune responses and five related to cell cycle control and signal transduction. In immunological responder patients, increased protein levels of the chemokine receptor CXCR4 and the Fc-receptor CD32 were observed on cell membranes of CD8+ T and B cells and the monocyte population, respectively, confirming gene expression results.Our study contributes to finding new molecular markers associated with clinical outcome and better understanding of clinically relevant immunological responses induced by anti-tumor DC-vaccines. Influence of genetic predisposition in medical conditions is a relevant aspect to be evaluated related to the benefit of a specific drug or therapeutic approach. A patient's genetic predisposition or resistance can be revealed, at least in part, by high-throughput gene expression profile studies, including microarrays, in which gene expression can be determined, preferentially in an extended manner \u20133.Recently, immunotherapy has emerged as a reliable therapeutic alternative after decades of treating cancer mostly by chemo- and radiotherapy . The accin vitro and in vivo antitumor and memory immune responses in patients with advanced malignant melanoma. Our clinical data showed that more than 60% of DC-vaccinated patients develop a delayed-type hypersensitivity (DTH) response against antigens derived from TRIMEL, and were classified as immunological responder patients, showing a significant correlation with improved survival of stage IV melanoma patients (median: 33 months for DTH-positive) compared with a median of 11 months for stage IV/DTH-negative patients that were classified as non-responder patients [For the last ten years, a protocol using a unique DC-based vaccine, consisting in autologous monocyte-derived DCs loaded with an allogeneic heat shock (HS)-conditioned melanoma cell-derived lysate , obtained from three allogeneic melanoma cell lines, has been included in phase I/II clinical trials. In this approach TRIMEL lysate provides a reproducible pool of several potential TAAs to DCs, suitable to be used in a broad number of patients, independently of their major histocompatibility complex (MHC) haplotype or the availability of autologous tumor tissue. Moreover, the unique composition of TRIMEL after heat conditioning allows for danger signals to trigger the optimal maturation and activation of monocyte-derived DCs ,14. Morepatients , 14. In patients . Moreovepatients . Taken tpatients .In this work, we quantified gene and protein expression of advanced melanoma patients in response to the treatment with TRIMEL-loaded DCs, according to the timing of their treatment. Our results showed that seventeen genes were consistently overexpressed in responder patients during the vaccination protocol, and the protein products of ten of those genes have well-known immune-response-related functions. Importantly, we observed that the chemokine receptor CXCR4 and the receptor for the Fc portion of IgG, CD32, were overexpressed in the lymphocytes' cell membranes and in the monocyte population in immunological responder patients. Further studies are warranted to confirm these targets as predictive and/or follow-up biomarkers for melanoma patients.Twenty-eight patients with advanced malignant melanoma (7 stage III and 21 stage IV patients) were recruited and treated with TRIMEL-loaded DCs according to a previously described protocol . PeripheTranscriptome profiling of twelve melanoma patients  that showed considerably prolonged survival times , which even exceed the reported median survival obtained by stage IV/DTH-negative patients in a previously reported study  [In this way, time courses of 17 genes were identified that allowed us to distinguish between both groups of patients. Fifteen of these genes have known functions. Interestingly, ten (66.7%) are related to the immune response\u2014CLEC2D, CXCR4, FCGR2A, GIT2, MS4A7, PRDM1, PRDX3, SDCBP, SPG21 and VNN2\u2014 whereas five (33.3%) are associated with cell cycle and signal transduction\u2014CREB5, CSNK1A1, EIF4G2, STRN3 and TROVE2 Figure . Some ofTo confirm this data set, RNA samples from the same patients were assessed for transcriptome profiling  using the setting of a maximum of 50 interactions, and BioGrid (https://thebiogrid.org/), with no limitations of interactions. The platforms list all molecular interactions known from the available literature, which is admittedly a bias. The results from both platforms are listed for each of the 15 genes in https://cran.r-project.org/bin/windows/base/), and turned into a graphical display by using \u201cigraph\u201d (http://igraph.org/r/ a windows binary package). This strategy enabled us to identify molecular targets connecting two or more of the 15 genes, and thus giving rise to a molecular network , whereas genes CLEC2D, CXCR4, FCGR2A, GIT2, SDCBP, SPG21 and VNN2 showed this discrimination at later expression point (4th vaccination), also with perfect ROC curves parameters , but with a p value at the limit of the significance (p = 0.053), suggesting their potential use as predictive or follow-up biomarkers    . qRT-PCRTo investigate if gene expression profiles observed on the transcript level correlated with the protein level, cryopreserved peripheral blood mononuclear cells (PBMCs) obtained from five healthy donors and 19 patients\u2014nine immunological responders and 10 immunological non-responders\u2014were subjected to flow cytometry. From these patients, three had previously been studied by microarrays. Expression data of samples from different vaccination time points were merged for the flow cytometry analysis due to a low availability of samples because of clinical states in individual patients. For that we defined as pre-treatment, samples collected at leukapheresis time, and before 1st and 2nd vaccination; and as post-treatment, samples collected at 3rd, 4th vaccination and at DTH evaluation time point. These criteria were used considering previously reported immunological evaluations for TRIMEL-loaded DC immunotherapy treated patients, which showed a specific cytokine response in PBMC from both DTH+ and DTH- patient subgroups, collected after the 2nd immunization, and after a complete vaccination cycle .To discriminate between lymphocyte and monocyte populations, the side-scatter parameter (SSC), related with cellular complexity, was used combined with the CD45 marker, which is expressed in all hematopoietic cells except in red blood cells (RBCs) and platelets . Furthermore, CD4+ T-cells (CD3+/CD4+), CD8+ T-cells (CD3+/CD8+), B-cells (CD3\u2013/CD19+), Natural Killer (NK) cells (CD16+/CD56+) and monocyte populations (CD14+) were identified by use of respective differentiation markers. There were a few samples where it was not possible to evaluate all cell populations. In those cases, protein expression was not determined .Based on the results obtained for gene expression, and taking their immune response related functions into account, the proteins CXCR4, CD32 (which comprises FCGR2A) and CLEC2D were evaluated. Higher CXCR4 protein expression was observed in immunological responder patients compared to healthy donors and immunological non-responder patients, at pre- and post-treatment time points, in all cell types except for NK cells Figure . MoreoveOn the other hand, significantly higher CD32 protein expression in monocyte populations was observed in immunological responder compared to non-responder patients, at pre-treatment time points, which is consistent with its role as a predictive biomarker. Additionally, significant differences of CD32 expression were obtained between healthy donors and immunological responder patients, at pre- and post-treatment in NK-cells; and at post-treatment in B-cells. Similar significant differences were found between healthy donors and non-responder patients Figure . RegardiConcerning CLEC2D protein expression, despite the variability obtained, significant differences between healthy donors and immunological responder patients, at pre- and post-treatment in CD4+ and CD8+ T-cells, respectively, were observed .Finally, if we consider the expression levels of the three proteins evaluated\u2014CXCR4, CD32 and CLEC2D\u2014in each cell population, and in each patient, a clear profile related to the treatment response is still unresolved.Several strategies have been designed to identify multiple factors associated with an effective antitumor immune response against melanoma. Until 2010, no randomized clinical trials had provided evidence of improved survival for patients with advanced-stage metastatic melanoma . HoweverIn this context, we have previously reported a TRIMEL-loaded DC-based immunotherapy capable of extending the survival time in stage IV malignant melanoma patients that respond with a DTH+ immune reaction , compared to patients that do not develop this type of response (DTH-)  , 14. BesInterestingly, some of the candidate genes obtained in these expression profiles were the immune-related receptors CXCR4, CD32 and the ligand CLEC2D, which displays relevant immune response functions, such as migration, antibody signaling responses and activation. These tentative targets were further quantified at the protein level within the leukocyte populations of patients with advanced malignant melanoma, which confirmed their capacity to discriminate between immunological responder and non-responder patients to TRIMEL-loaded DC immunotherapy, not only at the end of the immunization protocol, but also before the treatment. We decided to analyze these three markers at protein level for several reasons: 1) mRNA expression levels of these genes were corroborated by qRT-PCR analysis; 2) the ROC curve analyses, for these genes showed a bias between responder and non-responder patients at later expression points, supporting their use as predictive or follow-up biomarkers; 3) the protein product for these genes are expressed in different immune cell populations; and 4) the availability of well characterized antibodies against these genes products for flow cytometry use.CXCR4 is a chemokine receptor belonging to the G protein-coupled receptor family and is specific for the ligand CXCL12. The CXCR4/CXCL12 complex plays a central role in neutrophil migration, and in the recruitment of leukocyte populations to sites of inflammation , 21. AltCD32, a membrane glycoprotein receptor for the IgG crystallizable portion (FCGR), has three isoforms, FCGR2A, B and C. FCGR2A is expressed in all cells of the myeloid lineage, but not in lymphocytes; FCGR2B is expressed in basophils, neutrophils, monocytes, macrophages, DC and B lymphocytes; while FCGR2C is expressed in neutrophils, monocytes and NK cells. CD32 is involved in endocytosis, phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), release of soluble mediators and immunomodulation, making it an interesting target for modulating the immune system. The A and C isoforms have intracellular domains with a pattern of tyrosine-based activation (ITAM), whereas the B isoform contains the inhibitory counterpart (ITIM) \u201325. ConsOn the other hand, CLEC2D protein is expressed in DC, NK-, T- and B-cells, and participates in activation of lymphocyte populations through its interaction with the CD161 receptor, a member of the C-type lectin superfamily, which is found in immature NK cells \u201328. SincThe present data show that there are consistent differences between immunological responder and non-responder patients to TRIMEL-loaded DC immunotherapy, using CXCR4 and CD32 as tentative biomarkers, although, we cannot discard the dependence of other potential additional factors, as exome mutations, polymorphisms in immune related genes or immunological changes induced by particular tumor mutations in each patient. Moreover, these data include more gene products\u2014CLEC2D, GIT2, MS4A7, PRDM1, PRDX3, SDCBP, SPG21 and VNN2\u2014as potential biological markers that warrant further confirmation, either in the previously analyzed patients or in a follow-up study of new patients. Additionally, protein network analysis provides a more sophisticated insight into possible interactions between the 15 genes differentially regulated by the vaccination process. For instance, JUN/JUNB, CDC42, ITGA4 and UBC appeared connecting from 4 to 9 out of the 15 genes, constituting a theoretically \u201cvaccination network\u201d that included the most affected molecular targets in response to the expression changes experimentally discovered during the vaccination period, giving them a potential as tentative targets for pharmacotherapies. To our knowledge, this is the first identification of candidate genes with predictive value for positive outcomes to a DC-based immunotherapy. Their clinical use as biomarkers depends on further assessments in melanoma patients undergoing TRIMEL-loaded DC therapy.Peripheral blood samples were collected from 28 advanced melanoma patients (seven stage III and 21 stage IV patients) who had been vaccinated with TRIMEL-loaded DCs and followed up according to a previously described protocol . BrieflyThe study was performed in agreement with the Helsinki Declaration, and approved by the Bioethical Committee for Human Research of the Faculty of Medicine, Universidad de Chile. All patients signed an informed consent. Clinical and pathological characteristics of the patient cohorts including age, gender and disease stage are summarized in Table RNA was isolated from 10 mL of patient blood, using the LeukoLOCK\u2122 total RNA Isolation System , according to manufacturer's instructions. Briefly, whole blood was collected in EDTA and passed through the LeukoLOCK filter. The filter captured the total leukocyte population, while plasma, platelets, and RBCs were eliminated. The filters were flushed with 3 mL of PBS to remove residual RBCs and then with 3 mL of RNAlater to stabilize leukocyte RNA. The leukocytes trapped in the filter were lysed and collected in a 15 mL conical tube. Thereafter, RNA was captured from the lysate on RNA binding beads, purified, treated with TURBO DNase and finally eluted.The quantity of RNA was assessed using the NanoDrop \u039dD-1000A spectrophotometer  and RNA quality was evaluated using the Agilent 2100 Bioanalyzer . Only isolates with RNA integrity number (RIN) values equal or higher than 7.0 were used for further studies. RIN was determined using the RIN algorithm of the Agilent 2100 expert software.\u00ae Beadarray Reader system, Illumina\u00ae BeadStudio Application software and R+ Bioconductor software. Related data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE106128 (GSM 2830134\u20132830180). The analysis considered standard normalizations, a cut-off at 20 intensity values and a p-value of \u2264 0.01 with respect to the expression level. Subsequently, all genes that displayed p-values of > 0.01 or showed expression changes between 0.5- and 2-fold during the treatment in all samples, were eliminated because they were considered too small to be biologically significant. As a result, 13,702 genes were eliminated and 7,628 were included in the final analysis. RNAs obtained from the leukapheresis procedure or from the first sample obtained for the inoculation protocol  were used as a fold change reference.Total RNA (0.25 \u03bcg or 0.5 \u03bcg) was reverse transcribed and amplified with the Illumina TotalPrep kit  according to the manufacturer's instructions. Briefly, GPL6884 - Illumina HumanWG-6 v3.0 expression bead chip  that entail six arrays per chip  were hybridized overnight with biotinylated cDNAs, washed, blocked and fluorescence-labeled using streptavidin-Cy3. Data were obtained and analyzed by the Illumina\u00ae Green PCR Master Mix (2x) reagent  or ABsolute SYBR Green ROX Mix (2x) . The PCR assay was standardized using an Applied Biosystems 7500 Real-Time PCR System and the SDS 1.3 program or with an ABI Prism 7900HT and the SDS 2.2.2 program . Primers were selected in such a way that annealing temperatures were in a narrow range around 60\u00b0C  was reverse-transcribed into cDNA using SuperScript\u2122 III Reverse Transcriptase  according to manufacturer's instructions. The qRT-PCR was performed with a 1:10 cDNA dilution, using Power SYBRund 60\u00b0C . PCR was in 2002 . SubsequGenes were selected according to the following rules: (i) The first value in the time series is similar for both clinical responders (DTH+/long time survivors) and non-responder (DTH-) groups; (ii) at least one of the last two values  differ between the patient groups; (iii) the expression dynamics are similar within the groups; (vi) the gene expression shows significant fold changes for the clinical responders, and no significant changes for non-responder patients.pg(t)R for non-responder patients by pg(t)N.These rules are implemented in form of a set of scores for each gene. To compute these scores, we used the expression dynamics  of the individual genes for each patient gip(t), where i is the gene index, p the index of the patient, and t the time point. R represents the group of clinical responder patients and N the group of non-responder patients. We denote the average expression dynamics for clinical responder patients by The set of scores includes:sim: The absolute difference between the average 1st vaccination values for clinical responder and non-responder patients: diff26: The Euclidean distance between the average time series clinical responder patients tscore: For this score we first compute the sign function of the logarithmic fold changes, i.e. 1 if the gene is up-regulated and \u22121 if the gene is down-regulated. Then sum over the group of clinical responder patients (and non-responder patients) is then computed for the 4th and 5th time points: If for the entire group  the fold changes are above one, this score assumes a large positive number. If all fold changes are below one, the score is negative. If some fold changes are above and some one, the value of the score is near zero.SignifR/SignifN: This score gives the number of fold changes above 2 or below 0.5 in all time steps and all patients of the same group .absFCR/absFCN: This score represents the sum of the absolute values of logarithmic fold changes, computed separately for clinical responder patients (absFCR) and non-responder patients (absFCRN), e.g. A small value of this score means that the fold change is not significant and similar for all time points and patients.NumSigPos: This score indicates in how many patients of a group  the fold change of the gene expression is significant (above 2) in at least one time point.NumSigNeg: This core is the analog to the previous one, with the difference that the significant down-regulations (below 0.5-fold) are considered. By combining NumSigPos and NumSigNeg we identify genes, which show strong up-regulation in all (or at least N) clinical responders, and no significant down-regulation in any patient of the clinical responder group.Using these scores, we then executed several searches in which a subset of the scores was used to find genes showing specific behavior, in particular:Thawed PBMCs were stained according to the manufacturer's recommendations, with the following antibodies, allophycocyanin (APC)-conjugated anti hCD32  and anti-hOCIL/CLEC2d ; fluorescein isothiocyanate (FITC)-conjugated anti-hCD14 (clone 61D3) and anti-hCD16 (clone eBioCB16) ; phycoerythrin (PE)-conjugated anti-hCD11c ; FITC/PE-conjugated anti-hCD3/CD4 (clone SK7/SK3) and anti-hCD3/CD8 (clone SK7/SK1) (BD Simultest); PE Cy7-conjugated anti-hCD184  and peridinin-chlorophyll protein (PerCP)-conjugated anti-hCD19 (clone HIB19) and anti-hCD45 (clone HI30) . To discard dead cells from the analysis, we used Propidium Iodide Staining Reagent Solution  according to manufacturer's instructions. Cells were then incubated with 2 mL of BD FACS Lysing Solution  for 5 min at room temperature, washed and suspended in 500 \u03bcL FACS buffer.Flow cytometry was performed on a FACSCanto II  of the Clinical Hospital of the Universidad de Chile. Analysis of FACS data was performed using the FlowJo software .https://string-db.org/) using the basic settings, with a maximum of 50 interactions, and BioGrid (https://thebiogrid.org/), with no limitations of interactions. Statistics were analyzed by GraphPad Prism software . The differences between conditions were evaluated by the nonparametric Mann\u2013Whitney test. All p-values are 2-tailed, and a p-value of <0.05 was considered statistically significant.Survival curves were calculated using the log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon test. Time series analysis was executed using custom software, implemented in Matlab. The evaluation of the diagnostic accuracy of the biomarkers was performed through the construction of ROC curves, by calculating the area under the curve (AUC) value and determining the optimal cut-off points to estimate the highest sensitivity and specificity altogether, as assessed by Youden's Index. Protein network analysis was performed through two platforms from the Internet: STRING ("}
+{"text": "A knowledgebase with systematic collection and curation of lncRNA-disease associations is critically important for further examining their underlying molecular mechanisms. In 2013, we presented the first release of LncRNADisease, representing a database for collection of experimental supported lncRNA-disease associations. Here, we describe an update of the database. The new developments in LncRNADisease 2.0 include (i) an over 40-fold lncRNA-disease association enhancement compared with the previous version; (ii) providing the transcriptional regulatory relationships among lncRNA, mRNA and miRNA; (iii) providing a confidence score for each lncRNA-disease association; (iv) integrating experimentally supported circular RNA disease associations. LncRNADisease 2.0 documents more than 200 000 lncRNA-disease associations. We expect that this database will continue to serve as a valuable source for potential clinical application related to lncRNAs. LncRNADisease 2.0 is freely available at  Large number of studies have indicated that long non-coding RNAs  are highly associated with the progression of a wide variety of diseases ,2. Over http://www.rnanut.net/lncrnadisease/.Previously, we developed LncRNADisease database , which iLncRNADisease 2.0 contains experimentally and/or computationally supported data. For experimentally supported data, we searched the PubMed (before 31 May 2018) using the following keywords: \u2018long non-coding RNA\u2019, \u2018lncRNA\u2019, \u2018lincRNA\u2019, \u2018circular RNA\u2019 and \u2018circRNA\u2019, in combination with \u2018disease\u2019, \u2018cancer\u2019 and \u2018tumor\u2019. Then, we retrieved the entries that describe the associations between lncRNAs/circRNAs and diseases from these publications. All selected literatures were manually curated by at least two researchers. More than 12 000 published literatures were curated, and 3878 literatures were recovered for lncRNA/circRNA-disease associations. We then separated those literatures covered in previous databases,  and weak experimental evidence (e.g. microarray and RNA sequencing) by a manual assignment. RNA-seq and microarray are high-throughput procedure for global gene expression profiling which may not exactly reflect the expression status of all gene. These diverse evidence contribute unequally to the identification of a specific lncRNA-disease association. A confidence score system was developed to evaluate the reliability of a specific lncRNA-disease association by integrating different evidence resources .t is the evidence type , n is the number of evidence and tw is the weight factor. We assumed that lncRNA-disease associations supported by more computational evidence should not be given higher confidence scores than those supported by experimental evidence. So, s, www and pw is set to 1, 0.75 and 0.25, respectively. The confidence scores of well-supported lncRNA-disease associations are close to 1.In principle, we assume that (i) experimentally supported evidence contributes more significantly to the confidence score than computationally supported evidences; (ii) strong experimental evidence are considered to be more reliable than weak experimental evidence; and (iii) the entries supported by more evidence should have significantly higher confidence scores than those supported by one evidence. Therefore, the confidence score depends on the evidence types and the number of evidence resources. The probability disjunction formula has been widely employed to measure combined scores in the case that multiple pieces of evidence exist ,23. The cis-regulatory function of lncRNAs (defined as pairs of lncRNA and mRNA located within a genomic window of 100 kb). MicroRNA was derived from miRBase , cardiovascular disease (11.6%) and neurodegeneration disease (7.3%) represent the top three classes of diseases. LncRNADisease 2.0 contains 10 564 experimentally supported lncRNA-disease associations and 1004 experimentally supported circRNA-disease associations across four species (Figure A user-friendly web interface was developed to present LncRNADisease 2.0. All data were managed by a relational database implemented with MySQL. The web interface for browsing and searching was implemented by PHP and JavaScript program. Apache Tomcat web server was used for the http server.Recent experimental technologies and computational prediction algorithms have been developed, leading to the expansion of many diverse lncRNA-disease associations. In LncRNADisease 2.0, an over 40-fold increase in lncRNA-disease association enhancement were obtained compared with the previous version Table . We compA user-friendly web interface was developed to present the LncRNADisease 2.0. Users can browse and search all lncRNA-disease associations in the database. LncRNADisease 2.0 also provided an option in the \u2018Search\u2019 page that allows users to filter lncRNA-disease associations by certain experimental methods. In LncRNADisease 2.0, each lncRNA-disease association entry contains detailed information, including gene symbol, gene category, disease information, regulatory relationship, PubMed information, etc. To facilitate users accessing disease information from external resources, the disease names were mapped to the DO and MeSH.LncRNAs play increasingly appreciated gene-regulatory roles, and can affect an abundant number of target genes by interacting with sponging miRNAs. To further annotation the functional implication of disease-related lncRNAs, we constructed and visualized lncRNA\u2013miRNA\u2013mRNA network in LncRNADisease 2.0. It was developed on the basis of Cytoscape web program.Emerging evidence suggests that deregulation of lncRNAs plays an important role in diseases. To date, substantial studies have documented numerous lncRNAs involved in the progression of pathological disorders. Most disease-related lncRNAs have been examined in independent studies, and lncRNA-disease associations are scattered in various resources. Comprehensive collection of these lncRNA-disease associations will provide scientists with a resource for disease research. Here, we provided the LncRNADisease 2.0 to curate these data and provided a platform to facilitate the study of lncRNA-disease associations. LncRNADisease 2.0 contains more lncRNA-disease associations. We expect that the number of disease-related lncRNAs will continue to increase in the future release. We will continually maintain and update LncRNADisease database and integrate more related datasets, such as genomic and epigenetic information. We expect that this database will continue to serve as a valuable source for potential clinical application related to lncRNAs."}
+{"text": "The sustainability problems to the SS has led to a glowing debate about what the full retirement age should be and if working longer is a plausible option for everyone or just for those who have some control over their retirement decisions . All ethno-racial groups have increased their average retirement age over the last years. However, Hispanics\u2019 retirement age is still lower even if they stated they plan to continue to work at retirement . Most studies about retirement timing have focused on middle-class Whites, and the prediction of planned or actual retirement separately. One of the lesser studied complexities of the retirement conundrum concerns ethno-racial differences and cultural-related predictors of retirement timing . This study seeks to extend the understanding of differences between Hispanics and non-Hispanics regarding the timing of retirement relative to when they thought they would retire by including a broad array of cultural and family related predictors. Multinomial regression models were used. The results indicate significant differences between Hispanics and non-Hispanic Whites. Taking care of grandchildren was a significant predictor among Hispanics but not among non-Hispanic Whites. For Hispanics taking care of grandchildren, for over 20 hrs., was associated with a decreased probability of stating they will never retire. The increase of one dependent was associated with an increased on the probability of retiring earlier than planned. The effect of one additional dependent was larger for non-Hispanics."}
+{"text": "\u0394TEMP values and time for temperature to reach maximum (\u0394TIME) were subjected to two-way ANOVA and Bonferroni's post-hoc tests .After local Ethics Committee approval , local anesthesia, rubber dam isolation, small occlusal preparations/minute pulp exposure (n=15) were performed in teeth requiring extraction for orthodontic reasons. A sterile probe of a temperature measurement system  was placed within the pulp chamber and the buccal surface was sequentially exposed to a LED LCU  using the following exposure modes: 10-s low or high, 5-s Turbo, and 60-s high. Afterwards, the teeth were extracted and K-type thermocouples were placed within the pulp chamber through the original access. The teeth were attached to an assembly simulating the in vitro than in vivo. No significant difference in \u0394TIME was observed between test conditions. A significant, positive relationship was observed between radiant exposure and \u0394TEMP for both conditions .Higher \u0394TEMP was observed in vitro model overestimated in vivo PT increase, in vitro PT rise was close to in vivo values for clinically relevant exposure modes.Although the  Most of those studies relied on in vitro methods on the attention of researchers and manufacturers to this possible issue, it is reasonable to assume that in vitro conditions do not fully reproduce the complex physiological mechanism involved in the real in vivo condition. As a consequence, in vitro analysis is expected not to be capable of precisely reproducing in vivo PT when intact teeth are exposed to a LED light using varying exposure modes. However, due to the lack of in vivo studies that evaluated PT changes during heat stimulus when most in vitro studies were published in the past, and because of differences between tested teeth and tooth condition among studies, no contemporary data are available to confirm how well an in vitro model can reproduce temperature changes seen in the in vivo model, when under thermal stimuli such as the exposure to light emitted from a powerful LED LCU. Recently, an in vivo methodology was published that measured PT within the pulp tissue of human premolars.\u2013Despite the important impact of these conclusions based on in vitro model is able to reproduce temperature increase (\u0394TEMP) values compared to the in vivo model, in anesthetized intact, unrestored, human upper premolars, in order to validate the in vitro methodology. The unique feature of this work was that the same premolar teeth tested for in vivo temperature rise were extracted for orthodontic treatment, and were subsequently tested in a clinically relevant in vitro system. In addition, the same LCU used in the in vivo analysis was also used for in vitro analysis. The tested alternative hypotheses were that (1) there are no significant differences in \u0394TEMP values and time for temperature to reach maximum (\u0394TIME) measurements between in vitro and in vivo models, and (2) both the in vivo and in vitro models show a direct, positive correlation between applied radiant exposure to intact facial tooth surface and both \u0394TEMP and \u0394TIME.Thus, the purpose of this study was to evaluate how similar an in vivo studies,\u2013After approval by the local Ethics Committee , study participants requiring extraction of upper right and left first premolars for orthodontic reasons were selected from the Orthodontic specialization program in Ponta Grossa, Brazil, and were recruited in February, 2013. The participants were seen between March and April, 2013. Inclusion and exclusion criteria were based on previous in vivo real-time temperature analysis within the pulp was evaluated following a method previously described in the literature.\u2013\u00ae LED LCU  using the following exposure modes (EMs): 10-s at low intensity (10-s/L); 10-s at high intensity (10-s/H); 5-s at Turbo intensity (5-s/T); and 60-s at high intensity (60-s/H). These exposure modes were selected because they are the most clinically relevant modes used for a wide variety of clinical applications. A 7-min time span between each exposure was allowed for the PT to return to baseline levels. The sequence of EMs was randomly determined and the operator was not aware of which mode was being used. The time of data acquisition when each light mode was applied was recorded using a digital time counter that started recording simultaneously to the beginning of real-time PT analysis, so that time of light activation and correlated temperature measurement could be precisely made. The probe was removed from the tooth at the end of the temperature data acquisition, and the tooth was extracted as planned. Radiographs were taken from the proximal side of the extracted tooth with the probe in position as it was intraorally in order to confirm the proper insertion depth and location of the probe within the pulp chamber during temperature measurement.The in vivo and in vitro analyses. This value was then multiplied by the light exposure duration in order to derive the value of radiant exposure applied to each tooth surface for each light output mode  connected to a 6-in integrating sphere , previously calibrated using a NIST-traceable light source was used to evaluate the spectral power of the tested EMs. In this regard, the LCU tip end was positioned at the entrance of the integrating sphere, so that all light emitted from the unit was captured. Wavelength-based, spectral and power emission during each EM were recorded using software  between 350 to 550 nm, which also provided the total emitted power value for that wavelength range. Radiant emittance values of each EM mW/cm2 were din vivo study were tested in the in vitro, so any possible difference between outcomes would be exclusively attributed to the differences between the two models. The extracted teeth were stored in 0.1% thymol  until the moment they were fixtured to and tested in the in vitro model previously established.in vivo environment: controlled intrapulpal physiologic baseline temperature of approximately 35\u00b0Cin vivo analysis. The thermocouple junction was placed in a similar position to that of the thermocouple used in the in vivo analysis. In order to assure similar placement, x-ray analyses were used to compare thermocouple positioning for each tooth between the in vivo and in vitro conditions. Occlusal access was sealed and stabilized using acid etching and a flowable composite . Small sections of 16-gage stainless steel tubing were attached to the root ends using acid etching using the same flowable composite. A section of flexible plastic tubing was connected to one tube end and a portion of the remaining end was coiled to increase surface area in contact with the warmed water in the Erlenmeyer flask in which it was immersed. The tubing continued through a hole in the plastic plate placed over the water-filled Erlenmeyer flask . In this manner, the temperature calibration of the main in vitro temperature measurement system could be achieved by adjusting the water offset temperature to match that of the NIST-traceable device. The real-time profile data of in vitro and in vivo PT increase were plotted into line graphs , which were used to determine \u0394TEMP and \u0394TIME.The thermocouple output was connected to a multi-channel thermocouple interface card , where it was electronically cold-junction compensated, digitized, and sent to software  on a personal computer. The software both visually and digitally recorded the thermocouple temperature in real-time, at a rate of 10 data points in vivo model, the \u03c4 obtained was 1.46 s, while a \u03c4 of 0.05 s was observed for the thermocouple used in the in vitro model. In other words, the time required for the temperature data acquisition system to provide a 1-degree Centigrade temperature change using the in vivo PT analysis was approximately 0.07 s, while 0.0029 s were required for the thermocouple to provide the same 1-degree Centigrade temperature change using the in vitro setup.Time constant (\u03c4) of each thermocouple was determined as previously described.in vivo and in vitro were subjected to two-way repeated measures ANOVA, followed by Bonferroni's post-hoc test at a pre-set alpha of 0.05. Linear regression analysis was performed to examine the relationship between applied radiant exposure level and \u0394TEMP, as well as between radiant exposure and \u0394TIME in both conditions. The comparison between the slopes of both regression lines obtained from the in vivo and in vitro data was performed by performing t-tests as well as by comparing possible overlaps in the 95% confidence intervals (CI) for the mean slope values. No overlap indicated a significant difference, and overlap suggested no significant difference. Post-hoc power analysis was performed for the statistical analysis of \u0394TEMP and \u0394TIME. All analyses were performed using statistical software on a personal computer .The \u0394TEMP and \u0394TIME data obtained in vivo study was adequately powered for EM and condition (in vitro and in vivo) factors . in vitro and in vivo \u0394TEMP values. Overall, the in vitro model recorded significantly higher \u0394TEMP values than the in vivo model, regardless of EM. Although a significant, positive relationship [in vivo: ] was observed between delivered radiant exposure and \u0394TEMP in both models, the slope of the regression line created in vitro was significantly higher than that observed in vivo , the in vivo and in vitro models (p<0.001), whereas the 10-s/L EM produced the lowest values (p<0.001). Despite the in vivo \u0394TEMP average values being lower than 5.5\u00b0C, some teeth exhibited higher \u0394TEMP values than that threshold temperature .Use of the 60-s/H EM caused the highest \u0394TEMP in both perature . On the in vivo model was compared to that of in vivo results, regardless of EM  was also observed between delivered radiant exposure and \u0394TIME for both test conditions .Despite such differences in \u0394TEMP values, no significant differences in \u0394TIME values were noted when the ss of EM . In thisnditions . No signin vivo and in vitro models, the time/temperature profiles  between delivered radiant exposure and \u0394TEMP, the significantly lower regression slope obtained from the in vivo results  than the well-known threshold temperature of 5.5\u00b0C, in all extracted teeth, while the same EM applied in vivo resulted in lower average increase (4.8\u00b11.0\u00b0C) than that threshold temperature.in vitro studies may overestimate the effects of LCU exposure on pulp chamber temperature increase when high radiant exposure values are delivered to the teeth.In the current study, although the in vivo and in vitro methodologies may be attributed to the physiological response of the pulp tissue against heat rise, some features in these methodologies may have contributed to the differences in the temperature rise within the pulp chamber. For instance, the Class I preparation in the in vivo method was sealed with provisional restorative material, while the Class I preparation in the in vitro methodology was sealed with flow resin composite to keep the simulated fluid flow within the pulp chamber. Because zinc-based cements, such as the provisional material used in the in vivo model, allow for greater heat transfer than do resin composites,Although much of the differences in PT rise between in vitro model is capable of detecting temperature changes as the delivered radiant exposure values increased in a similar pattern as that observed in the in vivo model. Therefore, the only flaw observed in the in vitro model is the overestimation of the temperature rise within the pulp chamber. In this regard, some studies have shown that the increase in the in vitro simulated fluid flow rate resulted in lower temperature rise within the pulp chamber.,,in vitro model could be a valuable alternative to compensate for such difference between in vivo and in vitro models. Further studies are required to determine the settings of in vitro methodologies to provide temperature increase values within the pulp chamber similar to those observed in vivo.Despite these differences, the current results demonstrated that the Within the limitations imposed by the methodologies used, the following conclusions may be made:in vitro model detected higher PT increase than the in vivo model, when the same teeth were exposed to the same exposure modes from the same Polywave\u00ae LED LCU;The In vitro PT increase values were close to in vivo values when clinically relevant exposure modes were delivered (between 7 and 12 J/cmin vivo and in vitro models, so the influence of varying exposure modes on PT increase was similar for both models.A significant, positive and non-parallel correlation was observed between delivered radiant exposure and PT increase for both"}
+{"text": "Among the many methods available for solubility enhancement, mesoporous carriers are generating significant industrial interest. Owing to the spatial confinement of drug molecules within the mesopore network, low solubility crystalline drugs can be converted into their amorphous counterparts, which exhibit higher solubility. This work aims to understand the impact of drug overloading, i.e., above theoretical monolayer surface coverage, within mesoporous silica on the release behaviour and the thermal properties of loaded drugs. The study also looks at the inclusion of hypromellose acetate succinate (HPMCAS) to improve amorphisation. Various techniques including DSC, TGA, SEM, assay and dissolution were employed to investigate critical formulation factors of drug-loaded mesoporous silica prepared at drug loads of 100\u2013300% of monolayer surface coverage, i.e., monolayer, double layer and triple layer coverage. A significant improvement in the dissolution of both Felodipine and Furosemide was obtained . However, incomplete drug release was also observed at low drug load in both drugs, possibly due to a reversible adsorption to mesoporous silica. The addition of a polymeric precipitation inhibitor HPMCAS to mesoporous silica did not promote amorphisation. In fact, a partial coating of HPMCAS was observed on the exterior surface of mesoporous silica particles, which resulted in slower release for both drugs. The most important properties of promising drug candidates for oral dosage form are aqueous solubility and intestinal permeability. Over 40% of drugs on the market are BCS class II and IV, which have low solubility. Furthermore, new chemical entities are even less soluble compared to marketed products with a projection of up to 70\u201390% of drug candidates in the pipeline suffer from low solubility . FollowiThe problem of low solubility can be addressed by using solubilisation techniques, namely solid dispersion systems, size reduction, salt formation, prodrug, liposomes, etc. Among those techniques, solid dispersions are preferred by the industry due to their practicality and low cost. This technique is mainly based on a so-called \u201camorphisation\u201d, whereby the crystalline drugs are converted into their high energy amorphous form, which exhibits a superior solubility in comparison with that of the original ones . Mesoporw/w) could lead to incomplete amorphisation, i.e., a small amount of crystalline drug will remain on the exterior surface of the generated particles [For loading a drug into mesoporous silica, there are several techniques, which can be categorised into two main approaches: solvent-free methods and solvent-based methods. Solvent-free methods are comprised of physical mixing followed by heating to melt the drug, co-milling between the drug and mesoporous materials, and using supercritical carbon dioxide. Although solvent-free loading methods offer apparent advantages, e.g., no requirement for checking the residual solvent in drug products and low environmental impact, these methods are still under investigation to exhibit better performance in terms of the loading efficiency and stability of thermolabile drugs. On the other hand, solvent-based approaches offer a practical and straightforward solution for drug amorphisation within mesoporous silica. Simply put, a drug is dissolved in a suitable solvent, e.g., ethanol, then mixed/impregnated with mesoporous silica. The solvent can be removed by appropriate drying techniques at the end of the process. There are various factors that influence drug loading into mesoporous silica, such as the type of solvent, drug load, accessible surface area, and the pore volume of the mesoporous silica. In general, solvent-based loading techniques, especially spray drying, produce drug-loaded mesoporous silica with a high loading efficiency compared to solvent-free techniques . Howeverarticles . Drug moarticles . Dening articles  studied articles  found thFelodipine and Furosemide are BCS class II and class IV drugs, respectively. Felodipine is mainly absorbed in the small intestine , i.e., a\u00ae XDP 3050  was kindly provided by W.R. Grace and Co. . Aqoat\u00ae (HPMCAS) was a generous gift from Harke Pharma . Sodium phosphate monobasic, sodium phosphate dibasic, sodium chloride, and sodium lauryl sulfate (SLS) were purchased from Sigma-Aldrich . Hydrochloric acid 37%, acetone and ethanol were purchased from Fisher Scientific . Deionised water was produced by Milli-Q Integral system .Felodipine (FELO) and Furosemide (FURO) were obtained from Discovery Fine Chemicals  and Chemical Point , respectively. Mesoporous silica Syloiddrug/wSyloid) to theoretically obtain monolayer adsorption  in mesoporous silica:Theoretical drug load was calculated based on an assumption that drug molecules would adsorb to the surface area of mesoporous silica particles in a packing geometry that increases the bonding between drug molecules and silica surface, i.e., to maximise the contact surface . The folSSA: Specific surface area of mesoporous silica (m2/g), e.g., 310 m2/g for Syloid XDP 3050 (in-house data measured by gas adsorption porosimetry).wM: Molecular weight of model drug (g/mol).MSA: Maximum projected contact surface area of single molecule (\u00c52): calculated using the two largest molecular dimensions of drug molecule (molecule .AN: Avogadro\u2019s number (6.022 \u00d7 1023).w/w): 10.8, 21.6, and 32.4% for FURO; 12.6, 25.2, and 37.8% for FELO to represent monolayer adsorption (100% surface coverage), double layer adsorption (200% surface coverage), and triple layer adsorption (300% surface coverage), respectively. The ternary Drug\u2013Syloid\u2013HPMCAS formulations were prepared in a similar fashion with the ratio of drug to HPMCAS at 10:1. Syloid was added to solutions of HPMCAS and either FELO or FURO in ethanol-acetone 50:50 (10 mg/mL). All of the suspensions were gently stirred for 12 h, then spray-dried at inlet temperature of 100 \u00b0C using a mini spray dryer Buchi B-290 and inert loop Buchi B-295  in closed mode with a nitrogen flow rate of 600 L/min, with a feed rate of 5 mL/min and a drying gas flow rate of 30 m3/h. Spray-dried FELO or FURO (prepared in the same procedure without the incorporation of either Syloid or HPMCAS), physical mixtures between Syloid and either FELO, or FURO with a ratio of 1:1 were used as control samples.Syloid was added to either FELO or FURO in ethanol (10 mg/mL) to form suspensions at various theoretical drug loads  in an Erweka DT 126 dissolution tester . Each sample containing 20 mg of FELO was filled into a HPMC hard-shell capsule and tested in 500 mL of pH 6.5 medium with 0.25% SLS at 37 \u00b0C (adapted from USP 36 monograph with a reduction of SLS concentration from 1.0 to 0.25%). Samples were withdrawn during a 120 min period at the following timepoints: 15, 30, 60, 90, and 120 min. The concentrations of dissolved FELO were determined according to a HPLC method described in United States Pharmacopoeia  with mobFor FURO, samples containing 40 mg was filled into a HPMC hard-shell capsule and tested in 900 mL of a pH 3.0 medium with 0.25% SLS at 37 \u00b0C. The medium was prepared by dissolving 2 g of sodium chloride and 2.5 g of SLS in 400 mL of deionised water, then adding 0.1 mL of hydrochloric acid 37% and diluting with deionised water to 1000.0 mL . The conThe thermal properties of samples were characterised by DSC instrument TA Q200 . Each sample was accurately weighed  into Tzero low-mass aluminium pan (sensitivity for a minimum sample size of 0.5 mg), and heated in the range of 50\u2013250 \u00b0C (for FELO) or 100\u2013300 \u00b0C (for FURO) at a scanning rate of 10 \u00b0C/min under nitrogen airflow of 50 mL/min. TA universal analysis 2000 software (version 4.5) was employed to analyse the resulting DSC thermograms.The surface of the drug-loaded mesoporous silica particles was examined by a Philips XL30 ESEM FEG  operating at 10 kV under a high vacuum. Prior to SEM imaging, samples were coated with gold by a sputter coater. Approximately 1 mg of each sample was placed onto a double-sided adhesive strip on a sample holder. SEM images were taken at 2000\u00d7 magnification.p value < 0.05. All results are presented as mean \u00b1 standard deviation where applicable.Statistical analysis was carried out using GraphPad Prism 7.03 software. Statistically significant difference was considered at a DSC analysis of Felodipine and Furosemide samples were presented in Sharp endothermic peaks at 222.8 \u00b1 0.8 \u00b0C and 223.9 \u00b1 0.3 \u00b0C were observed in DSC curves of Furosemide raw material and FURO-Syloid physical mixture respectively , which iTGA results  were useActual drug loads and loading efficiencies of Felodipine and Furosemide in mesoporous silica are presented in 2/g vs. 310 m2/g, respectively). Hence, despite the apparent difference in weight-per-weight drug load, after normalisation based on surface area, there is no difference in weight-per-surface area drug load between the two studies (0.542 \u00b1 0.004 \u00d7 10\u22123 g/m2 in comparison with 0.535 \u00b1 0.010 \u00d7 10\u22123 g/m2).Previously, Ambrogi et al.  studied 2/g and 1051 m2/g with a drug load of 25% [\u22123 compared to 0.428 \u00d7 10\u22123 g/m2 and 0.174 \u00d7 10\u22123/m2).For Felodipine-loaded mesoporous silica, there were two previous studies using solvent impregnation methods, in which silica had a specific surface area of 584 md of 25%  and 18.3In general, the drug loading process for mesoporous particles is usually established on a case-by-case basis due to the differences in loading solvent, solubility, targeted drug load, surface area, pore size, and pore volume of the mesoporous materials being used. The use of co-spray drying in the current study as a solvent-based technique produced comparable or slightly higher drug loads to those reported in the literature using traditional solvent-evaporation. However, the real added advantage of using co-spray drying lies in the better drug loading efficiency and shorter processing time. During spray drying, there is a possibility that solute/drug diffusion towards the particle centre might facilitate drug entrapment inside the pores. The findings of higher drug loading efficiency via spray drying are also in agreement with previous studies ,27.p < 0.05). Felodipine exhibited a low dissolution within 60 min with only 9.8%, 11.4%, and 15.5% dissolution for the physical mixture, raw materials, and spray-dried materials, respectively, compared to over 70% dissolution in any Felodipine-Syloid samples . A weak basic drug Felodipine in a loading solution can partially dissociate to form a negatively charged anion, which might be converted into a conjugation with an acidic polymer HPMCAS. Such a conjugation between Felodipine and HPCMAS could enhance the loading efficiency as there could be an extra amount of Felodipine staying within the polymer matrix. However, there were no significant improvements in loading efficiency for Furosemide with the presence of HPMCAS (p > 0.05). This result could be explained because Furosemide is an acidic drug, and, therefore, no conjugation was formed between Furosemide and HPMCAS in comparison to the synergistic effect in drug loading between Felodipine and HPMCAS . This polymer is only soluble at pH 5.5 or above, hence the partial coating of HPMCAS on the mesoporous silica was not dissolved at pH 3 acidic environment  and resulted in an adverse effect on the drug release of Furosemide (The inclusion of HPMCAS with Syloid did not help in enhancing the release rate or overall dissolution of Felodipine . In factrosemide .Furosemide-Syloid-HPMCAS still obtained 29% dissolution after 120 min as the drug was possibly released from silica particles through incomplete coating layers. A previous study  showed aSyloid enhanced the dissolution of both model poorly soluble drugs, Felodipine and Furosemide, due to amorphisation within its mesoporous network. Increasing the drug load or percentage of theoretical surface coverage increased the maximum attained dissolution. However, overloading could lead to the formation of surface nanocrystals, as observed in the thermal and morphological studies. Incomplete drug release happened at all drug loads, which could be due to the reversible adsorption phenomenon mentioned in previous reports. This was predominant at the lower drug loads as less drug is available to dissolve before reaching an adsorption equilibrium. In addition, overloading resulted in a decrease of loading efficiency for both tested model drugs. A new drug load based on the drug amount to specific surface area of materials could be used in order to enable the comparison between various types of mesoporous materials. The addition of HPMCAS at a low concentration prevented the complete amorphisation of the drugs. Furthermore, it also slowed down the drug release due to partial coating, which formed on the exterior surface of mesoporous silica particles. In the future, this concept of combining mesoporous silica and polymers in hybrid structures will be further investigated for potential application in sustained release drug delivery systems."}
+{"text": "We therefore grew well-characterized IDH1mut (n = 4) and IDH1wt (n = 4) GSC lines under normoxic (20%) and hypoxic (1.5%) culture conditions and harvested mRNA after 72 h. Transcriptome analyses were performed and hypoxia regulated genes were further analyzed using the expression and clinical data of the lower grade glioma cohort of The Cancer Genome Atlas (LGG TCGA) in a confirmatory approach and to test for possible survival associations. Results show that global expression changes were more pronounced in IDH1wt than in IDH1mut GSCs. However, when focusing on known hypoxia-regulated gene sets, enrichment analyses showed a comparable regulation in both IDH1mut and IDH1wt GSCs. Of 272 significantly up-regulated genes under hypoxic conditions in IDH1mut GSCs a hypoxia-related survival score (HRS-score) of five genes  was identified by the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm which was able to predict survival independent of age, 1p19q co-deletion status and WHO grade (II vs. III) in the LGG TCGA cohort and in the Rembrandt dataset. Altogether, we were able to identify and validate a novel hypoxia-related survival score in IDH1mut GSCs consisting of five hypoxia-regulated genes which was significantly associated with patient survival independent of known prognostic confounders.Glioma growth is often accompanied by a hypoxic microenvironment favorable for the induction and maintenance of the glioma stem cell (GSC) phenotype. Due to the paucity of cell models of Isocitrate Dehydrogenase 1 mutant (IDH1 In. Inwt anreported ,57. Hypoescribed . mRNA wamut GSCs has been shown by Kohanbash et al. [wt GSCs elevated clonogeneity as well as expression of certain stem cell markers has been shown in previous publications from our laboratory [The expression of several stem cell markers including CD133, SOX2, CD44, CSPG4, CD90, and nestin in the IDH1h et al. . Also foboratory ,60.mut GSCs as decribed by Balss et al. [(D)-2-hydroxyglutarate levels were measured in the cell culture supernatant of IDH1s et al. . In briewww.r-project.org). Inter-array normalization was conducted using vsn normalization in the vsn package. Differential gene expression was assessed by a paired test with the limma package. Survival analysis was conducted within the survival package. Gene set enrichment analysis for hypoxia related gene sets in the C2 (curated gene sets) and C5 (GO gene sets) collections of the MSigDB library was conducted by the GSVA package.1 \u03bcg of total RNA from normoxic and hypoxic cells was submitted to the Genomics Core Facilities of the German Cancer Research Center  for microarray analysis. After purification, reverse transcription into cDNA and labeling according to the Illumina protocol, samples were hybridized to Human HT-12 V.4.0 arrays . Raw data can be accessed in the NCBI GEO repository under the accession number GSE118683. Raw intensity data were obtained after image analysis of the fluorescent spot intensity reads. All preprocessing and normalization steps were performed in the \u201cR\u201d programming environment (available online: https://gdac.broadinstitute.org). Raw data were voom normalized by the help of the limma package. Of 515 patients, only IDHmut cases were considered for further clinical analysis (n = 395). In line with The Cancer Genome Atlas Research Network, we analyzed survival in the following IDH mutated subgroups: LGG with 1p/19q co-deletion (n = 159), LGG without 1p19q co-deletion (n = 236) and LGG WHO\u00b0 II (n = 214) and WHO\u00b0 III (n = 181) (https://gdoc.georgetown.edu/gdoc/). Based on the clinical data we selected patients with either astrocytoma or oligodendroglioma and completed survival data.RNAseq data from the TCGA Lower Grade Glioma cohort were downloaded from firehose (available online: n = 181) . The micLASSO (least absolute shrinkage and selection operator) is a linear regression which performs both, variable selection and regularization in a given dataset to fit a model that describes a linear correlation of a response variable  and several explanatory variables (here genes) . This maFurthermore, LASSO was adopted to fit Cox ph survival models and is implemented in the broadly used glmnet package within the statistical software environment R . Gene sep values for differences in survival between the groups were calculated with the log-rank (Mantel Cox) test, whereas the median of expression was used to dichotomize the cohort and to define \u201chigh\u201d vs. \u201clow\u201d. Univariate and multivariate Cox regression analyses were performed to determine the prognostic significance of selected candidate genes and the hypoxia score. A p-value \u2264 0.05 was considered significant. Student\u2019s t-Test was performed for the gene set enrichment analysis between GSCs cultured in normoxia and hypoxia for IDH1mut and IDH1wt.Statistical analyses in boxplots were performed using the \u201cPrism 5\u201d software ."}
+{"text": "Mitochondria are essential cellular organelles, controlling multiple signalling pathways critical for cell survival and cell death. Increasing evidence suggests that mitochondrial metabolism and functions are indispensable in tumorigenesis and cancer progression, rendering mitochondria and mitochondrial functions as plausible targets for anti-cancer therapeutics. In this review, we summarised the major strategies of selective targeting of mitochondria and their functions to combat cancer, including targeting mitochondrial metabolism, the electron transport chain and tricarboxylic acid cycle, mitochondrial redox signalling pathways, and ROS homeostasis. We highlight that delivering anti-cancer drugs into mitochondria exhibits enormous potential for future cancer therapeutic strategies, with a great advantage of potentially overcoming drug resistance. Mitocans, exemplified by mitochondrially targeted vitamin E succinate and tamoxifen (MitoTam), selectively target cancer cell mitochondria and efficiently kill multiple types of cancer cells by disrupting mitochondrial function, with MitoTam currently undergoing a clinical trial. Mitochondria are dynamic intracellular organelles with their own DNA . They have multiple important functions, including controlling adenosine triphosphate (ATP) generation, metabolic signalling, proliferation, redox homeostasis, and promotion/suppression of apoptotic signalling pathways. Genetic and/or metabolic alterations in mitochondria contribute to many human diseases, including cancer . AlthougWe have recently proposed the term \u2018mitocans\u2019, an acronym derived from the terms mitochondria and cancer, a group of compounds with anti-cancer activity exerted via their molecular targets within mitochondria, some mitocans being selective for malignant tissues . This clMitochondrial metabolism is highly complex and involves multiple functions and signalling pathways. The major functions of mitochondria are the production of ATP via OXPHOS and formation of metabolites needed to meet the bioenergetic and biosynthetic demands of the cell. Mitochondria are also central to a wide variety of vital cellular processes including apoptosis, maintenance of calcium homeostasis, redox signalling, steroid synthesis, and lipid metabolism. In addition, mitochondria have the ability to alter their bioenergetic and biosynthetic functions to meet the metabolic demands of a cell via a cross-talk with other sub-cellular organelles, in particular the nucleus, but also the endoplasmatic reticulum . AccumulMitochondrial ETC comprises four complexes (I\u2013IV) that transfer electrons and engage in redox reactions. Transfer of electrons by means of ETC is coupled to pumping of protons from the matrix to the intermembrane space by complexes I, III, and IV. Functional ETC supports OXPHOS activity and ATP generation that is essential for tumorigenesis . Since the majority of ATP in tumour cells is produced by mitochondria ,21, targMany ETC inhibitors, including metformin, tamoxifen, \u03b1-tocopheryl succinate (\u03b1-TOS) and 3-bromopyruvate (3BP), act by disrupting the function of respiratory complexes of ETC and by inducing production/accumulation of high levels of ROS to kill cancer cells ,25. MetfOur group reported mitochondrial complex II (CII) as a novel target for cancer therapy. We showed that \u03b1-TOS, an efficient anti-cancer agent, inhibits succinate quinone oxidoreductase (SQR) and succinate dehydrogenase (SDH) activity of mitochondrial CII by interacting with the proximal and distal ubiquinone (UbQ)-binding site ,34. GracOther than inhibition of CI or CII function, some anti-cancer compounds affect mitochondrial complex IV (CIV) or ATPase (CV) activity, inhibiting cancer cell respiration and ATP production. A small molecule VLX600 was reported to be active against colon cancer by disrupting the function of CIV, by suppressing the expression of its subunit 1, the COX-1 . TigecycThe TCA cycle, also known as the Krebs cycle, is located in the mitochondrial matrix in eukaryotic cells. It comprises a series of chemical reactions used by aerobic organisms to release stored energy via oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. The TCA cycle is a source of electrons that feed into ETC to drive the electrochemical proton gradient required for ATP generation. Its intermediates are used for biosynthesis of various macromolecules. This is exemplified by glutamine, a major carbon source that replenishes the TCA cycle intermediates and sustains their utilization for biosynthesis in tumour cells . It is cCancer cells efficiently use both glycolysis and OXPHOS for their energy needs. Moreover, malignant cells have the ability of flexibly switching between glycolysis and OXPHOS, and this feature plays a major role in multiple modes of resistance to oncogenic inhibition ,12. AgenHexokinase II (HKII) is a major isoform of enzyme overexpressed in cancer cells with an important role in maintaining glycolytic activity. It also associates with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane that has a function in apoptosis. As such, the inhibition of HKII will not only inhibit glycolysis but may also suppress the anti-apoptotic effect of the HKII\u2013VDAC interaction. FV-429, an inhibitor of hexokinase, strongly induced apoptosis in cancer cells by both inhibition of glycolysis via suppression of HKII and by impairing the mitochondrial function via interfering with the HKII-VDAC interaction, leading to activation of mitochondria-mediated apoptosis . It was In recent years, there has been an upsurge in research focusing on reprogramming cancer cells via the understanding of their metabolic \u2018signatures\u2019. Alterations in mitochondrial bioenergetics and impaired mitochondrial function may serve as effective targeting strategies such as in triple-negative breast cancer (TNBC), where hormone receptors are absent and endocrine therapy inefficient. Glucose starvation of MDA-MB-231 and MCF-7 breast cancer cells provoked a decreased mitochondrial respiration. Glucose starvation also sensitized MDA-MB-231 cells to apoptosis and decreased their migratory potential .Tumour cells can alter their redox balance and deregulate redox signalling to support malignant progression and to gain resistance to treatment . They inROS are short-lived molecules with unpaired electrons derived from partially reduced molecular oxygen that are constantly generated, transformed, and eliminated via a variety of cellular processes including metabolism, proliferation, differentiation, immune system regulation, and vascular remodelling . The lev2\u2212\u00b7or H2O2 using NADPH as a reductant [2 to generate O2\u2212 by means of univalent reduction of molecular oxygen resulting in electron leakage during mitochondrial respiration [Of high biological relevance, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are enzymes that catalyze the production of Oeductant . ETC usepiration ,59,60. Npiration  that is piration . Thus, lpiration . Anotherpiration . Furtherpiration . In addipiration .L and Bax) play pivotal roles. The intrinsic apoptotic signalling pathway is mediated by insertion of pro-apoptotic proteins Bax/Bak into the outer mitochondrial membrane. Subsequently, cytochrome c is released from the mitochondrial intermembrane space into the cytosol [L are anti-apoptotic proteins which prevent the release of cytochrome c and protect cells from apoptosis [L inhibitors with anti-cancer activity in a broad range of cancer types [L inhibitor (a BH3 mimetic), has been approved for use in patients with lymphoma and chronic lymphocytic leukaemia [The intrinsic apoptotic signalling pathway refers primarily to mitochondria-mediated apoptotic pathways, in which Bcl-2 family proteins  complexes can intercalate into mtDNA and induce mtDNA damage, followed by a decline of mitochondrial membrane potential, suppression of ATP generation, and disruption of mitochondrial energetics and metabolic status, eventually causing cancer cell apoptosis . AdditioAs mentioned, mitochondria are plausible targets for anti-cancer strategies. Agents that target mitochondrial metabolism, the ETC, apoptotic pathways as well as other mitochondrial-linked signalling pathways, show efficient anti-cancer potential. Many anti-cancer drugs  are already known to act within the membrane and the matrix of mitochondria ,104. DelA number of direct conjugates have been reported for mitochondrial delivery of anti-cancer drugs using various targeting moieties, including lipophilic cations  and peptides (mitochondria-penetrating peptide (MPP), mitochondria-targeting sequence (MTS) peptide, and Szeto-Schiller (SS) peptides) . In thisThere are multiple mechanisms and techniques to deliver drugs into mitochondria using the well-known approach based on a higher mitochondrial membrane potential of cancer cells compared to that of their cytosol and non-cancer cells, which allows selective targeting of cancer cell mitochondria . TriphenA number of researchers have used TPP+ conjugation to deliver anti-cancer drugs to mitochondria. Bryant and colleagues reported that Hsp90-TPP showed a 17-fold increase in mitochondrial accumulation than Hsp90 itself, and that \u201cmitochondrial Hsp90\u201d efficiently killed both primary and cultured acute myeloid leukaemia cells . Han andRecently, photodynamic therapy (PDT) has been proven to be a minimally invasive and highly efficient therapeutic strategy of cancer treatment. TPP was used in the development of a group of photosensitizers to enhance their cancer cell uptake efficacy and mitochondrial localization. Noh and colleagues developed MitDt, a mitochondrial targeting photodynamic therapeutic agent, by conjugating the heptamethine mesoposition of a cyanide dye with TPP. The PDT effects of MitDt are amplified after laser irradiation because mitochondria are susceptible to ROS which triggers anti-cancer effects . The catNanocarriers have been considered to carry drugs and deliver them to the target areas of tissues to enhance drug efficiency and reduce toxicity. Nanocarriers include micelles, polymers, carbon-based materials, liposomes, metallic nanoparticles, and dendrimers that all have been developed for applications, particularly in the field of chemotherapeutic drug delivery ,128. TheDoxorubicin (Dox) is one of the first choices of chemotherapeutic drugs applied to the nanocarrier delivery system. Liu and colleagues prepared Dox-loaded TPP-lonidamine self-assembled nanoparticles (NPs), which contain polyethylene glycol groups to enhance their circulation in blood for more extended periods. The NPs showed greater cytotoxicity in both drug-sensitive and drug-resistant cancer cells compared to Dox . Using aZhang et al. used glycyrrhetinic acid-attached graphene oxide with Dox as a model drug for dual targeting to mitochondria and the cell membrane due to its ability to interact with the mitochondrial respiratory chain and high binding affinity to protein kinase C (PKC) \u03b1, which is overexpressed in certain cancer types ,136,137.Lee and colleagues reported the formation of aggregates off a TPP-tagged coumarin probe (TPP-C) in an aqueous solution. With the encapsulation of Dox into the TPP-C NPs, the anti-cancer drug was efficiently delivered to the mitochondria and exerted considerable cytotoxicity toward cancer cells . LonidamBased on mesoporous silica nanoparticles (MSNs), a novel enzyme-responsive, multistage-targeted anti-cancer drug delivery system which possessed both CD44-targeting and mitochondrial-targeting properties was developed by Naz and colleagues . First, Mitochondria, with their various functions, have become novel targets for anti-cancer strategies. Targeting mitochondrial metabolism, including the electron transport chain function, the redox signalling pathways and ROS homeostasis, as well as apoptotic signalling pathways, have become a major focus for researchers . Mitocho"}
+{"text": "Replicative cellular senescence is the main cause of aging. It is important to note that early senescence is linked to tissue regeneration, whereas late senescence is known to trigger a chronically inflammatory phenotype. Despite the presence of various genome-wide studies, there is a lack of information on distinguishing early and late senescent phenotypes at the transcriptome level. Particularly, the changes in the noncoding RNA portion of the aging cell have not been fully elucidated. By utilising RNA sequencing data of fibroblasts, hereby, we are not only reporting changes in gene expression profiles and relevant biological processes in the early and late senescent phenotypes but also presenting significant differences in the expressions of many unravelled long noncoding RNAs (lncRNAs) and transcripts arisen from repetitive DNA. Our results indicate that, in addition to previously reported L1 elements, various LTR and DNA transposons, as well as members of the classical satellites including HSAT5 and \u03b1-satellites (ALR/Alpha), are expressed at higher levels in late senescence. Moreover, we revealed finer links between the expression levels of repeats with the genes located near them and known to be involved in cell cycle and senescence. Noncoding elements reported here provide a new perspective to be explored in further experimental studies. Aging is a natural process that is associated with many health issues, including\u2014but not limited to\u2014cardiovascular problems, neurodegenerative disorders, diabetes, liver disease, and cancer . Also, the decline of proper immune function with age was reported against infections, including the infection caused by the novel coronavirus SARS-Cov-2 . Even though the decline of functional tissue with age could be associated with the exposure to many external drivers such as inflammatory or genotoxic factors, cell division itself emerges as the main natural cause of aging. Many rounds of DNA replication result in an inevitable erosion of the telomeric ends of the chromosomes, triggering a DNA damage response that involves the p53 pathway, forcing the cell to exit the cell cycle and become senescent .Cellular senescence is characterised by distinct changes in the cell\u2019s cytological and nuclear architecture, as well as the factors that it is releasing to its environment. Senescent cells are known to gain a senescence-associated-secretory phenotype (SASP), which involves various interleukins and interferons, giving rise to chronic inflammation in the aged tissues . This inflammatory phenotype was shown to be highly influential on the onset of age-related diseases such as cancer and was undoubtedly linked to senescence-specific arrangements at the chromatin level. Importantly, the content and distribution of heterochromatin become altered along the way towards senescence, and senescence-associated-heterochromatic foci (SAHF) appear . Notably, SASP and SAHF do not present themselves right after the cell exits its cycle, and senescence is known to be a gradual process .Several studies have attempted to identify senescence-specific genes to shed light on the universal programming events in the cell towards the onset of full senescence. Using several in vitro senescence cell models, such studies highlighted the downregulation of cell cycle genes, upregulation of inflammation-related genes, and the genes involved in the biogenesis of extracellular matrix, as well as secretory network . In line with the drastic changes in gene expression profiles, the chromatin landscape is also transformed in the senescent cell. The methylation levels of certain histone residues  and the dysregulation in the activities of histone deacetylases (i.e. sirtuins) were found to be particularly important in promoting the senescent chromatin, which is associated with SAHF and the disorganisation of topological chromatin regions, including lamin-associated domains (LADs) . In addition to protein-coding genes, long noncoding RNAs (lncRNAs) were also implicated in senescence . The functions of various lncRNAs, including HOTAIR, , PANDA , MALAT-1 , and GUARDIN , were found to be particularly influential in maintaining the balance between cell proliferation and senescence. In addition to these, various novel lncRNA genes were also identified in a fibroblast senescence model .Nevertheless, the noncoding portion of RNAs emerging during senescence are not only limited to lncRNAs. MicroRNAs  and repetitive DNA transcripts  were also reported. There are more than a thousand types of repeat motifs identified in the human genome, altogether comprising more than 50% of our DNA . These elements, which are formed by tandem or interspersed repeat motifs and were originated from replication errors or ancient virus infections throughout our evolution , are particularly important in maintaining a healthy chromatin architecture. Among such elements are the satellites, DNA and LTR transposons, and LINE and SINE elements. Their expressions are tightly regulated and have been implicated in human embryonic development  and were additionally linked to proteasomal activity . Moreover, their aberrant expression was linked to cancer . The specific chromatin marks associated with these elements are thought to be the key to their participation in genomic regulation . HSATII and \u03b1-satellites, whose heterochromatinisation is essential for functional centromeres and kinetochores , were shown to become structurally extended in senescent cells . HSATII, which is also overexpressed in epithelial origin cancers , was reported to be upregulated in senescent cells . Moreover, the activities of transposons were shown to be relevant in senescence. The L1 family of the long interspersed nuclear elements (LINEs) is activated both in mouse and human replicative senescence . In terms of long terminal repeats (LTRs), only MusD was reported in an aging mouse , and whether any members of the LTR family contribute to this phenomenon in humans has not yet been studied.The studies mentioned above provided invaluable information on the transcriptome and chromatin signatures of cellular senescence in various models; however, there is still a paucity of knowledge on the differences between early and late cellular senescence, particularly at the level of lncRNA and repetitive DNA transcripts. It is important to understand the dynamics of the cell during the transition from early to late (deep) senescence because the outcomes of the two are vitally different. Early senescence is associated with wound repair and tissue regeneration , whereas late senescence promotes a chronically inflammatory phenotype that is linked to fibrosis  and cancer . Moreover, there has been no holistic genome-wide study that reports the transcripts stemming from the complete repeatome during senescence despite the efforts to study invidiual repeats .In this study, by employing a previously published  dataset (GSE109700) containing RNA-sequencing (RNA-seq) data from biological triplicates of early proliferating, early replicative senescent, and late replicative senescent LF1 human embryonic lung fibroblast cells with at least 70 million reads, we have quantified transcripts from protein-coding genes, lncRNAs, and repeat elements. We have also distinguished the transcriptomic signatures of early and late senescent cellular phenotypes, providing the full noncoding portion of the senescent transcriptome. The dataset we used was generated with the aim of detecting L1 transcripts and is therefore suitable for the quantification of transcripts from protein-coding genes and lncRNA genes, as well as all types of repetitive DNA at the same time. We were also able to confirm our key findings on an independent RNA-seq data (GSE63577) obtained from MRC5 fibroblasts . Our results highlight distinct biological processes and stage-specific lncRNAs, as well as previously unspecified repeat transcripts in the comparison of early and late senescent phenotypes versus the young and proliferating phase.\u201cfastq-dump \u2013gzip \u2013skip-technical \u2013readids \u2013dumpbase \u2013clip \u2013split-3\u201d. Human reference genome GRCh38 and the associated gene annotation in GTF file format (Release 32) were downloaded from the GENCODE project web sitehttps://www.gencodegenes.org. Repeat annotations were collected from RepeatMaskerhttp://repeatmaster.org. RNA-seq datasets were aligned to the reference genome with the Rsubread v1.34.7 package  of R v3.5.1 statistical computing environmenthttps://www.r-project.org/ with the following settings:\u201calign\u201d. We utilised SAMtools v1.3.1  in order to sort and index all BAM files generated in the alignment step.Sequencing reads of nine samples, including biological triplicates of cells that are in the early proliferating state and those that are in early and late replicative senescence, respectively, were downloaded from the Sequence Read Archive database  (SRA Accessions: SRP131506 and SRP050179) with SRA Tool Kit v.2.9.0, using the following command:\u201cfeatureCounts \u201d. Again, we utilised the featureCounts function in the R environment to quantify repeat expressions. However, repeat element features overlapping exons of protein-coding and lncRNA genes were filtered out from the annotation file before the quantification steps in order to minimise the ambiguity caused by their repetitive nature. We considered only uniquely mapped reads aligned to DNA, LINE, SINE, LTR, and satellite repeat regions. Repeat element and gene counts were then merged into a single count matrix for further downstream analysis.For the measurement of expression levels of protein-coding and lncRNA genes, we employed the featureCounts function  of the Rsubread package with the following command:Counts per million (CPM) values were calculated for protein-coding genes, lncRNAs, and repeat element families and subfamilies across all samples. We filtered out the features where expression levels\u2009 were CPM <1 in each condition and only considered samples where at least one condition has CPM >1 threshold in at least two replicates. To determine differentially expressed genomic features between two conditions, we made use of the EdgeR package v3.24.3 . In this step, trimmed mean of M-values (TMM) normalisation was applied to the filtered count values, and the dispersions were estimated with estimateDisp function for each comparison. For the calculation of false discovery rate (FDR) of each feature, appropriate contrast statistics were employed using exactTest function of edgeR. Next, all features were sorted based on an absolute log2 fold change value between conditions, and top-50 genes, lncRNAs, and repeat elements were determined individually. Gene ontology (GO) analysis on the differentially expressed genes was performed using DAVID\u2019s functional annotation tool .\u201cstrand\u201dand\u201cdistance\u201dparameters of the TEffectR::get_overlaps function were set as\u201cstrandness\u201dand\u201c5000\u201d, respectivelyIn order to identify statistically significant associations between repeat element expression and the transcription of adjacent genes across samples, we made use of the TEffectR package, which was recently developed by us . This tool takes sorted and indexed genome-aligned BAM files as input and predicts potential associations by establishing multiple linear regression models based on read count values of genes and repeats. We utilised the TEffectR package with default settings for DNA, LINE, SINE, LTR, and satellite repeat elements; theR statistical computation environment was utilised for all statistical analyses. We employed pheatmap package of Rhttps://CRAN.R- project.org/package=pheatmap to draw all the heatmaps, on which expression values were represented in the rows. Clustering was performed with the Euclidian method, and the prcomp function was used for principal component analysis (PCA). Other graphics were obtained using the ggplot2 packagehttps://ggplot2.tidyverse.org/.We analysed RNA-seq data from three biological replicates of young proliferating, early senescent, and late senescent LF1 human lung fibroblast cells (Supplementary Tables S1 and S2). The characterisation of their proliferating and senescent phenotypes at distinct early and late stages was previously done . Our PCA at the global transcriptome level showed that each biological group  clustered separately and that the triplicates were close to each other, indicating the high reliability of the dataset . The separation of the clusters was prominent not only for protein-coding genes but also for lncRNA, as well as repetitive DNA transcripts, implicating the contribution of all of these elements to different phases of senescence. To confirm that our analysis is correct and in line with the previous publication , we also analysed the expression levels of cell cycle control genes p21 and p16  and genes that are known to be associated with SASP . The expression of the cell cycle inhibitors p21 and p16 increased in the senescent phenotypes, and the SASP genes were only expressed at a mild level in the early senescent phenotype and at a higher level in late senescence, with the exception of IL6, which seems to be expressed at a higher level in early senescence . This agreed well with the previous publication .We analysed differentially expressed protein-coding genes, lncRNAs, and repetitive DNA transcripts and visualised the changes in their transcription using volcano plots . We realised that most of the changes in the protein-coding genes occurred when cells entered early senescence. Over 1500 genes were upregulated in the early senescent phenotype when compared to proliferating cells, and over 3500 of them were downregulated (|log2(Fold Change)|>1 and FDR < 0.05) . There was an obvious reduction in the number of downregulated genes when cells in late senescence were compared to early senescent phenotype. Moreover, most of the changes on the lncRNA and repetitive DNA transcripts were on the downregulation side, comparing the proliferating phenotype to the early senescent one, whereas an upregulation was more prominent in the early to late senescence transition. Indeed, there seemed to be a global upregulation in repeat expression in the late senescent phenotype. Considering the importance of repetitive DNA in the formation of chromatin architecture and the fact that they are normally silenced, it is likely that the major chromatin changes in the late senescent phenotype harbour repeat elements.Biological pathways that take stage during the transitions towards early and late senescence are generally revealed. De Cecco et al. (2019) employed Gene Set Enrichment Analysis (GSEA)  and pointed out that cytokine and toll-like receptor signalling, retinol, and olfactory pathways were implicated at distinct senescence phases. They performed a paired comparison for each group separately. To better visualise the changes in gene signatures and employ a more holistic approach, we generated a scatter plot where the x-axis denoted differentially expressed genes (|log2(Fold Change)|>1 and FDR < 0.05) in early senescence when compared to proliferating cells, and the y-axis denoted differentially expressed genes in late senescence in comparison to early senescence . Next, we performed a gene ontology analysis using DAVID  (Supplementary Table S3). There were 438 genes upregulated during the transition from early to late senescence. These genes were intriguingly downregulated in the transition to early senescence from the proliferating state. Along these genes were those related to cell division and proliferation, along with some others involved in SMAD, BMP, and \u03b2-catenin signalling pathways. Moreover, 107 genes that are involved in inflammatory response, cell adhesion, apoptotic signalling, cellular response to mechanical stimuli, roundabout signalling, and NF-\u03baB pathways were gradually increased as the progression towards late senescence continued . On the other hand, 214 genes linked to extracellular matrix organisation, angiogenesis, wound healing, MAPK, and TGF-\u03b2 pathways were only upregulated in the transition from the proliferating state to early senescence. Finally, 23 genes associated with extracellular exosome were continuously downregulated in early and late senescence . When differentially expressed lncRNAs were analysed with similar filters, it was realised that some lncRNAs were only upregulated in the late phenotype and some others were only upregulated in the early phenotype . Interestingly, almost all differentially expressed repeat elements were upregulated only during late senescence , consistent with what is presented in Figure 2b.We calculated the top-variable 50 transcripts for protein-coding genes, lncRNAs, and repeat transcripts and visualised these with heatmap representation . CDH1 (E-cadherin), WNT9B , SLC24A3 , GPRIN3 , and MYOZ2  genes were expressed at their highest levels in early senescent cells. On the other hand, STMN2 , CADPS , and ADD2  genes were expressed highly in late senescent cells . Moreover, we identified 18 lncRNAs to be expressed particularly highly in proliferating cells, 19 lncRNAs in early senescent cells, and 10 others in late senescent cells . Most of these lncRNAs were novel and not previously addressed in the literature on senescence and aging. When we checked the repetitive DNA transcripts, MER75 (DNA transposon), LTR70 (LTR transposon), LTR10B1 (LTR transposon), Charlie10 (DNA transposon), and L1MEa (LINE transposon) were expressed at their highest in proliferating cells . LTR1C1 and LTR12B (LTR transposons) were expressed highly in early senescent cells and most of the other top-variable repeats were expressed at their highest level in late senescent cells. The latter included members of the previously presented L1 (LINE) family , \u03b1 and HSAT5 satellites, as well as various members of the DNA and LTR transposons family. Our results on repeat transcripts indicate that repeat expression in late senescence is a broader phenomenon than previously described by De Cecco et al. (2019), with many previously unmentioned elements being dysregulated.Repeat motifs on DNA are known to modulate the expressions of proximal genes . To see if repeats might have a potential link with the expression statuses of nearby genes in the dataset we utilised, we employed TEffectR, a bioinformatics tool that predicts the associations between the expressions of the uniquely positioned repeats that are present in the proximity of genes using a linear regression model . We checked the repeats uniquely positioned within the 5kb upstream region of genes. Out of the significant repeat/gene associations, at least a dozen comprised genes that are involved in cell cycle, senescence, or aging. For example, a uniquely positioned LINE element L2b could explain 77.21% (P = 0.001) of the expression of its proximal gene FOXM1, which is a master regulator of the cell cycle . Another LINE element L1PA13 could explain 66.66% (P = 0.004) of the expression of its proximal gene NFKBIZ, which is known to promote SASP . The full list of relevant associations is presented in Table. These significant associations, which are worth exploring experimentally, could potentially imply the contribution of repeats to the genomic regulation of cellular senescence.In order to verify our key findings, we employed another dataset (GSE63577), where fibroblasts were cultured until they reached the late stage of replicative senescence . The authors in this study performed RNA-seq on fibroblasts, which were at different levels of senescence as determined by a population-doubling (PD) graph. Among the fibroblasts that they used, the RNA collection points of MRC5 fibroblasts were the only ones that resembled proliferating, early, and late senescence phases observed in LF1 cells in the De Cecco et al. (2019) study, even though the biochemical characterisation of senescence was not done as extensively.We quantified the transcripts and provided the count numbers and differential expression results in the Supplementary Tables S4 and S5. The PCA plot shows that the variation between triplicates was higher than the GSE109700 dataset , and we detected that the majority of differentially expressed genes emerged in the early to late senescence transition (PD62 vs. PD72) in this dataset. Still, the majority of upregulation of lncRNAs and repeat transcripts took place in late senescence , agreeing well with LF1 cells. The heatmaps  showed that some key transcripts followed similar trends with what was observed in the LF1 cells.We were able to verify important transcripts and highlighted particular signature changes in the expressions of lncRNAs and repeat elements in the MRC5 fibroblast dataset . Results were in line with the LF1 dataset . PURPL expression is already known to increase as the cells proceed into senescence , but LNC00607, AL353138.1 were not linked to senescence before. On the other hand, we are reporting that AC011503.2, HELLPAR, MIR924HG and TMPO-AS1 were expressed at the highest level in the proliferating state. Moreover, key repeats such as Charlie10a, L1MEa and LTR10B1 were linked with the young and proliferating state whereas classical satellites \u03b1- (ALR/Alpha) and HSAT5 as well as transposons HERVFH19-int and L1P4d were significantly linked to late cellular senescence. GO analysis of MRC5 senescence model revealed that cell proliferation, extracellular matrix, inflammatory response, cytokine activity, growth factor activity, cell-cell signalling, angiogenesis, MAPK pathway and wound healing were pronounced in agreement with LF1 cells . However, the quarters (senescence stages) where these pathways appeared were not exactly same in comparison to LF1 cells.Finally, we compared the expression correlations in MRC5 cells (not shown in Table) with the correlations obtained from LF1 cells and realised that correlations between L2 and NPIPB3 ; GSDMB and MIRB ; FER1L14; and MER1B  were significant along with an insignificant trend seen between NFKBIZ and L1PA13 .The plastic nature of genomic expression and the progressing events of the regulatory mechanisms are essential to many contexts in biology. Replicative cellular senescence is perhaps one of the most fundamental phenomena occurring naturally at different paces in every living organism. The high number of diseases associated with age and the cellular senescence, including cancer and neurodegenerative disorders, and the fine-tuned balance in which early senescence is particularly adjusted for tissue regeneration and development, while late senescence is disease-inducing, bring about the need for revealing the finer details of this phenomenon. Our comparative and holistic analysis on proliferating cells and those that are in early and late replicative senescence confirms some of the previously published findings and sheds further light on the formerly unreported dimensions of genomic regulation towards senescence. Our results on the gene expression signatures of inflammatory response, cytokine activity, and cell adhesion agree well with De Cecco et al. (2019), but we also posit that SMAD, BMP, TGF-\u03b2, \u03b2-catenin, and NF-\u03baB signalling pathways are involved at distinct stages of senescence using the same dataset. The exosome activity pathway seems to be continuously downregulated as cells proceed into senescence. More interestingly, pathways related to tissue regeneration such as wound healing and angiogenesis were only enriched in cells of early senescence but not in late senescence. Agreeing well with this, genes involved in growth factor and cytokine/inflammation activity appeared in different categories, suggesting their stage-specific expression in the early and late stages of senescence.An interesting feature realised in this study was that most of the differential expressions of noncoding RNA transcripts were downregulated in early senescence and upregulated in late senescence . It is known that proper heterochromatin on repetitive DNA is maintained through human development, and this is established right after the first phase of preimplantation (u20fda). Early senescent cells comply well with this fact, perhaps sharing characteristics of the developmental period required during regeneration. Indeed, cell lineage differentiation needs a pause in the cell cycle , and our results indicate that when cells first enter replicative senescence, they resemble this period in terms of the expression regulation of the noncoding portion; most of the noncoding RNAs (particularly repeats) were still repressed, but cell cycle was halted. Still, some lncRNAs  and particular repeats  were linked to the proliferating state, and their functions are yet to be characterised for cell cycle regulation. On the other hand, given that unstable repeat transcripts were linked to genomic instability , the relatively high level of repeat transcripts at deep/late senescence stage could be a potential factor manifesting cancerous events in such cells . In addition to studies that linked HSATII and L1 (LINE transposon) expression to cellular senescence , our study states that \u03b1-satellites (ALR/Alpha) and HSAT5 are specifically expressed in late senescence. HSATII expression was below the 1 CPM threshold in our analysis but showed a slight upregulation that was not statistically significant in late senescence. According to the DFAM database , most of the HSAT5 sequences are located at pericentromeres, which are essential components of cellular heterochromatin . Moreover, we report that previously presented L1 transcripts are not the only transposon-originated RNAs abundant in the senescent transcriptome and that the expressions of many LTR elements and DNA transposons also become activated. As opposed to L1s in general, L1MEa expression was at its highest in the proliferating state, and this emphasises the fact that not all members of the L1 family could be linked to senescence, which is different than previous perceptions in the field. We found that L1P4d is particularly expressed in late senescence. In addition, previously unmentioned LTR retrotransposons such as HERVFH19-int and LTR25-int expressions were also associated with late senescence. Even though no specific contributions were reported for these particular LTR transposons in the literature before, LTR activity has generally been linked to age-related diseases such as autoimmune disorders, neurological conditions, and cancer .We verified the above-mentioned lncRNAs and repeat transcripts in two independent datasets (GSE109700 and GSE63577) with the caveat that the second dataset (GSE63577) displayed a higher variation between triplicates. Also, the RNA sequencing library preparation methods of the two datasets were different. De Cecco et al. (GSE109700) provided a more extensive characterisation of the cells in displaying their proliferating, early, and late senescent features biochemically. Still, the verification of key noncoding transcripts at distinct stages of senescence in both datasets emphasises their emergence in the senescence phenomenon.In conclusion, our report illuminates further dimensions of the transcriptomic changes linked to senescence and provides the distinguishing features of the early senescent cells in comparison to late senescence in terms of activated biological processes and noncoding transcripts. LncRNAs and repeat-arisen transcripts, most of which were reported in terms of their link to senescence for the first time here, could be investigated further to uncover the hidden complex mechanisms of senescence or serve as markers to predict cellular age. The effects of repetitive DNA expression could be rather global, but the significant links between the proximal genes we found also suggests that there are finer details at the individual gene level. Specific transcripts expressed at different stages of senescence and listed in this report could be useful for future studies investigating the age-related changes of the cellular microenvironment and genomic integrity affecting the behaviour of cancer with age. Also, future innovations in wound repair and tissue regeneration could benefit from the genes listed as specific for the early senescent phenotype.Supplementary figures and tables can be downloaded from http://dx.doi.org/10.17632/839nknw58h.1."}
+{"text": "Current treatment options for both unicompartmental knee arthroplasty (UKA) and total knee arthroplasty (TKA) are still controversial with no consistent results in which one is superior to others. This is the first study to examine and analyze the following related data available in patients receiving either UKA or TKA from the National Health Research Database (NHIRD) in Taiwan.  The database was searched from NHIRD, pooling one million random patients. Patients' age, gender, and comorbidities were analyzed in either UKA or TKA between January 2005 and December 2013, or up until death. For the patients that had received bilateral surgeries, further subgrouping was divided into TKA to TKA, UKA to UKA, TKA to UKA, and UKA to TKA to analyze the completion rate curve. Additional analysis of the order codes 64202B, 64053B, and 64198B was defined as failures, and the related failure rate curves were analyzed separately within ten years. Finally, infection-related codes were analyzed. n = 276 UKA; n = 5903 TKA) were selected. Age (p < 0.0001) and gender (p = 0.037) had significant differences, with more young population and males having UKA than TKA. Most comorbidities had no significant difference. For the bilateral surgery analysis, the UKA to UKA group had the fastest completion rate (p < 0.001) and UKA to TKA was the slowest. There were no significant differences in the failure rates of 64202B, 64053B, and 64198B.  6,179 patients ( Most UKA and TKA are appropriate solutions to treat patients with osteoarthritis or osteonecrosis. UKA to UKA is the quickest bilateral completion surgery, and UKA has a higher chance of undergoing revision surgery than TKA. Severe stages of osteoarthritis (OA) and osteonecrosis (ON) often lead to hip or knee arthroplasty . In TaiwIn Taiwan, previous studies were conducted using Taiwan's national database focused more on TKA, such as the cost comparison between staged versus simultaneous bilateral TKA  and a 15Datasets were retrieved from the National Health Insurance Research Database (NHIRD) in Taiwan, which represents most population, if not all, as it covers over 99% of the Taiwan population (approximately 23 million residents) . By provThis study is a population-based database cohort study, analyzing patients receiving UKA or TKA between January 2005 and December 2013, or up until death. Additional analysis is shown in All characteristics of the patients were available from the database. The index date was set prior to the day each patient received their first completion of UKA or TKA recorded by the administrative insurance database. The end of the follow-up was death or until December 31, 2013.p value < 0.05 was considered statistically significant. Descriptive statistics were performed for demographic characteristics . For assessing the risks of receiving another arthroplasty surgery in the future, a 95% confidence interval was used to express the correlation and statistically significant difference. In analyzing the failure rate from patients' first surgery to their follow-up surgery, the Kaplan-Meier estimator was used. The log-rank test was used to find the differences in failure rate and completion rate.This study utilizes SAS  for data integration and statistical analysis. A two-tailed test of p < 0.0001) and gender (p = 0.037) showed significant differences from the total number of patients that underwent TKA and UKA. In patients that were 60 years old or older, 73% of patients had UKA while almost 90% of patients had TKA. Both groups had more females than males; however, the ratio of males in UKA was higher than TKA. Lastly, in comorbidities and complications, the peripheral vascular disease had a significant difference (p = 0.045) while the others had no significant differences. Diabetes mellitus (type I and II) had the highest proportion out of the other categories where 14.8% of the UKA patients and 17.7% of the TKA patients were diabetic.Figures p = 0.0004) in p = 0.3382). In p = 0.9586). In p = 0.8969).Figures 3The current treatment options for knee arthroplasty are still controversial where both UKA and TKA are both utilized to treat OA and ON. No consistent results have been shown in previous literatures; however, the use of both UKA and TKA has been increasing worldwide . To our Our study in In this study, distribution in females was more than males in both groups but the ratio of males in UKA was more. This could possibly be explained by more male workers that were younger than in TKA. Lin and colleagues showed a similar trend from their previous study of a 15-year retrospective study in Taiwan . Their dp = 0.219) in our study between TKA and UKA, the total numbers for diabetic patients  were more than other comorbidities and complications. In the United States, a 15-year study claimed a total of 8.55% diabetic patients from a total of 750,000 joint replacement patients [For comorbidities and complications, hip and knee replacements were common among diabetic patients. Even if there were no significant differences (patients  that havpatients .n = 578) and TKA to TKA (n = 768) using their national registry database in Norway from 1994 to 2011. Overall, with a rate of rerevision from UKA to TKA of 12% and TKA to TKA of 13%, they were both comparable with a ten-year survival rate of 82% and 81%, respectively. In conclusion, they claimed that both UKA to TKA and TKA to TKA had similar outcomes in terms of survival, functional outcome, level of pain, satisfaction in patients, and changes in health-related quality of life. In another study using the Australian Orthopaedic Association National Joint Replacement Registry from 1999 to 2008 [Previous studies have shown several revision rate percentages; however, what was often seen was the UKA to TKA revisions that were done frequently \u201323. UKA  to 2008 , they clIn our study, the completion rate of UKA to UKA is faster than other completion groups. If UKA to UKA was mostly contralateral surgeries, this could be explained that UKA had a faster recovery rate, less tissue damage, lower pain, and faster recovery time than TKA . If UKA There are some possible scenarios with the recommended revision of UKA, such as an early case of aseptic loosening of a single component, liner change in case of mobile-bearing dislocation with a revision to a thicker polyethylene, isolated polyethylene wear when diagnosed early, revised before metal-on-metal wear or osteolysis. The recommended revision of TKA is commonly known for its deep infection. Therefore, subgrouping of revision of total knee replacement, arthrotomy for acute septic joint, and removal of the prosthesis were necessary to further analyze its failure rate within the eight years, analyzing the possible revision surgery for not only UKA but also TKA as well.UKA has a higher failure rate than TKA in this study, with a failure rate of TKA and UKA, 1.74% and 2.9%, respectively. This could be explained by the continuous cartilage degeneration from the remaining cartilage after UKA was completed. In patients that had done TKA, the whole cartilage had been replaced with artificial and no further cartilage wear would appear. Therefore, the implant survival rate of TKA was higher than UKA. Dyrhovden et al.  also conThis study had two main limitations, due to the funding that used one million random sampling database LHID 2005. With the small number of samples of the UKA patients in comparison to a few thousands of TKA patients, this may affect the statistical analysis power. In suggestion to future research with additional funding, more researchers should enter the Ministry of Health and Welfare database to find out the true sample size comparison between UKA and TKA. In addition, even though our study had further analyzed the revision surgery, the patient's following related surgery may be the ipsilateral or contralateral knee. However, due to limited data not available in the database, this cannot be further analyzed.To our knowledge, this is the first population-based database cohort study that reports the following related surgeries of UKA and TKA in Taiwan. UKA to UKA is the quickest bilateral completion surgery while UKA has a higher chance of undergoing revision surgery than TKA. While there is no consensus when it comes to comparing UKA and TKA, both have their own inherent complications and compensations with their ultimate goal of improved longevity and optimal function to treat patients with osteoarthritis or osteonecrosis."}
+{"text": "Minimally invasive medical procedures, such as endovascular catheterization, have considerably reduced procedure time and associated complications. However, many regions inside the body, such as in the brain vasculature, still remain inaccessible due to the lack of appropriate guidance technologies. Here, experimentally and through numerical simulations, we show that tethered ultra-flexible endovascular microscopic probes can be transported through tortuous vascular networks with minimal external intervention by harnessing hydrokinetic energy. Dynamic steering at bifurcations is performed by deformation of the probe head using magnetic actuation. We developed an endovascular microrobotic toolkit with a cross-sectional area that is orders of magnitude smaller than the smallest catheter currently available. Our technology has the potential to improve state-of-the-art practices as it enhances the reachability, reduces the risk of iatrogenic damage, significantly increases the speed of robot-assisted interventions, and enables the deployment of multiple leads simultaneously through a standard needle injection and saline perfusion. The navigation of catheters through blood vessels requires flexible guiding wires that are pushable and tractable at the same time. Pancaldi et al. rely on hydrodynamic forces and magnetic torque in order to access even rather small capillaries with an ultraflexible magnetomechanical probe. Recent work has also demonstrated long-term recording and stimulation of brain dynamics using endovascular stent electrode arrays7. However, the majority of the brain is still inaccessible because the existing tools are bulky, and navigating through the minuscule and tortuous cerebral vasculature without causing tissue damage is extremely challenging. While technological progress in microengineering has introduced a variety of microdevices that can perform optical, thermal, electrical and chemical interrogation and modulation of the nervous system15, conventional navigation techniques are incapable of transporting miniaturized tethered devices deep into the microvasculature primarily due to mechanical limitations. The invention of a methodology that provides rapid and safe passage for microdevices regardless of the complexity of the trajectory may become instrumental for translational medicine and neuroscience research.The cardiovascular system oxygenates the entire body through an exquisitely interconnected fluid network, potentially providing physicians and scientists minimally invasive access to any target tissue. Taking advantage of this opportunity, the diagnosis and treatment of prominent abnormalities and diseases in the brain such as tumours, aneurysms, stroke, and arteriovenous malformations have been routinely accomplished using catheterization techniques22. Seminal work has demonstrated the feasibility of using external magnetic fields to remotely control the pose of elastic magnetic rods33. However, navigating gently through increasingly intricate vascular networks, while avoiding lesions, requires the development of microscopic probes (\u00b5-probes) as small as blood cells with extraordinary flexibility. This is particularly important for neurological interventions where accessing distal and tortuous vessels prohibitively increase the operation time and risk of intraoperative tissue damage. The bending stiffness of a rectangular beam scales cubically with the thickness of the material and, thus, reducing the dimensions of even high-modulus slender structures dramatically increases their flexibility. Furthermore, friction forces progressively increase with the distance, making it harder to push structures forward inside small vessels. As a result, state-of-the-art navigation techniques are not suitable for the advancement and steering of miniaturized endovascular devices.Advancement of conventional catheters relies on manual adjustment of the curvature of their distal tip. To automate the navigation process, robotics research has introduced continuum devices with active control over body deformation through cable-driven mechanisms, concentric tube systems or devices that possess tuneable mechanical properties such as shape-memory and variable stiffnessB, as low as 5\u2009mT. The persistent presence of flow ensures safe advancement of the \u00b5-probe until the next bifurcation without external intervention and with minimal contact with the walls, promoting autonomous passage through unknown or highly structured channels. In addition, in order to significantly reduce the size of the electromechanical \u00b5-probes, we abandoned the conventional two-step navigation paradigm that is based on the use of a flexible guidewire for the advancement of the functional catheter. Instead, we designed and engineered endovascular microrobotic devices with cross-sectional area as small as 25\u2009\u00d7\u20094\u2009\u03bcm2 that travel effortlessly in the vessels as if they were untethered while taking full advantage of the electrical and fluidic tethers. The physical principles of the navigation strategy have been systematically explored inside microfluidic devices with relevance to clinical challenges and using an experimentally validated computational model, which together led to the development of a repertoire of design and wireless control strategies. Finally, we demonstrate the feasibility of operating (multiple) microengineered \u00b5-probes at the target locations for making physical measurements in custom-made biomimetic phantoms and ex vivo local injection of chemicals inside the vasculature of a rabbit ear.Here, we introduce a robotic navigation strategy that relies solely on the ability of the blood flow to transport devices in vessels with arbitrary tortuosity. The method is based on the exploitation of elastohydrodynamic coupling between flexible structures and the surrounding fluid. Harnessing viscous stresses and pressure for propulsion while controlling the heading with spatially homogenous magnetic fields nullifies the need for proximal pushing. This strategy allows autonomous flow-driven transportation of \u00b5-probes along three-dimensional (3D) trajectories close to the speed of flow Fig.\u00a0. The tor34, ultra-flexible and ultra-lightweight filaments cannot simply be pushed into a stream because axial compressive loads will no longer be effectively transmitted due to bending of the \u00b5-probe. For this purpose, we developed a hydromechanical insertion system that seamlessly couples with the vasculature and keeps the \u00b5-probe under tension during the entire operation. The proximal extremity of the \u00b5-probe was attached to a rigid rod that slid across a sealing gasket at the rear end of a custom-made syringe barrel, and the motion of the rod was controlled by a linear positioner. Gentle pulling of the rod ensured proper loading of the \u00b5-probe into the barrel. Saline solution was pumped inside the system through an intake, applying tensile stress to the relaxed filament. Percutaneous cannulation in conjunction with controlled perfusion was performed for the fast and versatile deployment of the \u00b5-probe  ribbons with 200-\u00b5m width as generic structures for the study of fluid-body interactions under the influence of flow. Coating the complete surface of the ribbon with a 100-nm-thick gold film exaggerates the stiffness of functional \u00b5-probes with patterned electrical circuits. As a result, we guarantee that the navigation results are representative for all electronic \u00b5-probes presented in this work. Cylindrical magnetic heads with varying sizes, diameter from 40 to 350\u2009\u00b5m, were fabricated from a hard-magnetic elastomer composite using a moulding process. The experiments were performed inside biomimetic vascular networks made from photopolymer or elastomer using 3D printing and sacrificial moulding, respectively. A Newtonian fluid matching the viscosity of blood was pumped into the channels. We regulated the average fluid velocity, \u22121 and \u22121, which corresponded to 0.15 We realized a tortuous fluidic scenario to replicate challenging vascular trajectories as shown in Fig.\u00a0entclass1pt{minima35.The sustained tension on the \u00b5-probe provided by the viscous stresses differentiate the proposed deployment system from conventional strategies. Standard push-based endovascular devices rely on the outward wall contact to be able to transmit the axial force along the structure in non-linear trajectories Fig.\u00a0, left. TE\u2009=\u20093\u2009GPa). To reproduce the advancement of the filament inside the channel, we iteratively added material at the tip and solved the equations for the full structure. This strategy resulted in a much shorter simulation time compared to translating the whole \u00b5-probe as it requires significantly fewer iterations for convergence. Furthermore, this formulation was less prone to mechanical instabilities. A penalty algorithm was implemented in order to account for contact between the walls of the channel and the \u00b5-probe. A force normal to the channel that depended quadratically on the penetration depth was applied on each node of the filament that violated the non-penetration condition. Snapshots of numerical simulations are given in Fig.\u00a0We developed a computational model to gain more insight on the navigation mechanism and for rapid testing of robotic control strategies  simulations visualized the 3D streamlines inside the channel and quantified the distribution of flow velocity Fig.\u00a0. The \u00b5-pytip, right before entering a bifurcation is a reliable indicator for the direction the \u00b5-probe is going to take  and used this prototype for the following characterization experiments, unless stated otherwise. Upon application of the magnetic field, the head rotated in order to align with the direction of the field, which changed the distribution of hydrodynamic forces and elastic stresses, leading to a new equilibrium configuration , which constrained the lift-induced upwards motion of the structure and served as a support point to further bend the distal end towards the direction of the magnetic field. At higher B, magnetic head rotated further, which resulted in the entry of the \u00b5-probe tip into the bottom half of the channel. We recapitulated the \u00b5-probe deformation under hydrodynamic forces and magnetic torque using numerical simulations , making it challenging to be implemented on miniaturized devices. Our flow sensors were operated in pulsed mode, by measuring the time-of-flight (TOF) between the injection of current to the heater and the detection of peak resistance at the sensor. This method requires a high sampling rate, which does not pose a trade-off with miniaturization.After establishing the navigation strategy, we explored whether we could dynamically record physiological parameters such as electric potential, temperature, or flow characteristics using \u00b5-probes. PI substrate was used to fabricate the main chassis while titanium and platinum strips were deposited to form electrodes and electrical circuits, respectively. As a proof of concept, we engineered a \u00b5-probe that uses convective heat transfer to measure the characteristics of local fluid flow. Upon injection of current into the heater circuit, the generated heat is transferred to the sensor circuit at a rate determined by the velocity profile of the fluid. The fabricated electronic \u00b5-probes consisted of a 0.1\u2009mm\u2009\u00d7\u20092\u2009mm heating element and a 200\u2009\u03bcm\u2009\u00d7\u2009500\u2009\u03bcm sensor component, positioned 50\u2009\u03bcm apart from each other Fig.\u00a0. Convent2 cross-sectional area at varying fluid velocities. The data showed an exponential decrease in the TOF with increasing \u03c4wall, which captures the effects of channel geometry on the thermal convection. The wall shear stress was computed from the fully developed flow profile, which was extracted using the Fourier sum approach43  \u00b5-catheters with an outer diameter as small as 120\u2009\u03bcm and several cm-long Fig.\u00a0. Upon pe44. On the other hand, interventional neuroradiologists usually perform endovascular operations using the visual feedback provided by a fluoroscope. To this end, we verified the visibility  of the magnetic head inside an ex vivo rabbit head and an anthropomorphic head phantom using two different fluoroscopes that are regularly used in the operating room  and Kapton (Young\u2019s modulus: 4\u2009GPa). Devices can be fabricated from medical-grade polymers that are used to manufacture commercially available catheters such as polyurethane and polyethylene. The design paradigm can also be adapted to hydrogels such as gelatin and N-isopropylacrylamide (NIPAAm), opening up the floor for biodegradable electronic devices55 and programmable soft micromachines56.The \u00b5-probes were fabricated using conventional clean-room technology, thus, they can be produced in large numbers and decorated with sophisticated electronic circuits57. Homogenous magnetic fields are generated within a volumetric space of 94\u2009\u00d7\u200953\u2009\u00d7\u200922\u2009mm3 with a maximum B of 50, 60, and 85\u2009mT, respectively. The current flowing through the coils is provided by three sets of power supplies  and servo controllers , which are modulated using an analog/digital I/O PCi Express board . The off-the-shelf motorized stepper-motor linear positioner controls the forward and backward motion of the connecting rod and, as a result, the position of the \u03bc-probes. Teleoperation of \u00b5-probes was performed using a 3D mouse (3DConnexion) and keyboard. The fluid flow was provided by a peristaltic pump . The oscillations in pressure were dampened using a Windkessel element. A solution of 42.5\u2009wt% of glycerol (Sigma-Aldrich) in deionized water was used as a blood analogue. Visualization of the \u03bc-probes inside the phantoms was performed using CMOS camera . We built an illumination system from smartphone backlights  were printed using a 3D stereolithography printer (Formlabs 2) from a transparent resin   CFD simulations of the flow inside the vessels were performed using an open-source software (OpenFOAM). The CAD design of the phantoms was used to extract the boundaries of the vessels. The cartesian2DMesh module of cfMesh was used to create the two-dimensional meshes of the channels with boundary layer elements. The initialisation of the flow was done using potentialFoam and the incompressible steady-state laminar Navier-Stokes equations were solved with simpleFoam. 3D CFD and FEM simulations were performed using COMSOL Multiphysics software.\u22122 and developed by AZ 726 MIF) has been used as mask for sputtering (Alliance Concept AC450) a 100-nm-thick layer of gold in stripes of 250-\u00b5m width and 9-cm length, followed by lift-off in acetone. The \u00b5-probe borders have been then laser cut (Optec MM200\u2010USP) and detachment from the wafer has been done manually. The fabrication of the flow sensors involves the sputtering of titanium and platinum layers serving for adhesion and electrical conduction, respectively. To add the two serpentines, designated as temperature sensor and heater, another 25-nm thick layer of platinum has been sputtered over the wafer, after surface activation by argon plasma (Alliance Concept AC450). The shapes of the traces and serpentines were defined using spin-coating and removal of positive photoresists . The electrical circuits were sandwiched between PI layers.Devices were fabricated using standard microfabrication techniques. The details are provided in Supplementary Note\u00a0\u22121. The magnetic head was attached to the distal end of the Kapton ribbon using epoxy and manual positioners (Thorlabs). The electrical measurements were performed using a sensitive dual-channel source measure unit (Keysight B2902A).The magnetic head for all \u00b5-probes was fabricated from a composite of PDMS and Neodymium Boron Iron (NdFeB) microparticles with an average diameter of 5\u2009\u03bcm , mixed at a mass fraction of 1:1. The structures were cured at 65\u2009\u00b0C in the oven in a custom-made mould. The size of the cylindrical magnetic heads varied from 40 to 350\u2009\u03bcm in diameter and from 100\u2009\u03bcm to 3\u2009mm in length. Magnetization was performed using an impulse magnetizer (Magnet-Physik) at a field strength of 3500\u2009kA\u2009m61. In brief, a 40-\u03bcm-diameter tungsten wire (Goodfellow) was immersed in PDMS (10:1 ratio) and heated with the electrical current at a density of 320\u2009A\u2009mm\u22122. Thermal dissipation provided by Joule\u2019s heating ensured local polymerization around the wire. The thickness of the PDMS \u00b5-catheter was determined by the time of polymerization  was added to the PBS solution to avoid adhesion of the PDMS tubes to the barrel of the insertion system. Mean perfusion flow was maintained at a rate of 90\u2009\u03bcL\u2009s\u22121. A total of nine ears were used throughout the experiments. The ears were obtained from a local farm .Navigation experiments were performed with ex vivo Rex rabbit  ears immediately following their delivery. The ears were perfused with phosphate-buffered saline (PBS) solution 1X (Sigma Aldrich) in the central artery shortly after excision. The \u03bc-probes were introduced into the central ear artery using the insertion device and a 21\u2009G hypodermic needle. A 3\u2009\u00d7\u20091062. In brief, Luciferin-luciferase ATP reaction buffer (ATP mix) was prepared using 200\u2009\u03bcg\u2009mL\u22121 of luciferin, 40\u2009\u03bcg\u2009mL\u22121 of luciferase, 2.4\u2009mg\u2009mL\u22121 of bovine serum albumin, and 2.4\u2009mg\u2009mL\u22121 MgSO4. 100\u2009\u00b5L ATP mix were added into the well containing the samples previously agitated for 15\u2009min, 1200 RPM at room temperature before measuring platelet aggregation using absorbance. Four independent experiments were performed for both tests using blood from four different donors (2.75\u2009\u00d7\u2009105\u2009\u00b5L\u22121 to 3.66\u2009\u00d7\u2009105\u2009\u00b5L\u22121 \u2013 PRP concentration among donors). Collagen-I (10\u2009\u00b5g\u2009mL\u22121) was used as positive control and PRP incubated with PBS was used as negative control. All protocols were approved by the Swiss ethics committee (project-ID 2017-00732).Fresh human blood samples were collected from the Health Point at EPFL. Platelet-rich plasma  was added to the wells containing the devices, and the plate was agitated for 15\u2009min, 1200 RPM at room temperature. Platelet aggregation was measured using a plate reader . For the platelet activation test, the ATP release from activated platelets was measured using luciferin-luciferase reaction, as previously describedX-ray images of the magnetic structures were taken using two different fluoroscopes, Canon Alphenix Sky+ with 200-\u00b5m resolution and Canon Alphenix Core+ with 76-\u00b5m resolution, inside an ex vivo rabbit head or an anthropomorphic head phantom.Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Reporting Summary"}
+{"text": "MYO3A, MYO15A and COL9A3, with a resolution rate of 50% (9/18 patients). The study identified significant genetic differentiation in novel population-specific gene variants at FOXD4L2, DHRS2L6, RPL3L and VTN between HI patients and controls. These gene variants are found in functional/co-expressed interactive networks with other known HI-associated genes and in the same pathways with VTN being a hub protein, that is, focal adhesion pathway and regulation of the actin cytoskeleton . The results suggest that these novel population-specific gene variants are possible modifiers of the HI phenotypes. We found a high proportion of ancestral allele versus derived at low HI patients-specific minor allele frequency in the range of 0.0\u20130.1. The results showed a relatively low pickup rate of PLP variants in known genes in this group of Cameroonian patients with NSHI. In addition, findings may signal an evolutionary enrichment of some variants of HI genes in patients, as the result of polygenic adaptation, and suggest the possibility of multigenic influence on the phenotype of congenital HI, which deserves further investigations. There is scarcity of known gene variants of hearing impairment (HI) in African populations. This knowledge deficit is ultimately affecting the development of genetic diagnoses. We used whole exome sequencing to investigate gene variants, pathways of interactive genes and the fractions of ancestral overderived alleles for 159 HI genes among 18 Cameroonian patients with non-syndromic HI (NSHI) and 129 ethnically matched controls. Pathogenic and likely pathogenic (PLP) variants were found in  More than 80% of people affected by HI live in low- and middle-income countries. The highest incidence rate is found in the sub-Saharan Africa (SSA): up to 6 per 1000 compared with about 1 per 1000 in Europeans or North Americans (.Hearing impairment (HI) is a common sensory impairment, affecting nearly 500 million people worldwide . The burmericans .HI may be due to genetic, environmental and/or unknown factors. About 50% of congenital HI cases in high-income countries are due to genetic causes . For exaGJB2 and GJB6 have been shown to account for most of genetic-associated HI among European, Asian and North American populations   .Advanced genomic technologies, particularly targeted enrichment genome analysis by next-generation sequencing (NGS), have now made it possible for efficient determination of other genetic contributors without necessarily understanding the pathways involved. However, NGS has demonstrated a consistently low pickup rate for isolated cases of HI in individuals of African ancestry ,25. Thisin silico PLP variants and then examine the fractions of ancestral to derived alleles for 159 known HI-associated genes  is associated with Usher syndrome type IIA and was defined as benign; HSD17B4 (MIM:601860) is associated with Perrault syndrome (MIM:233400) that has a clinical presentation not found in the patients; while MYO1A has been excluded as a HI-associated gene  was performed with data from Cameroonian controls and HI patients along with those of eight other African populations . CamerooP-value of <0\u00b705/159, which is 3\u00b71 \u00d7 10\u20134, and the permutation adjusted P-value was also obtained after 100\u2009000 permutations. We investigated frequencies difference in population-specific gene variants, using an approach that is similar to gene burden analysis .Following WES, aggregated SNP frequencies within 159 known HI genes were analyzed for unusual differences between the patients and controls using Fisher\u2019s combined probability . The significance level for difference between cases and controls had a analysis , a well-in silico candidate variants in RPL3L  and VTN , additionally indicated genetic differentiation between HI patients and controls  files  files .VTN, RPL3L, FOXD4L6 and DHRS2L6) with population-specific candidate variants were queried for their functional or co-expressed interactive subnetwork, and therefore, the associated pathways and molecular functions of the resulting subnetwork. The protein products of VTN and RPL3L are indicated to functionally interact in 42 other protein\u2013protein pairings , the networks interactions revealed major associated pathways, which notably included osteoclast differentiation \u00a0The four identified genes  and regulation of the actin cytoskeleton , as indicated in binding and catalytic activity, as indicated in Moreover, the interactions of the protein products of ed genes  were inved genes  and the ed genes . VTN is COL9A, substitution of the uncharged non-polar amino acid GLY136 with the uncharged polar amino acid SER136 renders the protein structure inflexible and may impact binding interactions, stability and conformation of the protein. For MYO3A, substitution of the positively charged and hydrophilic amino acid HIS142 with the uncharged polar and hydrophobic amino acid TRP142 could impact binding interactions and the stability of structure and conformation of the protein (Molecular dynamic (MD) simulations  showed t protein .GJB2 among patients, which translates into a high proportion of derived alleles within the gene   of genetic HI in 61 Egyptian families  . MYO3A wi family  and has i family . A recese family . On the n family  and amonn family . Similar Morocco  and Tuni Morocco , and varfamilies . At leasorldwide ,40.COL9A3 were found in two patients in this study  of congenital HI will likely apply to populations of African ancestries. A much lower resolution rate of about 25% in 116 HI genes was observed among African Americans . Using targeted panel sequencing of 116 HI genes, we found a higher pickup rate of 70% in a modest sample of 10 multiplex Cameroonian families in which HI was associated to c origin . These smericans . TherefoFOXD4L6, DHRS4L2, RPL3L and VTN) of which RPL3L and VTN indicate genetic differentiation between patients with HI and controls. Importantly, the protein products of VTN and RPL3L are shown to interact with other HI genes  bins]. To our knowledge, this analysis is the first to explore such an approach in HI research. This result suggests that rare variants may have evolved conservatively from the evolutionary lineage and supports the possibility of multigenic and polygenic adaptation influencing congenital HI. A point of interest is that the proportion of derived alleles in ulations ,21,47 anulations . This fissive HI . Future The present study has a few limitations: first, the modest sample size of the HI individuals. Indeed, with the extreme genetic heterogeneity, larger sample sizes are needed to capture the genetic profile that is representative of the population of Cameroon. Second, the study was performed on isolated HI cases and without the possibility of variant segregation studies within the family or in trio. Lastly, purposely designed future functional analysis should interrogate the effects of PLP on protein production and functions as well as explore the hypothesis of multigenic causes in congenital HI, using appropriate cellular or animal models. We used multiple tools instead of direct Sanger sequencing to validate variants, which is possibly another strength of this study. Indeed, studies have shown that a single round of Sanger sequencing is more likely to incorrectly refute a true-positive variant from NGS than to correctly identify a false-positive variant from NGS ,53. TherMYO3A, MYO15A and COL9A3. We additionally reported novel population-specific variants in four genes  of which RPL3L and VTN interacted with other HI genes, with VTN being a hub protein, suggesting a possible modifier action in the HI phenotypes. Lastly, we reported for the first time, differential frequencies of ancestral alleles versus derived alleles in known HI genes among patients versus controls. These findings may signal an evolutionary enrichment of some variants of HI genes in patients as the result of polygenic adaptation and suggest the possibility of multigenic influence on the phenotype of congenital HI, which deserves further investigations.This WES investigation identified PLP variants associated with HI in 9/18 patients in three known genes: The study was performed in accordance with the Declaration of Helsinki. It was granted ethics approval by the Cameroon National Ethics Committee  as well as the University of Cape Town Human Research Ethics Committee . Written informed consent was obtained from patients 18\u00a0years or older or from parents/guardians for minors, accompanied by verbal assent from the minor.GJB2 and GJB6 mutations as previously reported  clinics in Cameroon. The patients were examined by qualified medical geneticists and ENT specialists. Detailed family and medical histories were obtained from the patients and their parents. In this study, 18 Cameroonian patients presenting with NSHI of either putative genetic origin  or of unknown origin were selected from a cohort of 582 patients  on the DNA of 18 Cameroonian patients living with HI and 129 controls from the same ethnolinguistic background. DNA concentration was quantified using the QuantiFlour dsDNA System on a Quantas Fluorometer ; 50\u00a0ng of genomic DNA was used for library preparation following an Illumina Nextera Rapid Capture Exome kit  that uses Nextera transposomes. The libraries were then hybridized with a 37\u00a0M probe pool to enrich the sequences and were then sent for WES using the Illumina HiSeq 2500 (Illumina), 100\u00a0bp run format, with an average read depth of 30X.The WES data was subjected to quality control using FastQC  and SoleAfter high confidence variants were obtained with the VariantMetaCaller, the resulting Variant Call Format (VCF) file was split into two groups: 18 HI patients and 129 controls. ANNOtate VARiation (ANNOVAR)  was usedWe double-checked the presence and quality of variants discovered against all sample BAM files by using FastQC  to ensurA statistical test of difference was performed to detect the possibility of unusual genetic difference between the hearing-impaired patient and control groups. The aggregated SNP frequency for all SNPs in a given gene was computed. In this regard, SNPs were mapped to their associated genes using dbSNP database and differences in the aggregate SNP frequencies were determined using Fisher\u2019s combined probability .Population structure was analyzed based on PCA using smartpca ,87. The SNP ancestral alleles were downloaded from Ensembl and included 59 comparative alignments of 32 species ,89. The The relationship between the fraction of derived allele and minor allele frequency in each gene was investigated. To do so, the alternative alleles were categorized into 6 bins  with respect to the patient and control group frequencies, and the fractions of derived alleles in each bin were independently computed. The fraction of ancestral/derived alleles for all known and candidate HI genes was further computed. This was done by aggregating the fraction of ancestral/derived alleles at SNP-based level to gene, taking into consideration all SNPs located within the gene\u2019s downstream or upstream region .A comprehensive human protein\u2013protein Interaction (PPI) network ,92,93 waTo enable a community network analysis, the list of candidate gene variants was combined with those of the 159 known HI-associated genes  obtainedQ14050 and MYO3A has UniProt protein identifier Q8NEV4). The web server generated 1436 amino acids out of 1610 amino acids, with the missing structure of 174 amino acids on the tail end of the protein structure. The tertiary structures of the COL9A- and MYO3A-encoded proteins were generated using the I-tasser homology web server (MD simulations were conducted to assess the effect of novel variants on protein functions. Amino acid sequences were obtained from UniProt (COL9A has UniProt protein identifier b server . All MD b server  using Amb server ). The syb server  and a prb server . The shob server ,106. Theb server . Then, tSupplementary_Figure_1_ddaa225Click here for additional data file.Supplementary_Figure_2_ddaa225Click here for additional data file.Table_S1_ddaa225Click here for additional data file.Table_S2_ddaa225Click here for additional data file.Table_S3_ddaa225Click here for additional data file."}
+{"text": "Currently, tRNA-derived small RNAs (tsRNAs) are recognized as a novel and potential type of non-coding RNAs (ncRNAs), which participate in various cellular processes and play an essential role in cancer progression. However, tsRNAs involvement in colorectal cancer (CRC) progression remains unclear.Sequencing analyses were performed to explore the tsRNAs with differential expression in CRC. Gain- and loss-of functions of 5\u2019tiRNA-His-GTG were performed in CRC cells and xenograft tumor to discover its role in the progression of CRC. Hypoxia culture and hypoxia inducible factor 1 subunit alpha (HIF1\u03b1) inhibitors were performed to uncover the biogenesis of 5\u2019tiRNA-His-GTG. The regulation of 5\u2019tiRNA-His-GTG for large tumor suppressor kinase 2 (LATS2) were identified by luciferase reporter assay, western blot, and rescue experiments.Here, our study uncovered the profile of tsRNAs in human CRC tissues and confirmed a specific tRNA half, 5\u2019tiRNA-His-GTG, is upregulated in CRC tissues. Then, in vitro and in vivo experiments revealed the oncogenic role of 5\u2019tiRNA-His-GTG in CRC and found that targeting 5\u2019tiRNA-His-GTG can induce cell apoptosis. Mechanistically, the generation of 5\u2019tiRNA-His-GTG seems to be a responsive process of tumor hypoxic microenvironment, and it is regulated via the HIF1\u03b1/angiogenin (ANG) axis. Remarkably, LATS2 was found to be an important and major target of 5\u2019tiRNA-His-GTG, which renders 5\u2019tiRNA-His-GTG to \u201cturn off\u201d hippo signaling pathway and finally promotes the expression of pro-proliferation and anti-apoptosis related genes.In summary, the findings revealed a specific 5\u2019tiRNA-His-GTG-engaged pathway in CRC progression and provided clues to design a novel therapeutic target in CRC.The online version contains supplementary material available at 10.1186/s13046-021-01836-7. CurrFor decades, non-coding RNAs (ncRNAs) have been shown to play critical roles in both cellular function and human disease. The emerging roles of ncRNAs in cancer development have also been well discovered. Some ncRNAs are stable and detectable in various human bio-fluids, which can be regarded as potential non-invasive biomarkers in cancers . AdvanceTo date, an explosion of studies have focused on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), piwi-interacting RNA (piRNAs), and circular RNAs (circRNAs), however, researchers rarely concerned tRNA, which is an ancient and well-known adapter that carries amino acids to the ribosome. Intriguingly, recent studies have identified tRNA can be cleaved under stress conditions \u201310, and Both subtypes of tsRNA are closely related to the development of tumors , 20, 21.Despite the emerging interest in tsRNAs, the biogenesis and mechanism of tsRNAs remain largely unknown. In the present study, we identified that 5\u2019tiRNA-His-GTG plays an oncogenic role in CRC progression. Regulated by the hypoxia inducible factor 1 subunit alpha (HIF1\u03b1)/ANG axis, 5\u2019tiRNA-His-GTG interacts with large tumor suppressor kinase 2 (LATS2) to suppress hippo signaling. Strategies to disrupt the 5\u2019tiRNA-His-GTG-engaged pathway might be developed for treatment of colorectal cancer.We obtained 25 paired CRC tissues and adjacent normal tissues from Ren-ji Hospital, Shanghai Jiao-Tong University School of Medicine . Patients were diagnosed clinically and pathologically with colorectal cancer  was used to remove 3\u2032-aminoacyl, 3\u2032-cP, phosphorylate 5\u2032-OH, and demethylate m1A, m1G, and m3C to promote efficient cDNA reverse transcription. After sequentially ligating 3\u2032 and 5\u2032 small RNA adapters , cDNA was synthesized and amplified using Illumina\u2019s proprietary reverse transcription (RT) primers and amplification primers. Subsequently, PCR amplified fragments of ~\u2009135\u2013160\u2009bp were extracted and purified from the polyacrylamide gel and used for library construction. Finally, the completed libraries were quantified using Agilent 2100 Bioanalyzer . The libraries were denatured and diluted to a loading volume of 1.3\u2009mL and loaded at a concentration of 1.8 pM onto a reagent cartridge sequenced on an Illumina NextSeq 500 system using a NextSeq 500/550\u2009V2 kit , according to the manufacturer\u2019s instructions. Illumina NextSeq 500 raw sequencing read data that passed the Illumina chastity filter were used for subsequent analysis. Trimmed reads  were aligned to mature-tRNA and pre-tRNA reference sequences. Statistical analysis of the alignment results was applied to retain the valid sequences for subsequent tRF & tiRNA expression profiling and differential expression analysis. The RNA sequence data have been deposited in the NCBI Gene Expression Omnibus (GEO) database and are accessible through the GEO Series accession number GSE140327.2, 5% CO2, and 94% N2 in a hypoxia chamber .Human colorectal cancer cell lines HCT116, LoVo, RKO, SW1116, Caco2, SW480, and DLD1 were purchased from American Type Culture Collection (ATCC). These cell lines were tested for mycoplasma contamination before use to ensure that they were mycoplasma-free. The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 or Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) medium supplemented with 10% fetal bovine serum . To induce hypoxia, cells were incubated with 1% OTotal RNA was extracted from cells and tissues using the Trizol reagent . A Cytoplasmic and Nuclear RNA Purification Kit  was used to separate RNA from the cytoplasm and nucleus. RNA Pretreatment Kit  and rtStar\u2122 First-Strand cDNA Synthesis Kit  were used to build specific cDNA libraries for the quantification of tsRNA in tissues. mRNA and 5\u2019tiRNA-His-GTG were reverse transcribed to cDNA using a PrimeScript RT Reagent Kit  and a Bulge-Loop miRNA qRT-PCR Starter Kit  in cells, respectively. Mature tRNA-His-GTG was reverse transcribed to cDNA using rtStar\u2122 tRNA-optimized First-Strand cDNA Synthesis Kit . Then, qPCR was performed with SYBR Premix Ex Taq (Takara). The expression levels of tsRNAs and tRNA-His-GTG were normalized to that of U6, and the expression levels of mRNAs were normalized to that of \u03b2-actin. The primer used for detecting tRNA-His-GTG was purchased from Arraystar (#: AS-NR-001H-1-074), and the details of the other primers are shown in Table SANG, LATS2, AGO1, AGO2, AGO3, and AGO4 were purchased from Genepharma Technology . The transient transfection of RNA oligonucleotides was performed using the DharmaFECT 1 siRNA transfection reagent . All the plasmids were obtained from Generay Biotechnology  and were transfected into CRC cells using the FuGENE transfection reagent . The sequences of siRNAs and the details of the RNA oligonucleotides are listed in Table SCells were seeded into plates overnight before transfection. The synthetic single-strand mimic , 50\u2009nM), inhibitor , and small interfering RNAs  against human Cell proliferation was determined by using a Cell Counting Kit 8 (CCK8)  according to the manufacturer\u2019s instructions. Treated cells were seeded in 96-well plates at an initial density of 1500 cells (HCT116) or 2000 cells  per well. For the colony formation assay, transfected cells (750\u20131000 cells per well) were cultured in 6-well plates for 8\u201310\u2009days, and then fixed with 4% formaldehyde and stained using 0.1% crystal violet. The relative colony formation ability was determined using ImageJ . The cell apoptosis assay was performed by using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I . Terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL) staining of tissue sections, used a TUNEL staining Kit  according to the manufacturer\u2019s instructions.Western blotting analysis was performed using standard procedures. An anti-\u03b2-actin antibody  was used as a reference control. The following antibodies were obtained from the indicated sources: Anti-HIF-1\u03b1 (#: 36169), anti-cleaved-PARP (#: 5625), anti-cleaved-Caspase9 (#: 9505), anti-PARP (#: 9532), anti-Caspase9 (#: 9502), anti-LATS2 (#: 5888), anti-Phospho-YAP-S127 (#: 13008), and anti-YAP (#: 14074) were obtained from Cell Signaling Technology (USA). Anti-ANG (#: ab10600) was obtained from Abcam (UK). The HIF-1\u03b1 inhibitors LW6 (#: CSN20474) and 2-ME (#: CSN19253) were purchased from CSNpharm (USA).ANG promoter region, as well as the 3\u2032 untranslated region (UTR) region of LATS2 and 5\u2019tiRNA-His-GTG mimic. The sequence of the ANG promoter was cloned into vector pGL3-basic. The pGL3-ANG and Rluc plasmids were co-transfected into HCT116 cells, treated with a HIF-1\u03b1 inhibitor and DMSO, respectively, and cultured in a hypoxia chamber. The 3\u2019UTR sequence of LATS2 was cloned into vector pmirGLO-basic. The negative control (NC) mimic or 5\u2019tiRNA-His-GTG mimic were co-transfected with the wild-type or mutant vectors. After 48\u2009h, the cells were harvested, and a luciferase assay was performed according to the manufacturer\u2019s protocol . The relative value of luciferase was detected using a FLUOstar Omega  and normalized to the value of Renilla luciferase activity.Dual-luciferase reporter assays were used to evaluate the direct binding between HIF-1\u03b1 and the Cells were grown on a chamber slide, cultured in normoxia or hypoxia chambers separately for 48\u2009h, fixed with 4% paraformaldehyde, permeated with 0.3% Triton X-100, and blocked with 1% bovine serum albumin (BSA). The cells were then incubated with primary antibodies  and secondary antibodies  according to the manufacturer\u2019s protocol. Finally, the cell nuclei were counterstained using-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI). The images were collected using a fluorescent microscope .6 in 100\u2009\u03bcL phosphate-buffered saline (PBS)) were injected subcutaneously through the right axilla of 5-week-old male BALB/c nude mice (6 mice per group), which were purchased from VRL Animal Technology . Six days after tumor cell inoculation, mice were randomly divided into five groups, including PBS, NC antagomir (5\u2009nmol each), Scramble antagomir (5\u2009nmol each), 5\u2019tiRNA-His-GTG agomir (5\u2009nmol each), and 5\u2019tiRNA-His-GTG antagomir (5\u2009nmol each). The five groups of mice were treated with RNA-oligos by way of multiple-center intratumor injection four times every 3\u2009days. The tumor size was measured using calipers every 3\u2009days. After 3\u2009weeks, the mice were sacrificed, and xenografts were then collected for weight measurements and other experiments. The schematic diagram of the animal experiments is shown in Fig. SAll animal experiments were performed in strict accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of the Shanghai Research Center for Model Organisms Inc.; the IACUC permit number was 2019\u20130023. An antagomir is an RNA oligonucleotide with specific modifications suitable for in vivo experiments, and we confirmed that the 5\u2019tiRNA-His-GTG antagomir performs the same function as the 5\u2019tiRNA-His-GTG inhibitor in HCT116 cells  was separated by 15% urea acrylamide-polyacrylamide gels, then the separated gel was transferred onto the pre-wet Hybond-N+ nylon membrane (Millipore). The membrane was then UV-crosslinked at 1200\u2009mJ in Stratagene UV Stratalinker 1800  and hybridized with DIG-labeled DNA probes . The sequences of the northern probes are listed in Table Sp <\u20090.05 was considered statistically significant.All results were obtained from at least three independent experiments and are expressed as means \u00b1 SD. All analyses were performed using GraphPad Prism 7.0 . A two-tailed Student\u2019s t-test was used to compare normally distributed continuous variables between two groups. The relationship between 5\u2019tiRNA-His-GTG and tumor size was determined using Pearson\u2019s correlation coefficient. p-value <\u20090.05), with 39 upregulated tsRNAs and 18 downregulated tsRNAs  and tRFs , based on their enzyme cleavage sites Fig.\u00a0a. 5\u2019tiRNtsRNAs are heavily decorated by RNA modifications that interfere with cDNA synthesis, and many tsRNAs share the same base sequence; therefore, the current detection method for tsRNAs requires additional de-modification and 3\u2032/5\u2032-adaptor ligation Fig. d, left. 6 HCT116 cells were injected into the right axilla subcutaneous tissues of nude mice. 6\u2009days after inoculation, the tumors were treated with PBS, NC antagomir, Scramble antagomir, 5\u2019tiRNA-His-GTG agomir, or 5\u2019tiRNA-His-GTG antagomir via multiple-center intratumor injection five times every 3\u2009days. Tumor growth was measured every 3\u2009days and tumors were dissected at the end of the experiment. The size of the tumor in the 5\u2019tiRNA-His-GTG agomir group was larger than that in other control groups, and the results of tumor growth and tumor weights showed that the 5\u2019tiRNA-His-GTG agomir could increase the formation and growth of the transplanted tumors, while the 5\u2019tiRNA-His-GTG antagomir had the opposite effect  were determined using qRT-PCR, and the results showed that 5\u2019tiRNA-His-GTG is highly expressed in HCT116 and LoVo cells, and weakly in RKO cells Fig.\u00a0a. InhibiANG  and cannot fully bind to the LATS2 mRNA; in the AGO protein family, only AGO2 has strong enzymatic cleavage activity. Finally, rescue experiments showed that attenuation of LATS2 partly rescues 5\u2019tiRNA-His-GTG inhibition-mediated suppressive effects on CRC cells. This confirmed the role of LATS2 in 5\u2019tiRNA-His-GTG-mediated carcinogenesis.We further explored the mechanisms by which 5\u2019tiRNA-His-GTG contributes to colorectal carcinogenesis. Current research suggests that tsRNAs are involved in various cellular processes, including translational reprogramming , 36, apoOur study also has several limitations. First, although we found that four tiRNAs were upregulated in CRC tissues, only 5\u2019tiRNA-His-GTG was uncovered. The role of 3\u2019tiRNA-Ile-AAT, 3\u2019tiRNA-Lys-CTT, and 3\u2019tiRNA-Arg-TCT-2-1 need to be further studied. Second, ANG is well-known for promoting tumor angiogenesis under hypoxia , but it In conclusion, we identified 5\u2019tiRNA-His-GTG as an oncogenic tRNA half in CRC and further uncovered a novel pathway in which hypoxia-induced 5\u2019tiRNA-His-GTG suppresses LATS2, leading to CRC progression. Targeting 5\u2019tiRNA-His-GTG or its associated network could represent novel therapies for patients with CRC.Additional file 1: Table S1. Clinical information for the patients included in this study. Table S2. Primers used for real-time PCR assay. Table S3. Small interfering RNA (siRNA) sequences. Table S4. RNA oligonucleotide sequences. Table S5. Northern blot probe sequences. Table S6. Predicted target genes of 5\u2019tiRNA-His-GTG. Figure S1. Distribution of the 5\u2019tiRNA-His-GTG relative expression level in different groups. (a) The expression level of 5\u2019tiRNA-His-GTG showed no significant difference between early stage and advanced stage CRC. (b) There was no difference in the expression level of 5\u2019tiRNA-His-GTG above and below the age of 60 years old. (c) The expression level of 5\u2019tiRNA-His-GTG was not significantly different between males and females. ns: not significant. CRC, colorectal cancer. Figure S2. Sanger sequencing of the PCR products of qRT-PCR. (a) 3\u2019tiRNA-Lys-CTT. (b) 3\u2019tiRNA-Arg-TCT-2-1. (c) 3\u2019tiRNA-Ile-AAT. Figure S3. The 5\u2019tiRNA-His-GTG antagomir performs the same function as 5\u2019tiRNA-His-GTG inhibitor in HCT116 cells. Figure S4. Schematic diagram of animal experiments. Figure S5. qRT-PCR validation of different genes. Figure S6. 5\u2019tiRNA-His-GTG inhibition reduces anchorage-dependent growth and induces apoptosis in RKO cells. Figure S7. LW6 and 2-ME have no effect on HIF-1\u03b1/ANG/5\u2019tiRNA-His-GTG axis under normoxia in HCT116 cells. Figure S8. The expression level of LATS2, phospho-YAP (Ser127), and YAP in the xenograft tumors. Figure S9. The expression level of LATS2 upon different cell densities and various hypoxia time. Figure S10. The role of 5\u2019tiRNA-His-GTG inhibitor under hypoxic environment. Figure S11. The expression level of tRNA-His-GTG and 5\u2019tiRNA-His-GTG in various groups using qRT-PCR and northern blot."}
+{"text": "Background and Purpose: Prediction models for functional outcomes after ischemic stroke are useful for statistical analyses in clinical trials and guiding patient expectations. While there are models predicting dichotomous functional outcomes after ischemic stroke, there are no models that predict ordinal mRS outcomes. We aimed to create a model that predicts, at the time of hospital discharge, a patient's modified Rankin Scale (mRS) score on day 90 after ischemic stroke.Methods: We used data from three multi-center prospective studies: CRISP, DEFUSE 2, and DEFUSE 3 to derive and validate an ordinal logistic regression model that predicts the 90-day mRS score based on variables available during the stroke hospitalization. Forward selection was used to retain independent significant variables in the multivariable model.Results: The prediction model was derived using data on 297 stroke patients from the CRISP and DEFUSE 2 studies. National Institutes of Health Stroke Scale (NIHSS) at discharge and age were retained as significant (p < 0.001) independent predictors of the 90-day mRS score. When applied to the external validation set , the model accurately predicted the 90-day mRS score within one point for 78% of the patients in the validation cohort.Conclusions: A simple model using age and NIHSS score at time of discharge can predict 90-day mRS scores in patients with ischemic stroke. This model can be useful for prognostication in routine clinical care and to impute missing data in clinical trials. Prediction models of functional outcome after ischemic stroke can aid clinical decision making for providers, patients, and families by guiding rehabilitation goals, discharge planning, and patient expectations \u20133. They To address this need, we aimed to develop an ordinal logistic regression model that predicts the 90-day mRS score based on variables available at the time of hospital discharge.The data that support the findings of this study are available from the corresponding author upon reasonable request.This study used de-identified patient data from three prior studies: CRISP, DEFUSE 2, and DEFUSE 3 \u201310. CRISThe dependent variable was the mRS score obtained 90 days after the index event. For patients with missing 90-day mRS scores, 30-day mRS scores were carried forward. Patients missing both outcome measures were excluded from the study. Patients who died during the initial hospitalization were also excluded.Predictor variables included baseline characteristics , imaging measures , and clinical measures .R-splitters. After upsampling, variables that were associated with outcome at a p < 0.2 in univariate analysis were entered in a multivariable model and were retained if they reduced the AIC by seven or more points.The CRISP and DEFUSE 2 datasets were used as the derivation set. The ordinal outcome measure was the mRS score at 90 days. We used a proportional odds model, which estimates intercepts for each level, but assumes a common coefficient across ordered response categories. Validity of the proportional odds assumption was verified by trending univariate odds ratios for each cutoff and plotting partial residuals. The derivation dataset was \u201cupsampled\u201d to account for the relatively small number of participants with an mRS score of five at day 90. The upsampling method augments the minority class by sampling random observations from this class with replacement . We implemented upsampling using the upsample function in the package The model was internally and externally validated to assess model performance on unseen data, thereby mitigating possible overfitting of the model. The model was internally validated within the derivation set using five-fold cross-validation. Univariate screening and forward selection were repeated to derive a model for each fold. The model was externally validated using the DEFUSE 3 dataset.R2 (coefficient of determination), mean absolute error (MAE), and the percentage of predicted outcomes that fell within one point of the observed outcomes. As an additional measure of model performance, we ran the primary efficacy analysis of the DEFUSE 3 trial based on imputed 90-day mRS scores and compared it to the same analysis using observed 90-day mRS scores.Measures used to evaluate for model performance included t-test or Wilcoxon rank-sum test. We report two-sided results and used a p < 0.05 as a threshold for statistical significance.Proportions were compared using Fisher's exact test, and distributions of continuous and ordinal variables were compared using the All statistical analyses were performed using R software (version 3.6.2) and SAS software (version 9.4).p = 0.02), prevalence of hypertension , hemorrhagic transformation score (p = 0.01), NIHSS score at discharge , and infarct volume at early follow up  were different between the derivation and validation groups.There were 201 patients enrolled in the CRISP study, 130 in DEFUSE 2, and 182 in DEFUSE 3. In the derivation set (CRISP and DEFUSE 2), we excluded two patients with missing 30 and 90-day mRS outcome data, and 32 patients who died during their initial hospitalization. In the validation set (DEFUSE 3), we excluded 22 patients who died during their initial hospitalization. . The patp < 0.2) with the 90-day mRS score in univariate analyses, included age (p < 0.001), hypertension (p < 0.001), diabetes mellitus (p < 0.001), history of stroke (p = 0.12), hemorrhagic transformation (p < 0.001), NIHSS score at discharge (p < 0.001), infarct volume at early follow-up (p < 0.001). After forward selection, age and the NIHSS score at discharge remained as independent predictors of the 90-day mRS score in the multivariable ordinal regression model , and a mean of 80% of predictions were within one of the observed value. Additional results of the five-fold internal cross-validation are shown in R2 of 0.60, and a mean absolute error of 0.94 (95% CI 0.80\u20131.07). The model predicted 34% of the 90-day mRS scores correctly, 78% of the scores within one point of the observed value, 96% within two points of the observed value, and 99% within three points of the observed value. Additional performance metrics of the model in external validation are presented in p = 0.15, p = 0.75, With five-fold internal cross-validation, age and NIHSS were retained in each model and no other variable was retained in a majority of models. The mean The primary analysis of the DEFUSE 3 trial, a comparison of the distribution of 90-day mRS scores between patients in the endovascular treatment and control groups, was rerun with 90-day mRS scores predicted by the final model. The odds ratio for benefit from endovascular therapy using predicted mRS scores was 5.01 (95% CI 2.77\u20139.05), which is similar to the results of the same analysis performed using observed mRS scores .A prediction model based on two easily obtainable clinical variables, age and NIHSS score assessed at the time of hospital discharge, had moderately high accuracy for predicting a patient's 90-day modified Rankin Scale score. It accurately predicted the 90-day mRS within one point in ~ 80% of patients. The model's independent variables \u2014 age and NIHSS score \u2014 are corroborated by prior studies that have demonstrated that these factors are correlated with functional outcome after ischemic stroke \u201314. ThisSeveral prior studies have focused on predicting survival or other dichotomized functional outcomes after ischemic stroke , 6. A reOne of the relative advantages of an ordinal regression is that there is less information loss as compared to dichotomous outcome models. Multiple studies have shown that ordinal analyses increase statistical power and efficiency, and suggest that further clinical research could benefit from increased utilization of ordinal analyses where relevant \u201317. BecaSome researchers have cited concern about the use of ordinal regression models and the need to test for proportionality of the odds. To address this, we confirmed proportionality by visual inspection of the univariate odds ratios and partial residuals. In addition, we empirically demonstrated the model's performance by validating its accuracy in an external dataset.There are limitations to our study. First, our dataset is limited to patients from three endovascular stroke therapy studies and is therefore not representative of all patients with ischemic strokes. Specifically, the model may not apply to patients with mild strokes or strokes in the posterior circulation who were not eligible for the trials. The model may also not apply well to patients with pre-existing severe disability who were excluded from the studies. The model did perform similarly in patients who underwent endovascular therapy and those who did not, likely because the effect of endovascular therapy is captured in the NIHSS score at discharge, which is one of the prediction variables in the model , 21 FutuIn summary, our internally and externally validated model predicts the ordinal mRS score at 90 days after ischemic stroke with moderately good accuracy and could be used for prognostication in clinical practice and to impute missing data in clinical trials.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 the Stanford IRB (DEFUSE 2 and CRISP) and the University of Cincinnati IRB serving as the Central IRB for StrokeNet (DEFUSE 3). The patients/participants provided their written informed consent to participate in this study.ML and MM organized the database. MZ wrote the first draft of the manuscript. All authors contributed to conception and design of the study, statistical analysis, manuscript revision, read, and approved the submitted version.The study was funded by grants from the National Institute for Neurological Disorders and Stroke  and the Stanford Medical Scholars Research Program.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": "Achromobacter spp. are emerging pathogens in patients with cystic fibrosis (CF) and Achromobacter spp. caused infections are associated with more severe disease outcomes and high intrinsic antibiotic resistance. While conventional CF pathogens are studied extensively, little is known about the genetic determinants leading to antibiotic resistance and the genetic adaptation in Achromobacter spp. infections. Here, we analysed 101 Achromobacter spp. genomes from 51 patients with CF isolated during the course of up to 20 years of infection to identify within-host adaptation, mutational signatures and genetic variation associated with increased antibiotic resistance. We found that the same regulatory and inorganic ion transport genes were frequently mutated in persisting clone types within and between Achromobacter species, indicating convergent genetic adaptation. Genome-wide association study of six antibiotic resistance phenotypes revealed the enrichment of associated genes involved in inorganic ion transport, transcription gene enrichment in \u03b2-lactams, and energy production and translation gene enrichment in the trimethoprim/sulfonamide group. Overall, we provide insights into the pathogenomics of Achromobacter spp. infections in patients with CF airways. Since emerging pathogens are increasingly recognized as an important healthcare issue, our findings on evolution of antibiotic resistance and genetic adaptation can facilitate better understanding of disease progression and how mutational changes have implications for patients with CF. Achromobacter spp. whole-genome sequencing data is available at European Nucleotide Archive under study accession number PRJEB39108.Achromobacter species are increasingly detected in patients with cystic fibrosis (CF) in which they can cause chronic airway infections. However, the knowledge about how Achromobacter spp. genetically adapt to the human airway environment is lacking. To address these questions, we analysed 101 genomes of Achromobacter spp. from 51 Danish patients to investigate within-host genetic changes of Achromobacter spp. over up to 20\u2009years of airway infections. We identified convergent evolution patterns of regulatory and inorganic ion transport genes. We additionally found that genes involved in inorganic ion transport, transcription, and energy production and translation were associated with antibiotic resistance phenotypes. Altogether, our analysis revealed the principal genomic adaptation and antibiotic resistance development patterns of Achromobacter spp. during the infection in patients with CF airway. The gained knowledge from our work helps to better understand how changes in the Achromobacter spp. genomes impact the disease progression in patients with CF and facilitates the identification of the improved treatment strategies of CF airway infections.Achromobacter spp. are emerging pathogens causing chronic bacterial infections in patients with CF  with BacDist [A. ruhlandii, 87\u200a% (75.45\u201392.57\u200a%) for A. insuavis and 86\u200a% (75.95\u201393.61\u200a%) for A. xylosoxidans. Low-quality variants (<tenfold coverage or supported by <50\u200a%\u2009of mapped reads) or variants shared among all isolates were discarded by BacDist , A. insuavis (N=18), A. xylosoxidans (N=52), A. aegrifaciens (N=1) and a new genogroup (N=1). The latter two species were excluded from further analysis as the species contained only a single isolate. The remaining 99 Achromobacter spp. genomes were grouped to 61 lineages, which were defined as all isolates from one patient belonging to the same species and clone type .The genomes of 101 nfection . All isoAchromobacter spp. genomes available in the RefSeq database  together with our clinical Achromobacter spp. genomes. Our sequenced genomes were widely distributed across the genetic variability observed within the A. insuavis and A. xylosoxidans group; however, A. ruhlandii isolates, of which the majority (27/29) belonged to Danish epidemic strain (DES), reflected little genetic variability of A. ruhlandii species. Furthermore, our phylogenetic and average nucleotide identity (ANI) analysis revealed that Achromobacter spp. annotations are inconsistent among the RefSeq genomes and require corrections to improve species designation . This approach allowed us to account for the nature of the dataset where multiple clonal isolates from the same patient were available. The aggregated Achromobacter species-specific genes  present in all isolates of the respective species but not in the isolates from other species. Pan-genomes for each Achromobacter species were defined by using all bacterial isolates available [The aggregated pan-genome  containevailable  for the Achromobacter sp. isolates from different timepoints were available . The estimated substitution rates for A. ruhlandii (DES isolates which are known hypermutators [\u22126 (2.71\u00b710\u22126\u20135.39\u00b710\u22126) SNPs/year/site, 8.77\u00b710\u22127 (6.17\u00b710\u22127\u20131.13\u00b710\u22126) SNPs/year/site for A. xylosoxidans (0332-AX03DK11 hypermutator lineage [\u22126 SNPs/year/site was excluded), and 1.61\u00b710\u22127 SNPs/year/site for A. insuavis. These substitution rates correspond to an average of 21.5, 2.19 and 0.79 SNPs/year/genome for A. ruhlandii, A. xylosoxidans and A. insuavis, respectively .Within-patient bacterial substitution rate was estimated for lineages where three or more mutators ) were on lineage  with thede novo assembled genomes of the 99 Achromobacter spp. isolates we identified virulence (VFDB database) and antibiotic resistance (Resfinder database) gene orthologues. On average, bacterial isolates carried 2 (0\u20133) antibiotic resistance gene orthologues and 13 [catB10, which codes for chloramphenicol acetyltransferase. Interestingly, the gene was carried by all A. ruhlandii and nearly all (30/33) A. xylosoxidans lineages but none of the A. insuavis lineages  A. xylosoxidans lineages. All 13 A. insuavis isolates carried one of the following blaOXA genes: blaOXA-243, blaOXA-455, blaOXA-457, blaOXA-458 or blaOXA-459. All blaOXA genes carried by A. insuavis had \u226592\u200a% nucleotide identity. The latest isolate of DES (P7703-AX01DK01) appears to have acquired a new aph [From s and 13  virulenclineages . Further new aph -Id antib new aph .A. ruhlandii DES genomes was markedly lower (N=8) than other Achromobacter spp. isolates  (N=9), motility (N=7), and endotoxin (N=5) gene orthologs [The median number of virulence genes in nk test) . The majrthologs .Achromobacter spp., we first identified the genes, which were most frequently mutated within each species. Genes were defined as frequently mutated if they were among the 1\u200a% most commonly mutated genes within species. If more than 1\u200a% of the genes were mutated with the same frequency, those genes were also included in the analysis. A total of 27, 16 and 28 genes were identified as most frequently mutated for A. ruhlandii, A. insuavis and A. xylosoxidans, respectively .Although knowledge is lacking about many P. aeruginosa genes revealed three orthologues among the most frequently mutated Achromobacter genes: mexZ (WP_006389199.1), mexB (WP_024068614.1) and gyrA (WP_049072335.1).Orthologue search of a previously defined list of 52 CF-associated pathoadaptive Achromobacter species. One gene was frequently mutated in all three species (10 out of 26 lineages) and six genes were observed as most frequently mutated in two of the Achromobacter species . Furthermore, lineages only acquired non-synonymous mutations in the seven candidate pathoadaptive genes (Table S4).Seven genes were defined as candidate pathoadaptive genes in several  species , Table 2 species . LineageaxyZ \u2013 encodes TetR family transcriptional regulator of the RND-type efflux system and is associated with innate Achromobacter spp. antibiotic resistance [axyZ gene with highest antibiotic resistance increase against piperacillin/tazobactam  and meropenem .The only pathoadaptive gene, which was frequently mutated among all three species \u2013 sistance . FurtherN/dS) was significantly different between the 1\u200a% most frequently mutated genes and non-frequently mutated genes in Achromobacter spp. .The ratio of non-synonymous to synonymous substitutions .Finally, we investigated gene loss and acquisition patterns in 26 longitudinally collected Achromobacter species. Accordingly, we performed the association analysis for these ten antibiotics (see Methods for detailed information). Unitigs passed a 5\u200a% FDR corrected q-value threshold only for CHL, IPM, MEM, TZP, SMZ and SXT. Ten of the most significant unitigs were used for the six remainder association test analysis, resulting in 60 genes (50 unique genes) significantly associated with antibiotic resistance phenotypes  genes. The other four most abundantly associated genes belonged to transcription ; energy production and conversion ; translation, ribosomal structure and biogenesis , and defence mechanism  groups. Furthermore, transcription genes were enriched in the \u03b2-lactam  antibiotic group  while translation, ribosomal structure and biogenesis genes were 5.4\u00d7 enriched in trimethoprim/sulfonamides (SMZ and SXT) group (N=4). Defence mechanism genes were 9.2 and 9.0\u00d7 enriched in \u03b2-lactam and trimethoprim/sulfonamide groups, respectively while energy production and conversion genes were 2.9\u00d7 enriched in a trimethoprim/sulfonamide group.To test for associations between bacterial genetics and antibiotic resistance phenotypes we performed unitig-based DBGWAS analysis. Out of 21 antibiotics where the resistance was phenotypically tested, only ten antibiotics had both susceptible and resistant isolates from all three Of nine antibiotic resistance genes from the ResFinder four database which were present in the aggregated pan-genome, none were associated with antibiotic resistance phenotypes in the GWAS analysis. Furthermore, core and accessory genes were equally associated with antibiotic resistance phenotypes (Table S6).Achromobacter species are emerging pathogens causing chronic respiratory tract airway infections in patients with CF; however, the genetic epidemiology of these infections is little understood. We sequenced and analysed 101 genomes of Achromobacter spp. isolates from 51 patients with CF, which is the largest longitudinally collected Achromobacter spp. genome dataset available to date. This allowed us to investigate the population genomics and within-host adaptation, including genome-wide association analysis with antibiotic resistance phenotypes.Achromobacter spp. genomes from the RefSeq database revealed that our dataset well represented the genetic diversity of A. xylosoxidans and A. insuavis species. However, because of DES overrepresentation, A. ruhlandii isolates did not reflect the species genetic diversity. Furthermore, ANI together with core-genome-based phylogenetic analysis revealed that more than 10\u200a% of publicly available Achromobacter spp. genomes are supposedly misannotated in the RefSeq database, which we anticipated to unravel to ease future research on Achromobacter spp. (Table S2).Phylogenetic analysis of our dataset with 141 publicly available A. ruhlandii, A. insuavis and A. xylosoxidans which are similar to core genome size identified previously by Li et al. [A. ruhlandii isolates mostly belonged to DES, which is spread through patient-to-patient transmission [A. xylosoxidans or A. insuavis.From the aggregated pan-genome analysis we showed that core genome sizes were comparable between i et al. . Neverthsmission , therefoAchromobacter spp. are not known [A. ruhlandii dataset only consisted of hypermutator DES lineages, which led to a high substitution rate estimate, and our substitution rate estimate for A. insuavis was based on only one longitudinally sampled lineage. A. xylosoxidans (8.77\u00b710\u22127 SNPs/year per site) substitution rate is comparable to other Gram-negative bacterial species: P. aeruginosa (4.0\u00b710\u22127 SNPs/year per site) [Shigella sonnei (6.0\u00b710\u22127 SNPs/year per site) [Echerichia coli (2.26\u00b710\u22127 SNPs/year per site) [S. aureus (1.87\u00b710\u22126 SNPs/year per site) [Klebsiella pneumoniae (1.9\u00b710\u22126 SNPs/year per site) [A. xylosoxidans evolutionary rates are similar to other Gram-negative bacteria and the same models predicting evolutionary dynamics could be used [Unlike many bacterial species, substitution rates for ot known . Our A. er site) , Shigeller site) , Echericer site) . The suber site)  or Klebser site) . These f be used . FurtherblaOXA genes for Achromobacter species typing [blaOXA genes. Furthermore, we identified that A. insuavis can carry one out of several highly similar blaOXA genes, which might further complicate such species typing approach. We also show that none of the A. insuavis isolates carried catB10 chloramphenicol resistance gene orthologue; however, these identified resistance genes alone were not sufficient to explain the differences in antibiotic resistance phenotype between lineages and species.While it was previously suggested to use s typing , we showAchromobacter spp. carry several virulence factors with markedly less virulence genes in A. ruhlandii DES strain, which further supports the adaptive trade-off evolution hypothesis that virulence genes are not required or are selected against in chronic infections [cheW and cheY) and facilitating evasion of the host immune response  were obi et al. .A. ruhlandii isolates than A. insuavis or A. xylosoxidans isolates. This phenomenon might signal about the continuous adaptive evolution even in highly antibiotic-resistant strains such as DES [Achromobacter spp. reference genomes improved. Overall, our identified candidate pathoadaptive genes  are comparable to the observations in P. aeruginosa infecting patients with CF [Achromobacter spp. [Among the candidate pathoadaptive genes (frequently mutated genes), we identified multiple antibiotic resistance genes, which were markedly more frequently mutated among h as DES . Nonethe with CF  and otheter spp. Achromobacter spp. lineages seem to undergo the same amount of selective pressure, and we show from the seven pathoadaptive Achromobacter spp. gene analysis that lineages tended to either have acquired not more than two pathoadaptive mutations and be under stronger positive selection or have acquired mutations in more pathoadaptive genes and be under weaker/neutral selection. These findings are comparable to findings from genetic adaptation studies in P. aeruginosa from patients with CF airway [N/dS ratio in the 27, 16 and 28 most frequently mutated A. ruhlandii, A. insuavis and A. xylosoxidans genes confirms that there is strong selective pressure for changes in these genes during adaptation to the patients with CF airway. Altogether, dN/dS>1 in both frequently and non-frequently mutated genes show that more than the top 1\u200a% mutated genes are under selective pressure; nonetheless, a larger dataset is needed to identify more genes without sacrificing analysis accuracy.Furthermore, we highlight that not all F airway . OverallAxyZ (mexZ orthologue), which was the only candidate pathoadaptive gene in all three species, is involved in the development of multidrug resistance by regulating AxyXY-OprZ RND-type efflux system, hence is crucial during adaptation to the host environment [AxyZ orthologue mexZ in P. aeruginosa is established among pathoadaptive genes directly associated with increase in antibiotic resistance [axyZ could partially explain the increased tolerance to piperacillin/tazobactam and meropenem.ironment . AxyZ orsistance . FurtherP. aeruginosa; however, unlike in P. aeruginosa, no convergent loss or acquisition of gene clusters was observed [The observed gene loss and acquisition patterns were comparable to the ones observed in observed Achromobacter spp. isolates, we performed a k-mer based GWAS analysis. Limited number of bacterial isolates and high innate resistance to certain antibiotics restricted our analysis to only six successful GWAS associations, which revealed that inorganic ion transport genes contribute to antibiotic resistance development in all six antibiotics. Changes in inorganic ion transport genes (COG P) could have a secondary influence on antibiotic resistance as such changes help overcome the problem of iron deficiency in human airways allowing better intrinsically resistant bacteria survival despite the presence of antibiotics. Similar patterns were previously identified in other bacteria causing chronic infections in patients with CF [et al. [Achromobacter spp. develops resistance in vivo from our and other studies could be applied to improve the resistance phenotype prediction in the clinic.To further explore the differences in antibiotic susceptibility between  with CF . Another with CF . The obs with CF . Enrichm with CF . Similar [et al.  where itAchromobacter spp. during CF airway infections. Second, the lack of genome annotation and overall knowledge about Achromobacter spp. limited the interpretation of putative pathoadaptive genes and genes associated with resistance phenotypes. Finally, a single isolate at a given time point is not sufficient to completely reflect the genetic diversity of the bacterial population [Achromobacter spp.Our study has several limitations. First, even larger studies are necessary to further characterize and identify the genetic adaptation of pulation ; therefoAchromobacter spp. clinical isolates from patients with CF, we used a comprehensive analytical framework for thorough bacterial genomic data analysis. Thus, we identified pathoadaptive and antibiotic resistance genes in Achromobacter spp. causing CF airway infections. Furthermore, we showed that current knowledge about antibiotic resistance gene presence or mutations in those genes cannot sufficiently explain the resistance phenotypes, and GWAS offers a new approach of addressing this problem. The gained knowledge allows us to better understand the requirements for successful Achromobacter spp. adaptation during infection in airways of patients with CF, which could help accurately predict antibiotic susceptibility and clinical progression of Achromobacter spp. infections, and further the development of urgently needed optimized treatment strategies.In conclusion, by using the largest dataset to date of Click here for additional data file."}
+{"text": "Introduction: Schizophrenia is a mental disease with a profound impact on human health. Patients with schizophrenia have poor oral hygiene, increasing their risk of systemic diseases, such as respiratory infections, and declining their quality of life. Therefore, this study aims to assess the oral health status of inpatients with schizophrenia, analyze its related factors, and thus provide scientific evidence for further exploration of corresponding control strategies.Methods: A total of 425 inpatients older than 50 years with a diagnosis of schizophrenia from two psychiatric hospitals (mean age 58.49 \u00b1 5.72 years) were enrolled. The demographic data of the patients were checked on admission. Two independent dentists examined caries, missing teeth, and fillings. Mini-Mental State Examination (MMSE) and Global Deterioration Scale were performed as cognitive tests. Positive and Negative Syndrome Scale and Repeatable Battery for the Assessment of Neuropsychological Status rating scale were used to determine their mental status.Results: The average decayed, missing, and filled teeth index was 12.99 \u00b1 8.86. Linear regression analysis showed that the decayed, missing, and filled teeth index had a significantly positive relationship with age (p < 0.001) and smoking (p < 0.001) and a negative relationship with MMSE (p = 0.029). The missing teeth index had a positive relationship with age (p < 0.001), smoking (p < 0.001), and Global Deterioration Scale (p = 0.014) and a negative relationship with MMSE (p = 0.004).Conclusion: The oral health of elderly patients with schizophrenia is poor, which may be related to the cognitive level of patients and affect their quality of life. The focus should be provided to the oral care of patients with schizophrenia, and investment in their specialized oral treatment should be increased. The incidence of mental disorders has rapidly increased worldwide, inducing a series of severe family, social, and cultural problems and affecting the development of the economy. Schizophrenia is a complex mental disease with a profound impact on human health; it is a chronic mental disorder affecting 20 million people globally, as revealed by the \u201cGlobal Burden of Illness, Injury, and Risk Factors study\u201d in 2017 .per se and oral hygiene and health problems resulting from frequent medication and inadequate oral cleaning care  scale and the missing teeth (MT) index and concluded that tooth loss is a related factor of cognitive impairment or its deterioration . In accordance with the Chinese reference standard, they were classified as normal (18.5 \u2264 BMI < 24), lean (BMI < 18.5), overweight (24 \u2264 BMI < 28), and obese (BMI \u2265 28). The DMFT index was used to assess oral health, whereas cognitive and mental statuses were measured.We used a cross-sectional study in two psychiatric hospitals on elderly patients with schizophrenia. From 2018 to 2019, 425 hospitalized patients with schizophrenia diagnosed by the International Classification of Diseases, 10th Revision, included 265 from the Guangdong Hospital and 160 from the Wuhan Hospital . All lonThe Ethics Committee of the Institute of Psychology, Chinese Academy of Sciences, approved this study. Ethical approval was conducted in accordance with the latest version of the Helsinki Declaration (lines 96\u201398). Written informed consent was obtained from all participants.Two dentists had been trained before this study. Consistency was assessed by Kappa calibration, and the Kappa value was >0.8. The decayed teeth (DT: number of teeth or surfaces with caries in the mouth), MT , filled teeth (FT: number of teeth or surfaces that have been filled for caries), and DMFT indices were detected under artificial light in a dental chair.Cognitive function was evaluated with the MMSE and Global Deterioration Scale (GDSRANK) . MMSE isMental status was evaluated with PANSS  and Repet-tests. Mann\u2013Whitney U-test or Kruskal\u2013Wallis H-test was used for variables with non-normality or homogeneity of variance. The Kolmogorov\u2013Smirnov test was used to test normality. Counting variables were analyzed by the chi-squared test or Fisher's exact probability test. The relationship between metrological/classification data and DMFT was analyzed using the Pearson or Spearman correlation method. The risk factor analysis for DMFT and MT in elderly hospitalized schizophrenics was performed through linear regression. The statistical significance level was set at 0.05.IBM SPSS 24.0 was used to analyze the data with t = 3.855, p < 0.001). The total mean DT value was 4.61 \u00b1 5.03, the MT value was 7.82 \u00b1 8.39, and the FT value was 0.56 \u00b1 2.34. The total caries rate, missing rate, and filling rate were 83.1, 83.3, and 14.6%, respectively.The mean DMFT score of the 425 patients was 12.99 \u00b1 8.86, 14.25 \u00b1 8.93 for patients in Guangzhou and 10.89 \u00b1 8.37 for patients in Wuhan . Patientp = 0.001). p = 0.001), age (p < 0.001), education level (p = 0.038), and smoking status (p < 0.001) significantly influenced the DMFT scores of the patients. High DMFT scores were significantly associated with age and smoking. Female participants and those with high education levels showed significantly lower DMFT scores than their counterparts.The Kolmogorov\u2013Smirnov test showed that the DMFT and MT indices were not normal , total MMSE score , GDSRANK score , PANSS-N score , and PANSS-G score  but not with PANSS-P score , PANSS total score , or RBANS score .r = 0.43, p < 0.001), total MMSE score , GDSRANK score , PANSS-N score , PANSS-G score , PANSS total score , and RBANS score  but not with PANSS-P score .p < 0.001), smoking , and MMSE score  are risk factors for the DMFT index. The DMFT index increased with age and smoking and decreased with MMSE score ascending. The association between MMSE and DMFT scores remained significant after controlling for age and smoking (p < 0.05).As shown in p < 0,001), smoking , and GDSRANK score  are positive risk factors for MT index, whereas total MMSE score  is a negative risk factor of MT score. After controlling for age and smoking, total MMSE score and GDSRANK score remained significantly associated with the MT index (p < 0.05).As shown in Poor oral hygiene in elderly psychiatric inpatients may aggravate cognitive impairment and increase the risk of aspiration pneumonia infection, resulting in a high incidence of hospital infections. The oral health of these patients can be improved by implementing professional oral treatment or nursing during hospitalization . ImproveA large number of previous studies examined the relationship between the MT index and cognitive impairment of the elderly in the community or the general population , 28, 29,The prevalence rates of dental caries and tooth loss in elderly patients with schizophrenia were 83.1 and 83.3%, respectively, consistent with the previously reported caries rate of ~80.0% , 31 and Several published studies to date analyzed the oral health of inpatients with mental disorders by using the mean DMFT score, such as 20.6, 15.8, and 16.6 , 33, whiU = 10.532, p = 0.001), consistent with most previous results . The effect of age on oral health was confirmed by a large number of studies , which is in line with the finding of Vano et al. ; the Special Research Project for the novel coronavirus pneumonia funded by the Chengdu Science and Technology Bureau (grant number 2020-YF05-00171-SN); and the Science and Technology Department of Sichuan Province (grant number 2018sz0236).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 identity-by-descent (IBD)-based mixed model approach introduced in this study can detect quantitative trait loci (QTLs) referring to the parental origin and simultaneously account for multilevel relatedness of individuals within and across families. This unified approach is proved to be a powerful approach for all kinds of multiparental population (MPP) designs.IBD.MQMkin_F) is robust across a variety of MPP designs and allele segregation patterns in comparison to a widely used benchmark association mapping method, and in most cases, outperformed or behaved at least as well as other tools developed for specific MPP designs in terms of mapping power and resolution. Successful analyses of real data cases confirmed the wide applicability of our IBD-based mixed model methodology.Multiparental populations (MPPs) have become popular for quantitative trait loci (QTL) detection. Tools for QTL mapping in MPPs are mostly developed for specific MPPs and do not generalize well to other MPPs. We present an IBD-based mixed model approach for QTL mapping in all kinds of MPP designs, e.g., diallel, Nested Association Mapping (NAM), and Multiparental Advanced Generation Intercross (MAGIC) designs. The first step is to compute identity-by-descent (IBD) probabilities using a general Hidden Markov model framework, called reconstructing ancestry blocks bit by bit (RABBIT). Next, functions of IBD information are used as design matrices, or genetic predictors, in a mixed model approach to estimate variance components for multiallelic genetic effects associated with parents. Family-specific residual genetic effects are added, and a polygenic effect is structured by kinship relations between individuals. Case studies of simulated diallel, NAM, and MAGIC designs proved that the advanced IBD-based multi-QTL mixed model approach incorporating both kinship relations and family-specific residual variances (The online version contains supplementary material available at 10.1007/s00122-021-03919-7. MPP designs have their unique advantages for QTL mapping over biparental populations and association panels. Crossing two parents in a biparental population can balance allele frequencies and increase the chance to detect rare QTLs, but the narrow genetic diversity from only two parents limits the number of detected QTLs (Liu and Zeng QTL mapping models can be classified into family-based (or linkage) and population-based (or linkage disequilibrium) approaches based on the specific design  or IBD information as the basis for genetic predictors and how they account for individual relatedness. We simulated diallel, NAM, and MAGIC designs from four inbred Arabidopsis parents to evaluate the performance of our models and compared that performance where possible with that of alternative approaches, such as IciMapping  individual contributed by an offspring allele stemming from the  parent. We constructed two types of models, depending on how the genetic predictor was calculated. Specifically, in the IBS-based model, the genetic predictors are given by observed numbers of IBS alleles. In the IBD-based models, the genetic predictors are given by the expected numbers of IBD alleles, where the QTL allelic effect, We developed the QTL mapping methodology in the linear mixed model framework. First, a linkage map for the MPP design is required as input for IBD calculations and QTL identification procedure. Best is to construct a consensus map following a protocol of marker cleaning, grouping, ordering  with, respectively, homogeneous, or uniform (_U), and family-specific (_F) variance\u2013covariance (VCOV) structures on residual terms. The first model IBD.SQM_U can be expressed as:q. The The first two models are called IBD.SQM_F can be expressed as:The second model, QTL.SQM_F, as in composite interval mapping , By simulating QTLs with different segregation configurations in various types of MPP designs, we aim to test the performance of IBD-based models versus GWAS models, where we looked at mapping power and resolutions for major QTLs. In Section \u201cTo create offspring populations with realistic marker profiles and segregation ratios, we based our simulations on four real Arabidopsis inbred lines with known genomes. These four lines served as parents in crosses that simulated diallel, NAM, and four-way MAGIC designs. With this number of parents, we still obtain enough details for insightful simulations, while computation time per simulation remains low. Genomes for progenies were simulated by implementing a crossover process in which progenies inherited markers and QTLs from parents following one of the MPP designs.Phenotypes for the offspring individuals contained a contribution from alleles at three major QTLs, positioned on different chromosomes and at markers with varying genotypic configurations across the parents, to investigate the impact of the number of segregating families and allele frequencies on QTL detection. Details are given in Section\u00a0\u201cparent1), Br-0 (parent2), Got-7 (parent3), and Kas-2 (parent4). These parents were randomly selected from the Arabidopsis HapMap collection  on the \u2018true\u2019 simulated genome from each MPP design  value for the likelihood ratio test for the variance component of the QTL effects at that position should exceed the threshold of 4.2, obtained by the Bonferroni correction on all 462 markers on the consensus map at a genome-wide significance level of 0.01. Secondly, the distance between the true position of the simulated major QTL and the peak marker with the highest \u2212log10(p) value on the same chromosome should be within the QTL window size (20\u00a0cM). The mapping resolution of a major QTL in our study was indicated by the average genetic distance between the true simulated position and the detected position with the highest -log10(p) value on the same chromosome over 500 runs. A shorter distance indicates a higher mapping resolution.PLQ function in the software GAPL for background-controlled QTL mapping . The MQE model of mppR defines QTL effects for genomic positions at di-allelic, parental, and ancestral levels and chooses the type of QTL effect that produces the highest significance  and r is the rank of the fixed effects design matrix (related to the number of families); infoCriteria function from the package asremlPlus  were derived from crosses between four inbred parents  Fig.\u00a0B. The PCParental origins can be uncertain at multiple positions with non-segregating markers or missing genotypes. Using the pedigree information and genomes of parents and offspring, we reconstructed the parental origins represented by IBDs. We extracted the highest parental IBD probabilities at each marker position per progeny and averaged the maximum IBD probabilities of this position across all progenies. The averaged maximum IBD probabilities at all positions were above 0.9 in all simulated MPP designs demonstrating the high quality of the parental genomic reconstructions in the offspring populations.IBS.Kin identified 2, 3, and 5 positions as false QTLs in the respective simulated diallel, NAM, and MAGIC designs; among IBD-based models, only the advanced model IBD.MQMkin_F detected one false QTL on chromosome 5 in the simulated diallel design over 500 runs. The mapping tools ICIMapping and GAPL detected 3 and 4 false QTLs on chromosome 5 in simulated NAM and MAGIC\u00a0designs, respectively, while with mppR, 5 and 2 false QTLs were detected on chromosome 5 in simulated NAM and diallel. Therefore, no large deviations from the expected number of false positives were observed for the alternative mapping methods either.By comparing the QTL mapping results of the six models on simulated MPP designs, we evaluated the efficiency of using IBD information for genetic predictors and the validity of using the mixed model approach to account for the multilevel relatedness of offspring within and across families. Figure\u00a0IBD.Kin_F and IBD.MQMkin_F, incorporating polygenic effects, generally performed better than other models considering both mapping power and resolution. For the simQTL1 segregating in four out of six families, the mapping power by IBD.MQMkin_F was higher than that at simQTL2 and simQTL3, both segregating in three out six families.In the simulated diallel design, the IBD-based models IBS.Kin in improving mapping power and resolution for simQTL1 and simQTL2. Notably, the advanced IBD-based model IBD.MQMkin_F detected simQTL2, which is segregating in all three families, with the highest mapping power and resolution. For simQTL1, segregating in two out of three families, mapping power and resolution obtained from IBD.MQMkin_F were also higher than other models. However, for simQTL3 that segregated in only one family, IBD.MQMkin_F detected this QTL with slightly higher mapping power than other IBD-based models but the mapping resolution was lower than the IBS.Kin model.In the simulated NAM design, IBD-based models have advantages over the reference model simQTLs\u2014all of them were detected with relatively high mapping power and resolutions over diallel and NAM designs. Especially for simQTL1 with an expected genotype frequency of 0.5, both the advanced IBD-based model IBD.MQMkin_F and the reference IBS-based model IBS.Kin successfully detected this QTL with high mapping power and resolution. For simQTL2 and simQTL3, advanced models IBD.Kin_F and IBD.MQMkin_F performed better than the rest of the models considering both mapping power and resolutions.The simulated MAGIC design seems to be the most promising MPP for detecting all three IBD.MQMkin_F were comparable to ICIM-based approaches for simulated NAM and MAGIC designs, while the same IBD.MQMkin_F was comparable to the MQE model for simulated diallel and NAM designs.When comparing our IBD-based mixed model approach to alternative approaches from the literature, we observed see Fig.\u00a0 that ressimQTL1 and simQTL3 by IBD.MQMkin_F were higher than the MQE model using the mppR package, while no apparent difference occurred between IBD.MQMkin_F and MQE for simQTL2 in terms of mapping power. For the mapping resolution, IBD.MQMkin_F and other IBD-based models performed better than the MQE model on all simQTLs.In the simulated diallel design, the mapping power of IBD.MQM_F with JICIM approach in the software IciMappig and MQE model in the mppR. For simQTL1 and simQTL2, IBD.MQM_F performed better than both JICIM and MQE models with higher mapping power and resolution. However, for simQTL3 that segregated in only one of three families, IBD.MQMkin_F detected this QTL with lower mapping power and resolution than JICIM and MQE, because of the test for a variance component being underpowered for a single biparental population.In the simulated NAM design, we can compare IBD.MQMkin_F detected simQTL2 and simQTL3 with higher mapping power and resolution than the ICIM-based approach in the GAPL software. Both approaches detected simQTL1 with equally high mapping power, and the mapping resolution is slightly higher by using IBD.MQMkin_F than GAPL.For the simulated MAGIC design, we see that IBD.MQMkin_F is superior to other models because it detected most QTLs with increased mapping signal and the relatively small BIC value (Supplementary Table S1).The maize diallel design generated four families from four parents with multiple traits measured and analyzed in the previous study , we fitted these 7 detected QTLs in this advanced IBD-based mixed model to estimate the parental effects at those QTLs for trait GDDTAP. It shows that the parents of temperate type (B73 and B97) contribute negative effects at those QTLs while the other two parents of tropical type (CML254 and Ki14) contribute the positive effects at most of those detected QTLs.Coles et al.  reporteds et al. . Due to IBD.SQM_U and IBD.SQM_F  to estimate the parental effects (Supplementary Table S1). It shows that the parental effect of one beef type tomato (parent B) contributed negatively to the fruit shape at all three QTLs, and the other beef type tomato (parent A) show negative effects at two QTLs on chromosomes 2 and 9 on which the round type tomato (parent C) show positive effects at these two QTLs.The diallel F2 design of two beef tomatoes and one round tomato generated a population of diverse fruit shapes, and we identified some QTLs for fruit shape Fig.\u00a0D. In totIBD.MQMkin_F model to show that the three resistant parents , at the strongest QTL on chromosome 6, contributed negatively to the disease score. Tomato breeders have successfully fine-mapped this QTL as a strong resistance gene.In the connected NAM F2 design, a resistant parent crossed with four susceptible lines, and one of the susceptible lines was crossed with another two resistant lines. All five IBD-based mixed models identified two QTLs with strong signals on chromosomes 1 and 6 Fig.\u00a0E. BecausIBD.MQMkin_F detected most QTLs for location A, and all models detected three consistent QTLs for location B , compared to IBD.SQM_U and IBD.SQM_F, allowed us to detect more QTLs on chromosomes 5 and 9. IBD.Kin_F and IBD.MQMkin_F identified two linked QTLs on chromosomes 2 and 11. The advanced model IBD.MQMkin_F, among the five models, has the smallest BIC value (Supplementary Table S1).In the eight-way MAGIC F4 population, we mapped QTLs underlying fruit weight measured at two locations that we will refer to as A and B. The model IBD.MQMkin_F model. We fitted QTLs in the model IBD.MQMkin to estimate the percentage of phenotypic variation explained by the QTLs. The eight QTLs identified in our study slightly increased the explained percentage of phenotypic variation, from 51% in Pascual et al.  concerning mapping power and resolutions for major QTLs.We used the observed IBS with 5% of missing genotypes to estimate IBD probabilities and then compared IBS-based with IBD-based models. Parental origins can be ambiguous at non-segregating, missing, or mis-genotyped loci based on IBS information, whereas IBD probabilities inferred by the pedigree information and the whole genome can reduce uncertainty for genome origins  increased mapping power and resolutions for all simQTLs compared with IBD.SQM_F and IBD.MQM_F.Including only significant positions as cofactors from the initial genome-wide scans can lead to ignoring part of the genetic variance and missing heritability , multilevel relatedness exists between individuals as being full-sib, half-sib, and unrelated within and across families. A priori no population structure is expected in standard MAGIC designs, but MAGIC lines may still show complicated realized genetic relationships. Multilevel relatedness can be corrected by using a general QK model where the Q matrix accounts for family structure, and the pairwise relationship matrix K deals with the individual relatedness  offers us a robust and comprehensive solution to map QTLs in general MPP designs. In our simulation study, we observed no case where the IBD.MQMkin_F model performed significantly worse than other IBD-based models in terms of mapping power and resolution, and this model is also competitive with other tools developed for specific MPP designs. Most results from empirical MPP designs also show that the unified IBD.MQMkin_F model detected most QTLs with relatively small BIC, and the major QTLs were comparable to those identified by previous studies.To sum up, we can refer to conceptions from family-based and population-based mapping approaches to explain the efficiency of our approach. Family-based QTL mapping assumes QTL effects to be multiallelic and referring to parental origins. For the estimation of parental effects at the QTL, we need design matrices that are functions of IBD probabilities. Popular population-based mapping strategies employ the mixed model approach to deal with multilevel relatedness by imposing family-specific and kinship-based VCOV structures on the non-genetic residual and the polygenic term, respectively. Family-based and population-based mapping approaches complement each other, and their synthesis in an advanced IBD-based mixed model approach (Supplementary file1 (DOCX 2744 KB)Supplementary file2 (DOCX 20 KB)Below is the link to the electronic supplementary material."}
+{"text": "Self-catalyzed AlGaAsnanowires (NWs) and NWs with a GaAs quantumdot (QD) were monolithically grown on Si(111) substrates via solid-sourcemolecular beam epitaxy. This growth technique is advantageous in comparisonto the previously employed Au-catalyzed approach, as it removes Aucontamination issues and renders the structures compatible with complementarymetal\u2013oxide\u2013semiconductor (CMOS) technology applications.Structural studies reveal the self-formation of an Al-rich AlGaAsshell, thicker at the NW base and thinning towards the tip, with theopposite behavior observed for the NW core. Wide alloy fluctuationsin the shell region are also noticed. AlGaAs NW structures with nominalAl contents of 10, 20, and 30% have strong room temperature photoluminescence,with emission in the range of 1.50\u20131.72 eV. Individual NWswith an embedded 4.9 nm-thick GaAs region exhibit clear QD behavior,with spatially localized emission, both exciton and biexciton recombinationlines, and an exciton line width of 490 \u03bceV at low temperature.Our results demonstrate the properties and behavior of the AlGaAsNWs and AlGaAs/GaAs NWQDs grown via the self-catalyzed approach forthe first time and exhibit their potential for a range of novel applications,including nanolasers and single-photon sources. Nevertheless, the successful growth of high-qualityternary NWs remains challenging, due to the sensitivity of the growthparameters and potential issues associated with inhomogeneous elementaldistribution in the alloy.8 Despite thesechallenges, a variety of III\u2013V ternary alloys have been realized.16 Among the various III\u2013V material platforms available, AlGaAscombined with GaAs has the significant advantage of nearly identicallattice constants and a broad range of band-gap tunability, varyingbetween 1.42 eV (GaAs) and 2.16 eV (AlAs) at room temperature (RT).17 The band gap is direct up to 1.98 eV, whichcorresponds to an Al content of \u223c45%.17 Therefore, the planar GaAs/AlGaAs material system has led to a widerange of applications over the past few decades, including high-electron-mobilitytransistors,18 quantum well (QW) infrareddetectors,19 and near-infrared laser diodes.20An essential step to expand the functionality range of NWsis thesynthesis of ternary III\u2013V alloys, allowing tunability of theband gap via the elemental composition. This provides an additionaland controllable degree of freedom in the band-gap engineering22 quantum emitters,24 light-emitting diodes,25 and detectors.26 The majority of these results are limited toa GaAs core/AlGaAs shell architecture. However, the less-investigated,pure AlGaAs ternary NWs are also of great interest. For example, AlGaAsNWs could be exploited as efficient light sources on a Si chip, witha tunable emission wavelength. Furthermore, AlGaAs NWs can also actas hosts for GaAs nanostructures. For instance, radial QWs incorporatedin pure AlGaAs NWs can enhance the lasing performance compared totheir GaAs/AlGaAs core/shell NW counterparts,30 where the GaAs core forms a low-band-gapcarrier reservoir and can also absorb lasing photons emitted by theGaAs QWs. Pure AlGaAs NWs have been exploited as hosts for quantumdots (QDs) embedded in their axis, allowing the integration of theQDs with the photonic cavity formed by the NW morphology. Specifically,AlGaAs/GaAs nanowire-quantum dots (NWQDs) with light emission in thespectral range of 600\u2013800 nm have been reported.33AlGaAs has been successfully incorporatedin GaAs NWs for a rangeof structures, using different growth techniques. Several groups haveimplemented the GaAs/AlGaAs material system in NWs for applicationsincluding lasers,38 and AlGaAs/GaAs NWQD33 systems have used theAu-catalyzed growth technique. This significantly hinders their potential,as fast solid diffusion of Au atoms renders them incompatible withexisting technology.39 Furthermore, ithas been shown that the optoelectronic properties and material qualityof self-catalyzed GaAs/AlGaAs core/shell NWs are considerably improvedin comparison to their Au-catalyzed counterparts and can match theperformance of planar structures.40 Hence,utilizing the self-catalyzed method to grow NWs on Si substrates viamolecular beam epitaxy (MBE) is a critical step required for incorporatingAlGaAs NWs in Si-based photonics.43 The MBE technique allowsprecise control of material deposition,44 thus enabling the realization of highly complex structures. It isnoted that the focus regarding self-catalyzed ternary NWs has beenmainly III\u2013V\u2013V alloys, including GaAsP,10 GaAsSb,12 GaNP,14 and InAsSb,15 in terms of growth and device application. Eventhough progress has been witnessed regarding the catalyst-free developmentof InGaAs NWs,16 the overall inspectionof III\u2013III\u2013V ternary nanowires is limited compared withIII\u2013V\u2013V nanowires, possibly due to the relatively highsolubility of group III elements in the droplet, which leads to thechemical consistency of the droplet seed changing even under smallalterations in the balance between group III elements, rendering thegrowth particularly sensitive.8To date, published reports on AlGaAs NWsInthis work, self-catalyzed AlGaAs NW growth on Si(111) substratesusing solid-source MBE is reported. A systematic study of the morphological,structural, and optical properties of the AlGaAs NWs is presented.Initially, the impact of Al incorporation into the NWs is investigated,in terms of morphology, structure, and compositional distribution.It is shown that the NWs adopt a core/shell structure by the spontaneousformation of an AlGaAs shell, with a much higher Al concentrationthan the core. In addition, the shell exhibits complex alloy fluctuations.The AlGaAs NWs display strong photoluminescence (PL) emission anddemonstrate band-gap tunability as a function of the Al content. Furthermore,AlGaAs NWs containing a single GaAs QD are fabricated, which exhibitspatially localized emission lines corresponding to exciton and biexcitonrecombination, with a low-temperature excitonic line width of 490\u03bceV, clearly demonstrating light emission by the quantum-confinedstates within the QD segment.4 cracker cells, on commercially available p-typeSi(111) substrates. Before growth, the substrates were subjected toannealing at 660 \u00b0C for 10 min in the growth chamber to removesurface contamination. The growth was initiated by the formation ofGa-catalyzed GaAs stems, approximately 600 nm long, grown for 10 min.Next, the Al flux was introduced in order to form Ga-catalyzed AlGaAsNWs, with the nominal composition varying between 10 and 40%. TheNW length ranged between 4 and 8 \u03bcm, depending on the Al composition;hence, the GaAs stems represent a very small fraction of the entirestructure. The NW growth took \u223c100 min, and the Ga and Al fluxesused were those needed to produce AlxGa1-xAs thin films of the same composition on GaAs(001) substrates. TheGa flux was adjusted to give a thin-film growth rate of 0.6 ML/s,as previously determined by GaAs film calibrations on GaAs(100) substrates.Unless stated otherwise, a stable As4 flux of 2.75 \u00d710\u20136 Torr was maintained, giving an As/Ga ratioof 15. Based on the information provided above, the V/III beam fluxratio of As/(Ga + Al) for the samples with 10, 20, 30, and 40% nominalAl contents is 14.053, 13.119, 12.134, and 11.075, respectively. TheNWQD samples were grown under similar conditions, except that theAl supply was interrupted for seven seconds. This allowed for thedeposition of Ga and As only, which led to the formation of a GaAssegment of narrower band gap in the NW axis that inherently acts asa QD. Following this stage, Al flux was resumed in order for the toppart of the NW to grow. Hence, the GaAs QD was fully embedded in thenanostructures.AlGaAs NW samples were grownby MBE using solid Al and Ga sourcesand As2. Low-temperature (6 K) micro-PL (\u03bcPL) measurementswere performed on single NWs transferred to a SiO2/Si substratewith a focused laser spot size of about 1 \u03bcm and excitationwavelength of 515 nm.Scanning electron microscopy (SEM) measurementswere conductedin a JEOL JSM IT-100, with the accelerating voltage set at 20 kV andat a tilt angle of 30\u00b0 from the top view. Scanning transmissionelectron microscopy (STEM) imaging and energy-dispersive X-ray spectroscopy(EDX) analysis were performed using a doubly corrected ARM200F microscope,operating at 200 kV. The NWs were mechanically transferred to holeycarbon grids for side-view analysis and the microtome sliced for thecross-section STEM analysis. Finally, PL measurements on ensemblesof NWs were carried out at RT using a Nanometrics RPM2000 system withan excitation wavelength of 635 nm and a power density of approximately500 W/cmFigure S1a,c in the SupportingInformation (SI)]. Two main morphologies of the vertical NWs are found. It is interestingto mention that, whilst the appearance of kinked structures is occasional,the frequency of the branched NW formation is proportional to theAl composition. Specifically, at low Al contents of 10\u201330%,the percentage of branched structures is low and does not exceed 15%.On the contrary, with increasing Al content (at 40%), the percentageof NWs that develop branches reaches 60%.46 Notably, the diameter of the NWs was also observed to increase withelevating Al content, due to the trend of this element to adhere tothe sidewall facets and incorporate in the lateral shell growth. Moredetails on this phenomenon and the analysis of these unique structureshave been described in a previous report by our group.46 Additionally, the influence of the V/III ratiowas examined, as this parameter is crucial for NW growth. Consequently,the As flux was modified in order to give V/III ratios of 15, 20,and 35. The growth is found to be very sensitive to this value, asits increase above 20 causes premature solidification of the droplets,leading to short structures with morphological deviations . In what follows, we focuson fully grown NWs with the optimum morphological configuration, achievedwith V/III ratios lower than 15, with the exact values that were mentionedearlier. Structural analysis and characterization were conducted forall of the acquired samples. Nevertheless, the NWs with 20% Al contentwere chosen as the most representative examples.Initially,the morphology of NWs with a nominal Al content varyingbetween 10 and 40% was investigated via SEM images. For the lowestAl concentration, the majority of the NWs adopted a vertically aligned,one-dimensional morphology 1a. Howev38Structural and compositionalproperties of the AlGaAs NWs werestudied by STEM. NWs with the Ga droplet still present, and with nokinking or branching, were selected for these measurements. Elemental-distributionEDX maps close to the tip of an AlGaAs NW with a nominal 20% Al contentare shown in 47 The higher Al composition in the shell is attributed tothe shorter diffusion length of Al, with a much higher activationenergy for surface diffusion compared to Ga.49 Consequently, it is preferential for Alto be incorporated at the side facets of the structure during simultaneousvapor\u2013solid growth, leading to the formation of the Al-richshell. It is interesting to note that alloy fluctuations in the shell,similar to the ones observed in the current work, with alternatingregions of different composition, have been previously reported andexploited for the realization of radial QWs.30 Furthermore, such alloy fluctuationshave led to the self-formation of QD-like emitters and related nanostructures,which provide a novel type of nonclassical light emitter.51 Additional EDX mappings of the shell can validate the bright contrastderived from higher Al content within the shell. These measurements,along with the relevant discussion, are presented in Figure S2a\u2013d in the SI. The origins of the observedphase separation have not been conclusively identified. It has beenreported in previous works that such phenomena are attributed to thekinetics of the adatoms and more specifically to the different diffusivitiesof Al and Ga during the growth procedure.47 This has also been indicated by the fact that similar phenomenacan be drastically reduced in core/shell NWs developed at lower temperatures.47 The above observations are in good agreementwith predictions regarding the temperature-related modifications ofthe adatom diffusion length.52 However,more work is required to fully interpret the interesting formationof these alloy fluctuations.NWs were sectioned perpendicular to the growthdirection usingan ultramicrotome for further characterization. All sections analyzedhere correspond to regions above the GaAs stems formed at the initialstages of the growth, i.e., approximately the first 600 nm of thestructures. It is important to mention that these GaAs areas are notincluded in this study. ADF cross-sectional image, taken from thebottom part of a NW, with 20% nominal Al composition 3a corrobFigure S4). 53 In contrast, the thinning of the shell is attributed tothe previously mentioned short diffusion length of Al, which resultsin Al adatoms preferentially adhering to the bottom region of theNW sidewalls, with less Al reaching the upper region of the NW. Asimilar explanation was proposed for an analogous observation in InGaAsNWs.54 A schematic representation of thismechanism is included in Section S3 ofthe SI. Another reason for the thickness variations is that the toppart of the NW is formed at the later stages of the growth. Hence,there is not sufficient time for the shell to be developed close tothe tip, as opposed to the base, where the material has been accumulatingfor a longer time. Eventually, the elemental mappings of the crosssection close to the NW tip for As, Ga, and Al are presented in The crystal quality of the AlGaAs NWs was studiedby atomicallyresolved ADF. Eg(x)= 1.422+ 1.2475x, where x is the Al fractionand Eg is the band-gap energy at RT.55 For the samples with 10, 20, and 30% nominalAl content, effective Al compositions are calculated to be 7 \u00b12, 13 \u00b1 3, and 22 \u00b1 4%, respectively. The errors in thesevalues are obtained by attributing the line width of the emissionto the distribution of the Al content in the structures. The presentobservation of strong RT emission that is tunable with Al contentdemonstrates the potential of the AlGaAs NWs as wavelength-tunablelight sources on Si. No RT emission was measured from the sample witha nominal 40% Al content, possibly due to the actual composition ofthis structure being close to, or even above, the value at which theband gap becomes indirect.RT PL was recorded forNW ensembles with 10\u201330% nominalAl composition and with the NWs attached to the original Si substrate.The spectra are presented in 56 Thespectra exhibit a series of sharp emission lines, whose intensityand wavelength vary along the NW axis. Due to the relatively low incidentlaser power (6 \u03bcW) used to acquire these spectra, these linesare attributed to recombination from a low density of localized states,produced by fluctuations in the AlGaAs composition. The peaks arerelatively weak in the vicinity of the NW ends. Although it is notpossible to distinguish the base and tip in these measurements, theweak emission at one end most likely results from the presence ofthe GaAs stem at the NW base, used to initiate the NW growth. It isinteresting to observe that in the case of the AlGaAs NWs with 30%nominal Al content, there are two emission bands being presented.The band at 680 nm is suspected to originate from the alloy fluctuationsin the AlGaAs shell that were described earlier (30 or QD-like emitters.51 This is also indicated in a previouswork, where emission from the alloy fluctuations in the AlGaAs shellof core/shell NWs was traced at roughly the same spectral region.50 The band at 720 nm is derived from the AlGaAsNW core emission. 6 K \u03bcPL measurementswere performed at positions along theaxis of single NWs to probe the spatial variation of the Al composition.  earlier 3b and haSection S4 ofthe SI, in order to better demonstrate the aforementioned observations.It has recently been reported that the WZ form of AlGaAs exhibitsa significantly lower direct band gap than the ZB form.57 With both forms present in a nanostructure,carriers may diffuse into the WZ segments prior to their recombination.The resultant lower PL emission energy would hence lead to a lowercalculated effective Al composition, as the ZB band-gap values wereused in the calculation. Second, it is possible that PL measurementsare sensitive to lower-band-gap Al-deficient regions of the NWs dueto carrier diffusion and capture before recombination occurs. Structuralstudies show the NWs to have a complex morphology (Section S4 ofthe SI.The composition values determinedfrom EDX for the cores of thethree NW samples are higher than the corresponding values determinedby both the RT ensemble PL and the low-temperature \u03bcPL of singleNWs. Specifically, the latter gives an effective Al composition significantlylower than the nominal value, whilst the former gives a higher Alcontent. To explain this difference, we consider two possible factors.First, polytypism may play a role. The polytypic nature of the crystalstructure has been previously established, with the observation ofthin WZ insertions of 4\u20136 monolayers within the dominant ZB-phaseNWs 3d. Anenrphology 2b,c, witAfter having described the AlGaAs NW properties andbehavior, anotherinteresting topic of research is the suitability of NWs for hostingGaAs dots. For this reason, AlGaAs/GaAs NWQDs were grown via MBE,with a single GaAs QD incorporated in the center of AlGaAs NWs. TheQDs were deterministically embedded, axially in the AlGaAs NWs bystopping the supply of Al for 7 s, in order for the GaAs layers tobe deposited. STEM analysis and structural studies of a NWQD with20% nominal Al composition are shown in 58 give a free-particleground-state transition energy of 1.59 eV, equivalent to 775 nm. Whencorrected for an exciton binding energy of \u223c25 meV, typicalfor the GaAs\u2013AlGaAs NWQD system,59 an emission wavelength of 787 nm is obtained, consistent with theexperimentally observed emission peak at 791 nm. In addition, The optical analysis on NWQDsthat followed is presented in 59 Analytically, it can be seen that both resolvedlines exhibit an initial increase with power. The exciton line presentsa saturation and both lines decrease at higher powers. This is theexpected behavior of a zero-dimensional system with increasing averagecarrier occupancy.60 The activation energyassociated with this model is the separation between the ground stateand the first excited state of the QD. The value that is extractedfrom the fit to the experimental data is 3 meV. Nextnano simulationsgive the calculated separations of the ground and first excited QDstates of 1 and 0.6 meV for electrons and holes, respectively, inreasonable agreement with the extracted energy of 3 meV. It is notedthat the observed discrepancy between the experimentally extractedvalue and the simulated values of the separation energies can be attributedto crystal imperfections related to the GaAs QD and to the non-ideallyabrupt NW/QD interfaces. Details of the nextnano simulations are presentedin Section S5 of the SI.62 However, further works are needed to unambiguously verify the aforementionedphenomenon.The currentoptical spectroscopy results for the NWQD structureshow clear evidence for QD behavior at low temperatures. Emissionfrom a spatially localized region is observed, with different excitonicrecombination processes; these exhibit the expected saturation athigh pump powers consistent with fully quantized states. The temperaturedependence of the emission line width shows a behavior consistentwith that expected for a QD. The results reveal a relatively largeemission line width at low temperatures and a rapid quenching of theemission intensity with increasing temperature. The former most likelyarises from fluctuating carrier occupancies in nearby defect states.Consequently, improving the crystal purity of the structures wouldminimize this effect and adding effective surface passivation layerswould reduce the effects of surface states. Additionally, improvedtemperature stability would possibly result from the use of higherAl compositions in the shell of the NWs, since it is anticipated tolead to improvement of their emission properties.In summary, MBE growth of self-catalyzedAlGaAs NWs, with and withouta GaAs QD, and of varying Al content, on Si substrates has been reported.For low Al concentrations the NWs are vertically oriented, while increasingAl composition leads to complex morphologies. The spontaneous formationof an Al-rich AlGaAs shell is observed, with an inhomogeneous radialdistribution of Al. The shell is thicker at the bottom of the NW andthins towards the NW tip; the opposite behavior is observed for thecore. A strong RT emission and band-gap tunability from 1.50 to 1.72eV is achieved by varying the Al composition between 10 and 30%. AGaAs/AlGaAs single dot-in-wire structure shows clear QD behavior,exhibiting spatially localized emission and both exciton and biexcitontransitions. The exciton line width at 6 K is 490 \u03bceV. Theseresults demonstrate the potential of self-catalyzed AlGaAs NWs asthe host for QD emitters for applications including single-photonsources and lasers."}
+{"text": "BQAS-3F) has themaximum adsorption capacity for 2,4-dinitrophenol  (406 mgg\u20131). Electrostatic attraction and \u03c0\u2013\u03c0interaction are the main forces in adsorption. The adsorption kineticsstudies display that PANBQAS-3F can rapidly adsorb2,4-DNP in 10 min and achieve equilibrium within 20 min. The adsorptionprocess of 2,4-DNP by PANBQAS-3F follows the Langmuirmodel, demonstrating that the process is more consistent with monolayeradsorption. What is more, the adsorbent PANBQAS-3F can be reused after 10 adsorption/desorption cycles and still maintainsan excellent removal rate (99%). Otherwise, PANBQAS-3F was used in a continuous flow process and exhibited a removal rateof more than 96%, which certifies that PANBQAS-3F is an excellent adsorbent and can be utilized in practice.A series of biquaternaryammonium-functionalized fibers were developedto efficiently realize selective removal of phenolic compounds fromwater. Fourier transform infrared spectroscopy and X-ray photoelectronspectroscopy were employed to determine the successful preparationof functionalized fibers. Scanning electron microscopy, X-ray diffraction(XRD) patterns, and elemental analysis were used to analyze the microstructureand composition. First, the adsorption result shows that a fiber witha three-carbon alkyl chain (PAN Due to the low biodegradability,high solubility, and stability, 2,4-DNP has become a typical pollutantand has been banned by the American Environmental Protection Agencyand the European Union. In particular, the ingestion of 2,4-DNP maycause harmful effects on human health, like skin allergy, cardiovasculardiseases, and low concentration carcinogenesis to human health.2 Therefore, it is significant to develop efficientmethods for the removal of 2,4-DNP from wastewater before being dischargedto the environment.2,4-Dinitrophenol is widely used in industries processes,such as dyes, insecticides, preservatives, explosives, and paper bleaching.4 adsorption,7 electrochemicaldegradation,9 andbiochemical reactions10 have been explored.Among these approaches, adsorption methods have aroused considerableinterest owing to their simplicity, efficiency, and low cost.11 Till now, various adsorbents including silicagels,12 metal\u2013organic frameworks,6 and activated carbon14 have been used to remove 2,4-DNP from an aqueous solution. However,some of these adsorbents have the disadvantages of high cost, a tediouspreparation process, and unsatisfactory recyclability, which hindertheir practical applications. Thus, there still remains a great challengeto develop effective adsorption materials to overcome these defects.To achieve this purpose, many methods suchas photocatalysis,16 To date, many kinds of functionalized polyacrylonitrilefibers have been prepared and employed to remove organic pollutantsand to absorb and recognize metal.22 However, there are few reports on the application of functionalizedPANF on the removal of 2,4-DNP from the aqueous solutions, so it isof great significance and challenge to prepare efficient fiber absorbentsfor 2,4-DNP removal.A polyacrylonitrile fiber (PANF) is a low-cost material with alarge surface and high mechanical strength and contains numerous nitrilegroups, which can be easily transformed into various functionalizedmoieties.22.1\u20131). The corresponding expressions are 25 respectivelyW1 and W2 represent the weight of before and after modificationof the fiber, respectively, and M is the molecularweight caused by functional organic molecules . The modification degree of functionalized fibers canbe expressed by weight gain (%) and functionality , and PANBQAS-3F-10 (fiber recycled 10 times) samples were pulverizedby cutting and then prepared into KBr pellets. The FTIR spectra arepresented in \u20131, corresponding to the stretching vibrationsof C\u2261N and C=O,26 which arethe characteristic peaks of polyacrylonitrile fiber C\u2261N andC=O in methyl methacrylate or methyl acrylate, respectively.After modification, PANPF  decreased significantly  was used to demonstrate the successful preparation ofPAN2.2.3PF, and PANBQAS-3F show three peaks at 530.89, 397.96, and 284.38eV, corresponding to the characteristic peaks of O 1s, N 1s, and C1s, respectively. In addition, a new peak of PANBQAS-3F appears at 68.56 eV,27 attributed tothe characteristic peak of Br 3d, which indicates that small moleculesof bromine-containing quaternary ammonium salt were successfully graftedonto a tertiary amine-functionalized fiber.To prove the successful functionalization, the chemicalcompositionof the original fiber and the modified fiber were analyzed by XPS.As shown in BQAS-3F are shown in BQAS-3F show three absorption peakscorresponding to the functionalized groups C\u2013O/C\u2013N (284.57eV), C\u2261N (285.99 eV), and C=O (288.37 eV) 3 and N+ groups, respectively,30 which further proves the successful graftingof a quaternary ammonium-functionalized fiber  3a.28,29 ed fiber 3c.2.2.4PF  is slightly lower thanthat of PANF. The mechanical strength of PANBQAS-3F still retains 71% of the fracture strength of PANF. In conclusion,the strength of the fiber is slightly reduced during the modification,but the mechanical strength is still retained high. Moreover, theadsorption operation is generally carried out at room temperature;the adsorption process is mild and the damage to the fiber is minimal. This is due to the fact thatphenolic compounds can be dissociated into phenolic oxygen anionsin an aqueous solution and can bind to the positive ion adsorptionsites on biquaternary ammonium-functionalized fibers. Second, theadsorption capacity of PANBQAS-3F for phenolic compoundsincreases with acidity enhancement of these compounds . This isbecausethe higher the acidity, the easier it is for compounds to ionize hydrogenions to form phenoxy anions in an aqueous solution, and the strongerthe electrostatic attraction is to show the better adsorption capacity.In addition, comparing the adsorption ability of PANBQAS-3F to different aromatic compounds, the adsorption amount of naphthaleneis greater than benzene and naphthalene-1-phenol is greater than phenol,which indicates that there is a \u03c0\u2013\u03c0 interactionbetween the functionalized fiber and the target compound.Benzene, toluene,nitrobenzene, naphthalene, 4-methylphenol,phenol, naphthalene-1-phenol, 2-dichlorophenol, and 4-NP were selectedas target compounds to systematically investigate the adsorption selectivityof PANbstances 4. The exKa of the compound may be themain factor affecting the adsorption of the functionalized fiber tothe target compound. So more acidic phenolic compounds 2,4-DNP (pKa = 4.08) and 2,4,6-dinitrophenol (pKa = 0.38), were used to explore the adsorptionmechanism of functionalized fibers. The results are shown in BQAS-3F increaseswith a decrease in pKa, which illustratesthat the electrostatic attraction of positive and negative chargesis the main force of adsorption. In the follow-up research, 2,4-DNP,which has a larger adsorption capacity and is relatively easy to obtain,was selected as the target compound.Fromthe abovementioned experiment, it can be seen that the acidityor the p2.3.3BQAS-3F to phenolic compounds wereinvestigated at different pH values, and the results are shown in BQAS-3F for phenoliccompounds can be regulated by changing the pH values of the solution,which determine the existing forms of phenolic compounds in water.First, PANBQAS-3F has better adsorption capacityfor 2,4-DNP than other compounds at different pH values because ofthe strong acidity and a relatively high degree of dissociation of2,4-DNP. In both acidic and alkaline conditions, 2,4-DNP exists inthe form of a phenoxy anion, which can interact well with functionalizedfibers. On the other hand, the acidity of 2,4-dichlorophenol and 4-NPis relatively weak, and the degree of dissociation is also weak underacidic conditions. They exist mostly in a molecular form, and theadsorption performance is poor under acidic conditions. However, negativeions can be formed under alkaline conditions, which enhances the interactionwith fibers and improves the adsorption capacity of the fiber. Phenolhas the weakest acidity and still exists in the form of a moleculeunder weak alkaline conditions, so the adsorption properties underdifferent pH values are very weak.In the paper, adsorptionproperties of PAN2.3.4BQAS-3Fs with different weight gains  were prepared, and their adsorption capacities for 2,4-DNPare shown in BQAS-3F fiber with a weight gain of 73.6% was selectedfor the experiment.A series of PAN2.3.5BQAS-3F for 2,4-dinitrophenol wasmeasured, and the results are shown in BQAS-3F was rapid; it took only 10 min forthe fiber to almost reach its saturated adsorption equilibrium (405mg g\u20131). The adsorption capacity did not furtherincrease after 20 min.The adsorptionkinetics of PANFigure S12). The pseudo-first-order t (min) isthe adsorptiontime, qe (mg g\u20131) isthe equilibrium adsorption of the adsorbent, qt (mg g\u20131) is the amount of the 2,4-DNP adsorbedby PANBQAS-3F at a given time, and k1 (min\u20131) and k2 (g mg\u20131 min\u20131) are pseudo-first-orderand pseudo-second-order rate constants, respectively. It can be observedthat the qe,exp value obtained from theexperiment (406.0 mg g\u20131) and the qe,cal value calculated based on pseudo-second-order (403.2mg g\u20131) are more close to each other than that ofpseudo-first-order, and the values of R2 for the pseudo-second-order (0.99989) are greater than the pseudo-first-ordermodel (0.9641), which indicates that the pseudo-second model is moresuitable for fitting experimental data. Therefore, the adsorptionof 2,4-DNP from aqueous to PANBQAS-3F should bea chemisorption process . The Langmuir isotherm model33 assumesthat the adsorption process is monolayer adsorption, that there isno interaction between the adsorbates and they are independent, andthe surface of the adsorbent is uniform. The Freundlich isotherm modelis not only suitable for single molecular layer adsorption but alsofor multilayer adsorption or uneven surface adsorption. All equationswere expressed as3633 correspondingfitting parameters are listed in R2 valueof the Langmuir model is 0.9992 larger than that of the Freundlichmodel R2 (0.7969), indicating that theadsorption process of PANBQAS-3F to 2,4-DNP is moreconsistent with the Langmuir model and closer to monolayer chemicaladsorption. The result shows that the adsorption is due to the interactionof electrostatic attraction and hydrogen bonding. The maximum adsorptioncapacity (qmax) calculated by the Langmuirisothermal model is 429.0 mg g\u20131, which indicatesthat PANBQAS-3F has more advantages than other adsorbents(Table S2).Langmuir and Freundlich isotherm models were thenused to fit theequilibrium adsorption data  exhibited better adsorption of 4-nitrophenolthan monoquaternary ammonium-functionalized fiber . Then, PANBQAS-3F was studied to investigate adsorption properties for 2,4-DNP. PANBQAS-3F shows excellent adsorption capacity (406.0 mgg \u20131) and reusability . The adsorption experiment data fittedwell with the pseudo-second-order kinetic model and the Langmuir adsorptionisotherm model, indicating that the adsorption process is chemisorptionof a monolayer. In addition, PANBQAS-3F exhibitedremarkable and obvious performance under continuous flow conditions,which can be referred to in the video in the Supporting Information.A series of monoquaternary ammonium- and biquaternaryammonium-functionalizedfibers were successfully prepared and used to remove 4-nitrophenolfrom water. The results showed that the biquaternary ammonium-functionalizedfiber .A polyacrylonitrilefiberwith a diameter of 20 \u00b1 0.5 \u03bcm  was cut into lengths of 5\u201310cm before use. 4.1.21H NMR (600 MHz) spectra wererecorded on a JEOL JNM ECZ600R instrument using tetramethylsilaneas an internal standard. 1H NMR (400 MHz) spectra wererecorded on a BRUKER-AVANCE III instrument using tetramethylsilaneas an internal standard.An AVATAR360 FTIR spectrometer(Thermo Nicolet) was employed to obtain FTIR spectra of the fibers.Elemental analysis (EA) data of the original and functionalized fiberswere obtained using an ElementarVario EL instrument. X-ray photoelectronspectroscopy (XPS) was obtained using a VersaProbe spectrometer (modelPHI-5000). X-ray powder diffraction (XRD) patterns were recorded ona D/MAX-2500 X-ray diffractometer (Rigaku Corporation). The thermalstability of fibers was investigated using an STA409PC TGA/DSC simultaneousthermal analyzer . The mechanical propertiesof the different fiber samples were tested using an electronic singlefiber strength tester LLY-6 . The surface morphology of the fibers was observed using amicroscope . The pH values were determinedusing a pH meter (Model PHS-25). A TU-1901 dual-beam ultraviolet visiblespectrophotometer (Persee) was employed to determine the concentrationsof various phenol solutions. 4.2QASFs were prepared through a two-step reaction,as shown in N,N-dimethyl-1,3-propanediamine was graftedonto the PANFto obtain the aminated fiber (PANPF). Then, the fiber furtherreacted with halogenated hydrocarbon through quaternization to getdifferent quaternary ammonium salt-functionalized fibers PANQASFs. The detailed preparations of functional organic molecules (RBr)and functionalized fibers are described in the Supporting Information(Scheme S1).The quaternary ammonium salt-functionalizedfiber PANN,N-dimethyl-1,3-propanediamine (20 mL), and deionized water (10 mL)were added to a three-neck flask. The mixture was stirred and refluxedfor 4.5 h. After that, the functionalized fiber was filtered and washedwith hot water (60\u201370 \u00b0C) until neutral. The fiber wasdried overnight at 60 \u00b0C under vacuum to give PANpF (Scheme S2).Step 1: Dried PANF (1.0 g), PF (1.00 g), corresponding RBr (2 mmol),and ethanol (20 mL) were added to a three-neck flask. The mixturewas stirred and refluxed for 4.0 h. After the reaction, the functionalizedfiber was filtered and washed with ethanol in a soxhlet extractorto remove unreacted small molecules. The fiber was dried overnightat 60 \u00b0C under vacuum to give PANQAS-1F, PANQAS-2F, and PANnBQAS-Fs (Schemes S3 and S4).Step 2: Dried PAN4.34.3.1PF, PANQAS-1F, PANQAS-2F, and PANnBQAS-Fs) (15 mg) wereimmersed in 25 mL of 4-nitrophenol (4-NP)  and stirred for 12 h. The concentrations of 4-NP beforeand after adsorption were determined by UV\u2013vis spectroscopy.Dried functionalized fibers (PAN4.3.2BQAS-3F (15 mg) was immersed in 40 mLof a 2,4-DNP solution  and thenthe mixture was stirred at required time and temperature. For kineticsresearch, after being stirred for a certain time at room temperature,the fiber was filtered and the remaining concentration of 2,4-DNPwas measured by UV\u2013vis spectroscopy. Similarly, the isothermand thermodynamic experiments were carried out at different initialconcentrations (25\u2013400 mg L\u20131) and temperatures, respectively.Dried PAN4.3.3BQAS-3F (0.25 g) was filled into a cylindricaltube with a length of 100 mm and a diameter of 5.7 mm, and then thesolution of 2,4-DNP with an initial concentration of 200 mg L\u20131 and pH = 6 was pumped through the cylindrical tubewith the functionalized fiber at a flow rate of 2 mL min\u20131 using a peristaltic pump, and the liquid was collected. The concentrationof 2,4-DNP in the effluent was determined by UV\u2013vis spectroscopy.DriedPAN4.3.4BQAS-3F(0.20 g) was filled into a cylindrical tube with a length of 100mm and a diameter of 5.7 mm, and then a 2,4-DNP solution  was pumped through the cylindrical tubecontaining the functionalized fiber at a flow rate of 2 mL min\u20131, and the effluent was collected. The absorbance of2,4-DNP in the effluent was determined by UV\u2013vis spectroscopy.Adsorption: First, dried PAN\u20131) wasused as an eluent and pumped through a cylindrical tube containingthe adsorbed functionalized fiber at a flow rate of 2 mL min\u20131. The liquid was collected in a receiving bottle at the same timeinterval, and the desorption ratio of the fiber at different timeswas studied. The concentration of 2,4-DNP in the effluent was determinedby UV\u2013vis spectroscopy. Such adsorption/desorption recyclingis used to determine the reuse performance of the functionalized fiber.Desorption: A NaBr solution (0.1 mmol L"}
+{"text": "Primary CNS tumors are rare. Coexistence of two glial tumors of different histological origins in the same patient is even rarer. Here we describe two unique cases of coexisting distinct glial tumors in opposite hemispheres.Patient 1 is a 38-year-old male who presented with a seizure in February/2016. MRI showed a left parietal and a right frontal infiltrating nonenhancing lesions. Both lesions were resected revealing an oligodendroglioma WHO grade-2 and an astrocytoma WHO grade-2. Patient 2 is a 34-year-old male who presented with a seizure in November/2021. MRI showed a left frontal and a right mesial temporal lobe infiltrating nonenhancing lesions. Both lesions were resected revealing an oligodendroglioma WHO grade-2 and a diffuse low-grade glioma, MAPK pathway-altered (BRAF V600E-mutant). Patient 1 underwent adjuvant treatment. Both patients are without recurrence to date.Two histologically distinct glial tumors may coexist, especially when they are non-contiguous. Pathological confirmation of each lesion is imperative for appropriate management. We highlight the different management of gliomas based on the new CNS WHO 2021 classification compared to its 2016 version, based on NCCN guidelines. Although more molecular markers are being incorporated into glioma classification, their clinical impact of it is yet to be determined. Primary Central Nervous System (CNS) tumors are rare and account for about 2% of all cancers with an overall incidence of 22 per 100,000 population \u201329. HereA 38-year-old male presented to the ED after experiencing a new onset GTC seizure in February 2018. MRI brain was eventually obtained revealing two infiltrating, expansile, and non-contiguous T2-FLAIR hyperintense lesions on the left parietal and right frontal lobes. Faint enhancement was seen in the posterior aspect of the left temporal lesion Figure 1He underwent a craniotomy and GTR of the left parietal lesion on 6/2018. Pathology revealed oligodendroglioma, WHO grade 2, IDH-mutant, 1p/19q-codeleted. One month later, he underwent a second craniotomy and GTR of the right frontal lesion. Pathology revealed an astrocytoma, WHO grade 2, IDH-mutant, ATRX-mutant, and 1p/19q-intact Figure\u00a02The patient did well postoperatively and remains seizure-free while on antiepileptic monotherapy. He developed chronic right homonymous hemianopsia but is otherwise cognitively and physically intact. The decision was made to treat the astrocytic right frontal lesion with intensity-modulated radiation therapy (IMRT)  followed by temozolomide for a total of 12 cycles to treat both pathologies. He is currently doing well without recurrence or worsening neurological symptoms at his last follow-up on 1/11/2022. He continues to work three jobs and is excited about getting married in February.A 34-year-old right-handed male presented to the emergency department (ED) in September 2021 after new-onset episodes of expressive aphasia, each lasting less than 2 minutes. While in the ED, he developed two generalized tonic-clonic (GTC) seizures. MRI brain demonstrated two expansile, non-enhancing, and non-contiguous T2-FLAIR hyperintense lesions on the left frontal and right mesial temporal lobes Figure 3He underwent an uncomplicated awake craniotomy and gross total resection (GTR) of the left frontal lesion with intraoperative MRI. Pathology was consistent with oligodendroglioma, WHO grade 2, IDH-mutant, 1p/19q-codeleted.After he recovered from the first surgery, he underwent a right temporal craniotomy and subtotal resection of the contralateral lesion on the right mesial temporal lobe. Morphologic and molecular findings from this specimen were most suggestive of diffuse low-grade glioma, MAPK pathway altered as the tumor showed evidence of BRAF mutation (V600E). The tumor is notably IDH wildtype, H3 wildtype, 1p/19q not co-deleted, and no evidence of CDKN2A deletion, indicating a favorable prognosis Figure\u00a04The patient did well after both surgeries and remained seizure-free while on monotherapy antiepileptic. He remained both clinically and radiographically stable at his 6-month follow-up visit. He is currently doing well without recurrence; the most recent follow-up was on 6/7/2022. He works full-time as an engineer but has transitioned to an office job as he had trouble with extreme temperatures. He feels back to his baseline with infrequent memory problems.Here we describe two unique cases of co-existing glial tumors from distinct cellular lineages on opposite brain hemispheres. Both patients initially presented following new-onset seizures. They both underwent two separate craniotomies for maximal safe resection of each of the lesions. Both patients had coexistence of oligodendroglial and an astrocytic tumor, one in each hemisphere.To our knowledge, this is the first report of co-existing glial tumors from distinct histologic and molecule profiles on opposite brain hemispheres. It is critical to recognize this as part of the differential diagnosis when evaluating patients with two discrete, non-contiguous lesions, as historically  these may have been misdiagnosed as gliomatosis and/or multifocal pathologies. A literature review conducted by Tunthanathip et\u00a0al. identified 6 cases of coexisting intracranial tumors and summarized the author\u2019s experiences. They then identified 65 similar cases described in the literature. They noted that the most common association was a meningioma and pituitary adenoma, and that meningioma was present in most of their reported cases . AdditioThe strikingly different mutation and methylation profiles observed in the two tumors in each case 4 suggesAnother important point is the impact of the new 2021WHO classifications on management when compared to the 2016 WHO classification , 34. PriThis manuscript describes two rare cases of coexisting glial tumors in opposite brain hemispheres, and it illustrates the differences in glioma classification comparing the 2021 WHO classification to its previous 2016 version. It also highlights the difference in the management of brain tumors based on an integrated diagnosis strategy as recommended by NCCN guidelines.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.FP and MK contributed to the conception and design of the case report. IS wrote the first draft of the manuscript. FP, MK, and IS wrote sections of the manuscript. DC provided histologic imaging. MS contributed to the acquisition of the data, its analysis, and interpretation. All authors contributed to the manuscript revision, read, and approved the submitted version.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": "Both ideal tensile strength (ITS) and shear strength (ISS) are computed by applying homogeneous strain increments along high-symmetry directions  and low index shear planes ((100), (010), and (001)) of the orthorhombic lattice. We show that the ultimate mechanical properties of OEN are highly anisotropic during tensile loading, with ITS ranging from 4.5 GPa along [001] to 8.7 GPa along [100], and quite isotropic during the shear loading with ISS ranging from 7.4 to 8.9 GPa. During tensile test along [100] and [001], a modified structure close to OEN has been found. This modified structure is more stable than OEN under stress (or strain). We have characterized its elastic and ultimate properties under tensile loading. With ITS ranging from 7.6 GPa along [010] to 25.6 GPa along [001], this modified structure appears to be very anisotropic with exceptional strength along [001].The ultimate mechanical properties of MgSiOThe online version contains supplementary material available at 10.1007/s00269-022-01206-5. Only calculations at the atomic scale can predict the ultimate properties of these solids.The mechanical properties of crystals are usually controlled by defects. In the brittle field microcracks, pores, inclusions control toughness. In the ductile field, flow results from the motion of vacancies, dislocations, twins, disclinations, disconnections, etc. However, in some circumstances, these mechanisms cannot be activated and the mechanical properties of solids are ultimately controlled by the resistance of bonds. This is the case when plastic deformation mechanisms are inhibited by temperature being too low, or by kinetics reasons at high strain rates. The first approach to this problem was formulated by Frenkel  who pred3 is one of the most abundant planetary materials, present in planetary mantles, but also in interplanetary dust particles SiO3 orthoenstatite. From first-principles calculations, the ideal tensile strengths (ITS) and ideal shear strengths (ISS) are computed along high-symmetry directions [100], [010], and [001] and for homogeneous shear on (100), (010), and (001) planes .In this study, we investigate the elastic properties and mechanical stability of iron-free MgSiO3) unit cell.We performed the derivation of the anisotropic ideal strengths of OEN using the ADAIS free software, written by Zhang et al. . The ADA3 OEN, we performed tensile and shear tests by applying a homogenous strain incrementally by step of 0.5%. In practice, crystal atomic layers are moved along the tensile or shear directions  layers  is optimized. The ground-state properties, equilibrium lattice parameters, and elastic constants are given in Tables Tensile tests are performed along the [100], [010], and [001] directions. The evolution of the total strain energy as a function of the engineering strain is shown in Fig.\u00a0For both [100] and [001] tensile tests, we observe a discontinuity in the energy curves near 5% strain. However, for the test along [001], after this first discontinuity, the energy curve increases again until it bends at energy values much higher than those reached in the other two directions. The evolution of the energy for the [100] solicitation presents the same feature followed by a monotonous increase until a second energy drop, but of a larger amplitude. The test along [010] shows only one energy drop at 12%, after which the energy decreases continuously with strain, suggesting that above 12% strain, the OEN structure is no longer stable.The Cauchy stress can be obtained from the derivation of the energy  than the one of OEN which is the stable polymorph of MgSiO3 under these conditions. However, above 5% tensile strain along [001] and 8% strain along [100], this modified structure is more stable as shown by the energy drop  layers. However, the octahedron on Mg2 site of the modified structure is less distorted than the one of OEN, since, due to the exchange of two O3B oxygens, the bond lengths of both new O3B and O3A are equivalent and close to 2.35 . Normalized ITS are comparable  for [100], [010], and [001] Table . AlthougTo complete the ideal properties of OEN, we also performed simple shear deformation tests. Six additional simple shear deformation tests have been performed in this study. The evolutions of stress as a function of the engineering strain are shown in Fig.\u00a0In contrast to the previous calculations, more specifically the tensile tests along [100] and [001], we have not seen any formation of the modified structure during the shear tests.The initial slope of the stress strain curves is consistent with the elastic properties of OEN and we observe that the enstatite structure is maintained until a critical strain, associated with generally the maximum stress value. Ideal shear stresses (ISS) range from 7.39 GPa to 8.93 GPa Table  and normOverall, the stability of the structure mainly depends on Mg\u2013O bonds. Indeed, only the stress\u2013strain curve corresponding to the simple shear test along [100](001) shows a small discontinuity at 6% strain Fig.\u00a0b. HoweveFor the other shear tests, two behaviors are observed: one with a small hardening Fig.\u00a0c behavioThe jumps after 10% of strain on each stress\u2013strain curves Fig.\u00a0 are due In this work, the main objective is to investigate the mechanical response of OEN to applied strains until it becomes mechanically unstable. This point is reached after the linear elastic regime, which is exhibited by a parabolic regime in all energy\u2013strain curves. In consequences, in all stress\u2013strain curves, a linear regime is observed. This allows us to determine elastic moduli which, with the calculated lattice parameters, validate our calculations.OEN is quite isotropic under shear loading with ISS value normalized by the shear modulus close to 0.12. The structural analysis shows that the instability occurs when two Mg1\u2013O and Mg2\u2013O bonds are broken, in particular the bond of one Mg with an O3.During tensile tests, the first stress\u2013strain curve maximums correspond to the ITS of OEN, which are 8.7, 7.6, and 4.5 GPa along the [100], [010], and [001] directions, respectively. The ITS normalized by the Young\u2019s modulus are close to 0.05. During tensile loading along the shortest and the longest lattice parameters, instability appears rapidly during the test (close to 5% of strain). It is important to note that the instability occurs when the system undergoes structural modifications, like broken bonds, leading to structural reconstruction . Consequently, two O3B are exchanged with the Mg in site 2. This oxygen-bond exchange leads to the modified structure. We have characterized some properties of the modified structure by computing the elastic constants, ITS, and the powder X-ray diffraction pattern.This modified structure becomes more resistant against deformation, with two larger ITS values. Along the [100] direction, the strain reaches 10.5% before the instability occurs, with the ITS value being 1.7 times larger than the ITS value of OEN. Along the [001] direction, the instability is reached at 23.5% of strain, which corresponds to an ITS value 5.7 times larger than the ITS value of OEN. Along the [010] direction, the two structures exhibit the same ITS value with comparable ultimate strains (9.5\u201311%).After the case of cementite described by Jiang and Srinivasan , this stSupplementary file1 (DOCX 1654 KB)Below is the link to the electronic supplementary material."}
+{"text": "Study findings produce a cumulative understanding that biomedical HIV discourses and practices ignore structural violence embedded in Canada\u2019s social fabric, including legislation, policies and institutional practices that produce inequities and shape the social world of Black communities. Findings show that inequities in structural and social determinants of health such as food insecurity, financial and housing instability, homelessness, precarious immigration status, stigma, racial discrimination, anti-Black racism, criminalization of HIV non-disclosure, health systems barriers and privacy concerns intersect to constrain engagement and retention in HIV healthcare and ART adherence, contributing to the uncertainty of achieving and maintaining undetectability and violating their right to health. Biomedical discourses and practices, and inequities reduce Black people to a stigmatized, pathologized, and impoverished detectable viral underclass. Black people perceived as nonadherent to ART and maintain detectable viral loads are considered \u201cbad\u201d patients while privileged individuals who achieve undetectability are considered \u201cgood\u201d patients. An effective response to ending HIV/AIDS requires implementing policies and institutional practices that address inequities in structural and social determinants of health among ACB people.Biomedical advances in healthcare and antiretroviral treatment or therapy (ART) have transformed HIV/AIDS from a death sentence to a manageable chronic disease. Studies demonstrate that people living with HIV who adhere to antiretroviral therapy can achieve viral suppression or undetectability, which is fundamental for optimizing health outcomes, decreasing HIV-related mortality and morbidity, and preventing HIV transmission. African, Caribbean, and Black (ACB) communities in Canada remain structurally disadvantaged and bear a disproportionate burden of HIV despite biomedical advancements in HIV treatment and prevention. This institutional ethnography orients to the concept of \u2018structural violence\u2019 to illuminate how inequities shape the daily experiences of ACB people living with HIV across the HIV care cascade. We conducted textual analysis and in-depth interviews with ACB people living with HIV ( There are so many other barriers before we [Black people] get there [undetectable levels], and we cannot leave anyone behind.\u201d .This study is part of a larger institutional ethnographic project that explored how social relations shape the daily experiences of African, Caribbean, and Black people living with HIV across the HIV healthcare and treatment cascade , 2. BlacAchieving and maintaining an undetectable viral load for people aware of their HIV-positive status requires consistent ART adherence \u201312. At tDespite attentiveness to biomedical advancements and global initiatives aimed towards ending HIV, research focusing on the HIV care cascade has shown that inequities manifest through structural and social determinants of health (SDOH) \u201321 to neStructural violence is a concept for understanding the material harm, social injustices and\u00a0vulnerabilities to increased risk for diseases, suffering, lack of access to care, and poor health outcomes caused by broader social, political, economic and legal forces that determine people\u2019s social positioning and shape their social world . IntroduHealth scholars define structural violence as \u201ca host of offensives against human dignity, including extreme and relative poverty, social inequalities ranging from racism to gender inequality, and the more spectacular forms of violence that are uncontested human rights abuses\u201d . StructuMedical anthropologists and sociologists have argued that epidemics such as HIV and related consequences result from structural processes, forces, and forms of violence that interface to shape and constrain the agency of individuals \u201340. FarmGenerally, investigation of structural oppression and violence is rare. Most of the health research in Canada and globally focuses on individuals. More specifically, individuals are seen as the source of problems. In HIV/AIDS response, the impact of structural violence on people\u2019s ability to reach undetectability is rarely investigated because linkage, access, engagement, retention, and adherence to HIV healthcare and treatment are perceived as an individual\u2019s responsibility. HIV discourses, research, and healthcare\u00a0practices primarily focus on individuals\u2019 knowledge about HIV, lifestyle risk factors, behaviours and actions, and acute clinical care while largely ignoring structural factors constraining health practices among Black people , 42\u201344. This institutional ethnography (IE) study orients to the concept of structural violence to broaden the thinking and practices of HIV healthcare and treatment to establish a stronger relationship between individuals\u2019 material conditions and disease, inequities, SDOH, and human rights . The stuDeveloped by Dorothy E. Smith, IE is a \u201csociology for the people\u201d . The ontn\u2009=\u200920) and health workers (n\u2009=\u200915) involved in HIV healthcare delivery. Data collection began immediately upon receiving research ethics approval. All participants reviewed study information and provided informed consent to participate in the study. In some instances, pseudonyms were used to protect Black participants\u2019 privacy and professional roles to reference health workers.This article presents data collected between May 2019 and October 2020 from ACB people living with HIV  financial instability, b) immigration status, homelessness and housing instability, c) food insecurity, d) health systems barriers, e) stigma and discrimination, f) systemic and anti-Black racism, g) comorbidities and h) criminalization of HIV non-disclosure.In addition to HIV, Black participants reported that they lived with one or more comorbidities such as ulcers, diabetes, high blood pressure, high cholesterol level, and mental health issues, including depression, anxiety, stress, and trauma. Participants associated their experiences of mental health with history of trauma from political conflicts and colonialism in home countries, social situations such as substance use and addiction, and the challenges and violence they face navigating discriminatory systems including housing, employment, immigration, healthcare, and welfare. Key informants and healthcare providers reported that health system barriers such as the lack of comprehensive healthcare and drug coverage and culturally responsive services, patient-provider power dynamics and inadequate time constrain Black people\u2019s efforts to manage comorbidities and effectively achieve optimal health. Provincial public health insurance plan does not provide comprehensive coverage for all healthcare services.Time is an issue because you might have many questions and many things you want to discuss with your doctor. However, the doctor\u2019s rule is to bring one issue at a time, do not bring many issues. If I do not talk about all the other issues affecting me, then the doctor cannot pick out the cause of my condition. If they have more time with you to explore and listen to your narrative, they should be able to pick up [that] this headache is not just a migraine, there is something [else]. The person could be stressed because the child is not doing well in school or following up on their refugee papers. It might not have anything to do with HIV.When asked to reflect on healthcare delivery, Black people living with HIV emphasized the importance of \u201ctime\u201d. There was expectation that healthcare providers would allocate adequate time to consult about Black patients\u2019 overall health and subjective realities. However, Black people reported receiving suboptimal clinical care because the time allocated for consultation is often too short to adequately address their healthcare needs. The average time participants reported spending with their doctors was between 15 and 20\u2009minutes. Francis explained that the physician was often in a hurry to \u201cget him out of the door,\u201d even after waiting for a long time before seeing the doctor. Despite experiencing multiple health issues or concerns, participants expressed that they were subjected to \u201cone issue per visit\u201d by their healthcare providers. Eunice expressed:Healthcare providers reported that they see so many patients that they find it challenging allocating patients the time they need to talk about their health issues. A social worker stationed at an HIV clinic noted that the short clinical time allocated \u201cis very challenging, and sometimes patients seep through the crack.\u201d Under the Ontario Insurance Plan (OHIP) schedule of Benefits and Fees, \u2018fee for service\u2019 is the model that doctors bill and generate revenue for their services. While physicians can address as many issues as they want, the \u2018fee for service\u2019 model permits them to bill only one issue per patient per visit.n\u2009=\u200917) who primarily identified as recent immigrants to Canada indicated that lack of knowledge of how the Canadian healthcare system is organized obstructed their ability to navigate and access HIV-related healthcare services. Participants failed to look for general practitioners to treat their underlying conditions because of lack of knowledge of the roles played by different categories of healthcare professionals. Participants with HIV specialists as their sole healthcare provider expressed that they did not receive comprehensive care, including treatment for underlying health conditions. They noted that HIV specialists lacked adequate knowledge and willingness to address other health conditions. The HIV specialists prioritize monitoring viral loads and administering ART over assessing other underlying conditions and factors impacting Black people\u2019s health. Further, many healthcare providers lacked cultural competency and interest to address all the health needs of Black people. The power imbalance between Black people and their doctors prevented them from self-advocating for more consultation time and culturally responsive care. Brenda, who was volunteering at a community healthcare centre and self-identified as a Black immigrant living with HIV, elaborated:Most of my clients are newcomers. They do not know the system [and] the difference between a primary healthcare provider who is the GP [general practitioner] and an HIV specialist. If you have a primary healthcare provider who is vast in other medical areas than HIV, they can pick different things. Some [clients] may just see an HIV specialist who might just look at HIV, but they do not explore other things. Most HIV specialists just think about CD4 count, is your viral load undetectable, and that is it! Most clients that I see do not even have the information to know how to advocate for themselves, or they might have, but there is that power dynamics of feeling that this is a doctor and me I am just a patient, especially if the doctor is white.Black participants living with HIV  and housing instability (n\u2009=\u200913). Those experiencing housing instability lived in emergency shelters, were precariously housed in shared accommodations, assisted living or transitional housings. Black people associated their precarious housing situations to financial instability, high rental costs, unemployment, and precarious immigration status. Homelessness and housing instability constrained ART adherence. Precariously housed participants feared that routinely taking medication in the presence of roommates and other shelter residents could lead to involuntary disclosure of their HIV status and subject them to stigma, discrimination, and violence. At the time of the interview, all the Black people we interviewed reported that they relied on Ontario Disability Support Program (ODSP) to access HIV medication and extended healthcare benefits, including housing, transport, special diet, dental, and eye care. The ODSP is a program under the Ontario Ministry of Community and Social Services (MCSS) that provides \u201clonger-term income support for people with disabilities\u201d [We were only given about six hundred [CAD 600]. We could not use more than $600 [on rent]. So, the only option was for the four of us to share accommodation. We have to share the master bedroom with two people. The other roommates are in two smaller rooms. What this means is that these people don\u2019t know my status [HIV]. I cannot put my meds anywhere. My meds go in my laptop bag. I cannot take them openly when my alarm goes on at 8\u2009am. I have to take my meds in the bathroom. If my roommate is in the bathroom, I will wait for him to come out. Sometimes I forget. I have an alarm that reminds me that I need to take my medication at 8\u2009am and then another one at 9\u2009am. However, if I miss taking it and I go to work, you see that dynamic?Black participants in this study experienced homelessness  living in shelters reported that strict shelter regulations and practices made it hard to adhere to ART. Emergency shelter residents were required to vacate night-time shelters early in the morning every day and roam around until shelters reopen during evening hours. Such routines made it challenging for most participants to take medications as prescribed. Client support and accountability policies that organize the work practices of caseworkers produced perverse effects. Shelter rules requiring residents to report their daily itineraries to caseworkers every morning interfered with their medical appointments\u2019 schedules. Additionally, caseworkers whose roles included risk assessment and monitoring their clients\u2019 activities were seen as insensitive, having little compassion, and ignoring their clients\u2019 privacy and confidentiality needs. Participants expressed that those caseworkers forced them into involuntarily disclosing their health conditions and reasons for medical visits. Fear of breaking shelter regulation and losing accommodation forced participants who had early morning appointments that required them to leave the shelter before their caseworkers reported to work to miss their clinical visit.Black participants  were prescribed ART that must be taken with food. Black people could not access or afford healthier and culturally responsive food options necessary to maintain a healthy lifestyle and accommodate their health conditions, dietary restrictions and ART prescription requirement because of inadequate income and competing priorities. Participants on ART prescribed to be taken with food reported missing their dosages because of lack of food. Catherine expressed:One of the setbacks for African and Caribbean people adhering to their medication is that some medication works better if they take it with food and water. However, many people from Africa Caribbean backgrounds are barely surviving or have meagre income. They cannot access food to help the body boost the immune system\u2026Most of the time, the price of good quality food was outside the means of people of Caribbean and African descent.Black people reported that food insecurity constrained their efforts to adhere to ART and medical recommendations. Healthcare providers advise people living with HIV, and especially those with other medical conditions such as diabetes or hypertension to eat certain foods to stay healthy. Participants . However, the process of establishing and maintaining eligibility for benefits subjected applicants or recipients of ODSP to punitive income and financial security assessments. Recipients of ODSP are required to report their income for assessment. Under Section 5 6. Howeve.When I decide to remain on ODSP, I know the drugs will be expensive. If you are sure of getting the drug every month, your health is guaranteed. You are sure you will live well. You are not paying for it [ART]. I might be working every month. I think I\u2019m strong [and] money is coming. What if I am unable to work? What happens? Will I be able to pay for my medication? I might not use what I have to pay rent. I do not know if I will even be able to save enough to pay rent.Black participants expressed feeling trapped within the welfare system because of these draconian and punitive policies and institutional practices. Noting that their health and survival were dependent on guaranteed access to ART and extended social benefits, participants expressed fear and worry over the possibility of losing their ODSP eligibility as a result of getting gainful employment. Participants feared they would be forced to channel all income made to paying for medication and have nothing left to cover basic needs like food, housing, and clothing. Indicating that Black people living with HIV are subjected to precarious employment conditions, minimum wage, and premature dismissal because of their lack of or limited education, anti-Black racism and disability, participants expressed that they could not afford medication and other basic needs. Thus, the need to continue accessing drug benefits becomes the primary justification for remaining on ODSP. Lucy, who expressed her desire to work but feared losing ODSP, articulated:Participants who decided to seek employment rather than remain on ODSP expressed that they had to discontinue ART because they could not afford to pay for treatment out of pocket. Doing informal jobs and working odd hours also contributed to participants\u2019 non-adherence and inability to stay in care. For these reasons, many participants called for universal access to drug coverage, irrespective of their employment status. A participant stated: \u201cthat is why I said that medication should be there for everybody. The medication [insurance] should be 100% sure [universal] that you are getting your medication every month, even when working.\u201dYou leave ODSP and look for a job. Somehow in the paperwork, they are going to find out that you have HIV. Questions like, \u201cwhat have you been doing in the last three months?\u201d \u201cI was on disability.\u201d \u201cCan you tell us what form of disability you have so that we can accommodate you?\u201d They will not tell you why exactly they want to know, but they will tell you, \u201cSo that we can accommodate you.\u201d Once you have been honest and sincere with them, the company\u2019s accountant has to key in the insurance costs. You become gullible because the drugs are expensive.Key informants and healthcare providers commonly noted that racial discrimination, systemic and anti-Black racism, prejudice, and stigma resulting from discriminatory and punitive policies produced inequities in accessing social determinants of health including gainful employment and healthcare, and subjected them to poverty, financial instability, and fear of losing healthcare benefits, leading to continued dependency on ODSP and feeling of being trapped in the welfare system. Participants expressed that HIV status and history of living on social welfare benefits added another layer of stigma and discrimination, denying them an opportunity to access gainful employment. Esther elaborated:n\u2009=\u20099) recounted how they were increasingly policed and surveilled within healthcare settings because of being Black and the perception that they were dangerous, aggressive, and a public threat. Charles, who was dealing with mental health issues at the time of the interview described his experiences within a mental health institution:I started having mental health issues around the same time when I was diagnosed with HIV. It\u2019s been difficult for me to access healthcare. If I am going through an anxiety episode, I feel that as a person of colour, I may be looked at as maybe aggressive, rude, or dangerous once I enter the healthcare system. I get sort of feedback based on how people react around me. You are sitting down and looking up and seeing like three or four security guards suddenly sitting there. I feel that it has to do with the colour of my skin. While the alarm [surveillance and policing] is this high, for a [person] with mental health issues like me, in my head, I start asking, why are all these happening? Is it just me? What is going on here?Black participants living with HIV (n\u2009=\u200911) reported that they avoided, delayed, or stopped accessing healthcare because of fear that their HIV-positive status could be flagged in the immigration process and risk losing their immigration status, or face deportation or criminal charges. Uncertainties and confusion around when, where, and how to disclose one\u2019s HIV status to healthcare professionals resulted in Black people experiencing prejudice, stigma, and threats of criminal charges. Rose explained how a dentist threatened her son and herself with a lawsuit for failing to disclose\u00a0their HIV status:They [dental staff] are not supposed to discriminate against us. They [staff] were wrong at first because they did not give us the form to fill before attending to us, and they have no right to do that. We fear it affecting our permanent residence application because we just got into the country. We do not want any issue with the police [or] with the courts. You know the fear is there. We want to be law-abiding citizens, and all our claims are accepted, and we get our permanent residence.Black participants and healthcare providers indicated these oppressive and violent experiences of stigma, racial discrimination and anti-Black racism negatively impacted Black people\u2019s mental health, attitude, and behaviours towards seeking healthcare, and decision to adhere to ART. Black participants with precarious immigration status  because there is no realistic possibility of transmission; shall not prosecute where the person has not maintained a suppressed viral load but used condoms or engaged only in oral sex or was taking treatment as prescribed unless other risk factors are present, because there is likely no realistic possibility of transmission in such cases; and shall prosecute using non-sexual criminal offences instead of sexual offences where this would better align with the individual\u2019s situation, such as cases where the individual\u2019s conduct was less blameworthy; and must take into account whether a person living with HIV has sought or received services from public health authorities, to determine whether it is in the public interest to pursue criminal charges.Black people reported that they did not discuss their sexual health practice, risk, and needs with their healthcare providers due to ongoing HIV criminalization. In Canada, the criminal law is currently used to charge and prosecute people living with HIV who allegedly expose their sexual partners to HIV, fail to disclose known HIV-positive status before consensual sexual contact, or transmit HIV sexually , 68. Thetion Act  directinThe law says you cannot be criminalized if you have an undetectable HIV viral load. However, there is already a caveat there. What if somebody decided you are detectable? What if all these other circumstances and life struggles make the viral load spark up? It can spike up. When you are sick or when you are unwell. So, when I have HIV, I am still a criminal. The law does not say I am not. So, when providing sexual history, I do not want to tell them. Some want to ask you about sexuality, and I do not want to tell them right because there are all these things. I still have an immigration case going on. I do not know who will have access to medical information. Already the refugee board has a lot of my medical information.Black people highlighted their vulnerability and fear of exposure to HIV non-disclosure criminal charges. Participants were concerned about the uncertainty of reaching and maintaining undetectability due to the above-discussed structural inequities that constrained their retention in care and adherence to ART. This uncertainty produced fear and negatively influenced Black people\u2019s attitude towards healthcare systems and services. Black people also reported a lack of engagement in care and poor ART adherence due to concerns that accessing healthcare services would lead to the\u00a0flaging of their HIV status within the immigration system, leading to the impairment of legal status and deportation applications. Fred articulated:This study used institutional ethnography to explicate how structural violence embedded within social institutions and policies constrain retention in care, adherence to ART, and the ability of Black people living with HIV to reach undetectability. Mendenhall and colleagues state that \u201chow we think about disease pathologies affects how we design policies and deliver care to those most affected by social and economic inequities\u201d . This stThis study established that structural violence embedded within social structures such as laws, policies and institutional practices, and manifest through systemic inequities constrain Black people\u2019s retention in care and ART adherence, and ability to achieve and maintain undetectability, optimal health outcomes, and quality of life , 81, leaFood insecurity emerged as a significant barrier to ART adherence for Black people in this study. Food insecurity occurs \u201cwhenever the availability of nutritionally adequate and safe foods or the ability to acquire acceptable foods in socially acceptable ways is limited or uncertain\u201d . Food inHomelessness and housing instability for Black people which is compounded by structural violence resulting from the unjust and discriminatory labour market and housing policies, and manifested through systemic and anti-Black racism impact retention in care, ART adherence, and achieving undetectability for Black people . PrecariBlack people living with HIV experienced an array of comorbidities, including high blood pressure, diabetes, gastric conditions, cardiovascular diseases, and elevated mental health conditions. Comorbidities were compounded by structural inequities, including stigma, racial discrimination, anti-Black racism, poverty, housing instability, unemployment, financial instability, punitive and unjust policies, and healthcare practices. Research examining synergistic epidemics or syndemics has illuminated how structural factors produce and perpetuate structural vulnerabilities, resulting in increased incidences of comorbidities and burden of HIV, and worsening clinical outcomes among people living with HIV , 95. DruTime emerged as a structural determinant of health. Time shaped and constrained the health work process of retention in healthcare and adherence to ART and the overall health of ACB people. The time ACB people spent seeking healthcare services and the time medical professionals allocate to healthcare delivery relative to time each individual need to receive quality care were barriers to retention in care. Cumulatively, long wait times result from the time patients spent finding primary healthcare providers and specialists, booking appointments, processing referrals, and making clinical visits. The long wait and short consultation times are associated with the fee-for-service payment model and policy where healthcare providers bill each time a patient visits or revisits . The feePrescription drugs are a critical component of HIV healthcare and response. Despite progressive scientific advancements in HIV response, lack of access to ART remains a major barrier to healthcare retention and adherence to ART for ACB people living with HIV. Canada is the only country in the world with a universal health care system that does not include universal coverage for prescription drugs . InsteadFindings show that ACB participants who were all on social welfare assistance programs including ODSP at the time of the study wanted to engage in gainful employment to supplement the limited social benefits, escape poverty, have adequate access to social determinants of health and achieve optimal quality of life. Despite the inadequacy of social welfare benefits and readiness of welfare recipients to work, strict welfare policies and institutional practices regulating eligibility for social welfare benefits constrain Black people\u2019s integration into the labour market, leaving them trapped within the system. According to social welfare legislative policies and directives, social welfare beneficiaries who fail to declare their income risk losing their benefits and being criminalized . The orgPunitive and exclusionary law, policies, and institutional practices at the intersection of healthcare, public health, immigration, criminal justice system that punish, charge, and criminalize people living with HIV, including for non-disclosure of their positive status and failure to reach or sustain undetectable viral load level adversely impact retention in care and ART adherence. New prosecutorial directives stating that the federal and Ontario governments will no longer be prosecuting people living with HIV who consistently maintain an undetectable viral load for a period of six months raise essential questions around equity and social justice, considering that HIV criminalization legal frameworks apply to people who are not engaged in care and have detectable viral loads . The lawBiomedical discourses and evidence on ART and undetectability have informed the development of several national and global initiatives focusing on ending HIV/AIDS. The global 90\u201390-90 targets of the Joint United Nations Programme on HIV/AIDS (UNAIDS) aim to have 90% of all people living with HIV know their status, 90% of those diagnosed receive ART, with the third target focusing on having 90% of those on treatment achieve viral suppression by 2030 . In 2016Structural violence resulting from unjust welfare, housing, immigration, labour market and healthcare policies, and institutional practices subject Black people into permanent positions of homelessness, housing instability, food insecurity, inability to afford healthcare-related costs including transportation, and dependency on the social welfare system, violating their fundamental human rights . The vioTo end HIV/AIDS by 2030, \u201cleaving no one behind,\u201d governments must accelerate efforts to \u201cachieve universal access to comprehensive HIV prevention programmes, treatment, care and support\u201d , 117. ImThe harmful effects of structural violence on Black people\u2019s lives call for integrating trauma-informed knowledge and care into healthcare programs, services, and policies. There is growing evidence that trauma-informed HIV healthcare interventions improve healthcare access and retention, ART adherence, viral load suppression, health outcomes, and quality of life \u2013120. TheEliminating structural forms of violence and advancing social justice is timely, given the documented disproportionate impact of the COVID-19 pandemic on Black and racialized communities, particularly those living with HIV . The synBlack communities in Ontario are structurally disadvantaged. Biomedical advances in HIV healthcare and prevention practices, policies, and programs that aim to achieve \u201czero\u201d HIV transmission are not equitably benefiting Black communities. Structural violence ingrained in healthcare and health-related systems, legislative frameworks, policies, and institutional practices has produced inequities in accessing social determinants of health for Black communities living with HIV, leading to poor retention in care, ART adherence, health outcomes, and difficulty achieving and maintaining undetectability. These structural forms of violence result in a violation of Black people\u2019s right to health. Therefore, the practices surrounding HIV healthcare and the concept of undetectability should not be conceptualized as an individual responsibility but rather as human rights and social justice issues. As such, turning the tide on the HIV epidemic can only happen if structural factors perpetuating violence among Black communities and other vulnerable populations are identified and eliminated. Understanding how structural violence is embedded in the social fabric of Canada and produce inequities allows for identifying forces that influence HIV healthcare practices and health outcomes beyond the local and individual levels. Hence, recognizing the structural barriers to retention in care, adherence to ART, and maintaining viral suppression as acts of structural violence is a pathway to advancing equitable policies and institutional practices that enhance HIV healthcare and prevention practices, leaving no one behind. Therefore, achieving global targets that aim to end HIV/AIDS by 2030 and improve health outcomes calls for reforming and eliminating laws, policies, and institutional practices that produce structural and social inequities, and reinforce structural violations of human rights to health."}
+{"text": "The remarkable and unique characteristics of polyglycerols (PG) have made them an attractive candidate for many applications in the biomedical and pharmaceutical fields. The presence of multiple hydroxy groups on the flexible polyether backbone not only enables the further modification of the PG structure but also makes the polymer highly water-soluble and results in excellent biocompatibility. In this review, the polymerization routes leading to PG with different architectures are discussed. Moreover, we discuss the role of these polymers in different biomedical applications such as drug delivery systems, protein conjugation, and surface modification. Since the introduction of \u201cmacromolecules\u201d in the 1920\u2032s by Hermann Staudinger, the field of chemistry has been tremendously transformed, and the resulting polymer science has revolutionized modern life in many different aspects, especially in life sciences and medicine. The chemical foundation of polymers has led to the design of an endless variety of polymeric structures with different physical and chemical properties ,4, and aIn this review, we highlight the most common synthetic routes for obtaining polyglycerol with different architectures and highlight their application as carriers for active ingredients in different biomedical and pharmaceutical applications. A comprehensive and detailed overview of different polymerization methods of epoxide monomers has already been published elsewhere .2 type monomer  or Br\u00f8nsted acids (CF3COOH or CF3SO3H) .,36.p-tolIn 1994 Taton et al., reported for the first time the successful polymerization of EEGE by applying cesium hydroxide as an initiator in bulk polymerization. The reaction resulted in poly(ethoxyethyl glycidyl ether) (PEEGE) with a molecular weight in the range of 30 kDa and a relatively broad dispersity (PDI: 1.5) .tert-Butoxide (t-BuOK) [+/t-BuP4) [n) of about 300 [Changing the initiator to potassium or cesium alkoxide resulted in the synthesis of polymers with narrow molecular weight distribution . Until n(t-BuOK) ,40, pota(t-BuOK) ,42, alko(t-BuOK) , potassi(t-BuOK) , and BuL/t-BuP4)  have beebout 300 ,46. M\u00f6llbout 300 . The proWith the increase in temperature and in higher monomer to initiator ratios, this transfer reaction was more noticeable. In order to obtain polyethers with high molecular weights, it is generally assumed that the nucleophilicity of the active propagating chain-end has to be high enough to utilize the ring-opening of the epoxide ring but have relatively low basicity to prevent proton transfer and side-reactions .2) as a catalyst and varying the temperature. The results showed control over the branching of the polyglycerol backbone by varying the temperature and creating different protein-glycidol bioconjugates as an alternative to pegylated biostructures [Harth et al. developed an alternative method to synthesize semi-branched polyglycidols to the traditional ionic polymerizations. They investigated the homopolymerization of glycidol by employing Tin(II) trifluoromothenesulfate (Sn(OTf)ructures .2) to form polyesters has been proven to be controlled and pseudoliving [The bulk polymerization of cyclic esters using a catalyst such as Tin(II) 2-ethylhexanoate (Sn(Oct)doliving . Additiodoliving . The sysdoliving . The drudoliving .w/w loading capacity) and ensure its educated delivery into the stratum corneum, viable epidermis, and upper epidermis when compared with Protopic\u00ae\u00ae (containing 0.03% w/w of TAC) [Another system developed by Zabihi et al., followed a similar strategy to synthesize poly(glycidol-lactide) up to 43 kDa . Further of TAC) . On the  of TAC) . The cat of TAC) . The sys of TAC) .To obtain high molecular weight LPG, coordination-type polymerizations using organometallic catalysts were also conducted using EEGE as a monomer ,54,55.Haout et al. prepared LPG with high molecular weights of about 1 kDa by applying diethylzinc and water as an initiating system. However, poor control over the molecular weight leads to higher dispersities (PDI = 1.46\u20131.80) .4NBr and triisobutylaluminium (i-Bu)3Al, Carlotti and Deffieux et al. reported a polymerization strategy, which was a breakthrough for the polymerization of many functional epoxides including EEEGE [i-Bu)3 Al are combined [iso-butyl groups can cause lower molecular weights and ill-defined chain-ends, for the preparation of high molecular weight polyethers with defined structures, this \u201cactivated monomer\u201d polymerization is a widely studied and applied method in comparison to the conventional AROP [In 2007, using a dual initiating system composed of tetraoctylammonium bromide (Oct)ng EEEGE . This stng EEEGE . This pocombined . Gervaiscombined . Althougnal AROP ,57,58. Inal AROP .HPG is characterized by its high water-solubility and end group tune-functionality. Hence, its derivatives can be designed and functionalized to be used as a drug delivery platform when modified to include biodegradability to avoid in vivo accumulation and release its cargo and hydrophobicity to encapsulate hydrophobic cargos as additional properties. Nevertheless, HPG can also be integrated into other supramolecular structures such as polymeric micelles or nanogels to form drug delivery systems. As HPG-based drug delivery systems have been intensively discussed in previous reviews , we woulZhong et al. developed amphiphilic block copolymers that included sulfated HPG as its hydrophilic part, attached with a disulfide-bearing linker to polycaprolactone as the hydrophobic part . The ampN-isopropylacrylamide\u2013dendritic polyglycerol NG that was semi-interpenetrated with 2-acrylamido-2-methylpropane sulfonic acid or (2-dimethylamino) ethyl methacrylate [Baabur-Cohen et al. developed two polymeric carriers that have different supramolecular assembly architectures for the combination drug therapy of paclitaxel (PTX) and doxorubicin (DOX) . The druacrylate . The tNGacrylate . Miceli acrylate . They shacrylate . The NGsacrylate .Hofmann et al. developed polyethylene glycol (PEG)-substituted liposomes by developing multi-functional lipids based on the AROP of protected glycidols (EEGE and isopropylidene glyceryl glycidyl ether (IGG)) initiated by cholesterol and 1,2-bis-n-alkyl glyceryl ether. Due to the multi-functionality of these liposomes, they can be further functionalized. The authors used rhodamine B as an example for further modification . For selLPG was recently thoroughly studied as an alternative to PEG for the conjugation of biopharmaceuticals due to its structural similarity to PEG. In the last three decades, PEG has been the \u201cgold standard\u201d for conjugation to biopharmaceuticals (a process known as PEGylation) to address their intrinsic shortcomings such as instability, immunogenicity , and a sN-terminally to Interleukin-4 [In a study conducted by Abu Lila et al. liposomes were modified with PEG and LPG. It was observed that replacing LPGylated liposomes enhances the in vivo performance in comparison to PEGylated counterparts, as LPG-modified liposomes did not induce accelerated blood clearance (ABC), a limitation PEGylated liposomes have upon repeated administration, which negatively affects their pharmaceutical activity . The endleukin-4 , and Analeukin-4  and to Ileukin-4 . These sViral diseases are one of the major threats to our health, which are associated with morbidity, mortality, and serious socioeconomic consequences . VaccinaA primary mechanism that several viruses have developed for binding and delivering the viral genome is initial multivalent binding to different heparan sulfate proteoglycans (HSPGs) at the host cell surface ,93,94. HTheir biocompatibility and multi-functionality, and a plethora of different obtainable structures, make polyglycerols a promising platform for designing novel multivalent virus inhibitors. Bhatia et al., investigated the role of the flexibility of the scaffold in the inhibition activity in a systematic comparison between functionalized LPG and HPG with similar molecular weights for inhibition of IAV . They ob50 values from 0.03 to 374 nM from the flexible backbone (LPGS) to less flexible ones (dendronized and HPGS) was observed. Knowing that all the polymers had the same density of negative charges, it was concluded that the more flexible the backbone, the more it can change conformation and shield the virus surface. To further evaluate the role of the scaffold\u2019s flexibility, Mohammadifar and Ahmadi et al. used HPGS to form 2D constructs by reversibly fixing HPG on a graphene sheet, crosslinking them together to form a 2D construct, separation from graphene sheet, followed by sulfation. This system was then compared with sulfated 3D NGs in viral infection inhibition. Due to higher flexibility, the 2D system outperformed the 3D system by showing four times better inhibition of HSV-1 and SARS-CoV-2 [Dey et al. made this observation for the inhibition of HSV-1 by NGs. They prepared two classes of NGs based on sulfated HPG (HPGS) and used LPG or HPG as cross-linkers to develop scaffolds with distinctive rigidities. They further proved the flexibility differences between F-NGs and R-NGs by atomic force microscopy (AFlinearM), in which the negatively charged NGs were coated on a positively charged mica substrate, and it was observed that the F- NG, which was made with LPG linker, exhibited a smaller height and higher width in comparison to R-NG cross-linked with HPG. It was additionally observed that flexible NG had increased antiviral activity due to better shielding of the virus interaction with the cell surface. Their mathematical modeling supported this data by showing that to sterically shield a virus particle, six rigid NG are needed while only three of the soft NG of the same size are needed . Pouyan RS-CoV-2 .To investigate other heparin-like characteristics of the HPGS, Ferraro and Silberreis et al. investigated the anti-inflammatory properties of this scaffold. They observed that HPGS has anti-inflammatory activity and can regulate the complement response as good as heparin , and muc11) and compared the surface characteristics such as cell fouling, protein fouling, and chemical stability with the MI-dPG surface modified with commercially available amine terminated PEG (HO-PEG-NH2). The quartz crystal microbalance measurements with dissipation monitoring (QCM-D) revealed that the post functionalized surfaces with LPG outperformed the PEG modified surfaces in protein antifouling properties. In a follow-up study, the authors investigated the applicability of these coatings in regard to reducing shear and biomaterial-induced thrombosis on continuous-flow ventricular assist devices [11 showed no cytotoxicity up until 5 mg.mL\u22121 on A549 cell lines. In another study attempting to develop antifouling surfaces based on PG, the authors reported a simple and solvent-free surface-initiated polymerization from MI-dPG-coated TiO2 (hydrophilic) and polydimethylsiloxane  [2 and PDMS led to a slight decrease in the cell number on the respective surface. However, after grafting dPG, a drastic decrease (>95%) in the cell number was observed for both cell lines and investigated surfaces. Their approach provided a successful strategy for developing a highly biocompatible but cell-repelling surface coating [Unspecific biofouling or nonspecific protein adsorption on surfaces present a serious problem in biomedical applications such as medical implants, biosensors, and surgical equipment . Due to  devices . They obophobic) . They pe coating . Figure Functional polymers are an indispensable tool in medicine and life sciences. PG, with different structures and properties such as water-solubility, biocompatibility, and multi-functionality, has become one of the most studied and applied polymers in these fields . In the PGs have been successfully applied in targeted drug delivery systems as carriers and micelles. Furthermore, moieties with redox or pH-sensitive groups have been successfully introduced to the backbone, making these systems degradable under biological conditions. Due to similar characteristics of LPG to the gold standard PEG, it has been conjugated to biomacromolecules such as proteins to increase their stability, solubility, and half-life.We believe that the characteristics, ease of synthesis, scalability, multi-functionality, and structural versatility of PGs and PG-based systems can make a significant impact on the development and application of these materials both in vitro and in vivo."}
+{"text": "When the measurements made by the experienced surgeon and the inexperienced surgeon were compared, there was no significant difference between panoramic radiography and cone beam computed tomography. In situations where bone measurements are required for deciding on all-on-4 or one of its configurations (M-4 and V-4), it was found that panoramic radiography gives significantly incorrect results compared to cone beam computed tomography (p<0.05). Cone beam computed tomography is more reliable than panoramic radiography and eliminates the margin of error in the planning of all-on-4 or its variations to be made by either an experienced or an inexperienced oral surgeon.This study is aimed at analyzing the difference between the measurements made according to certain anatomical signs of the maxillary jaw using panoramic radiography and cone beam computed tomography (CBCT) to decide whether to use all-on-4, M-4, or V-4 configuration to prevent complications caused by incorrect measurements during the presurgical planning stage of the placement of implants in the all-on-4 technique. A retrospective study was conducted with 50 patients with upper edentulous jaws suitable for the all-on-4 technique, who underwent preoperative panoramic radiography and cone beam computed tomography evaluation for dental implant surgery. The shortest vertical distances between anatomical structures were measured. Measurements were made independently by two oral and maxillofacial surgeons, one experienced and the other inexperienced. A statistically significant difference was found between the mean values according to gender ( Dental implants are regarded as the main treatment option in terms of rehabilitation of edentulous jaws because of their stable results and satisfactory success rates , 2. In tIn treatment planning, the constantly used imaging methods are panoramic radiography, intraoral radiography, computed tomography (CT), and cone beam computed tomography (CBCT) . From thEven though a substantial number of publications are available on the applications of CBCT and panoramic radiography in dental implantology, there is still debate regarding the ideal imaging method for presurgical implant planning .Therefore, the present study is aimed at analyzing the difference between the measurements made according to certain anatomical signs of the maxillary jaw using panoramic radiography and CBCT to decide whether to use all-on-4, M-4, or V-4 configuration to prevent complications caused by incorrect measurements during the presurgical planning stage of the placement of implants in the all-on-4 technique. It is also aimed at comparing the measurements of a senior (experienced) and a junior (inexperienced) oral surgeon to evaluate whether experience significantly affects making an accurate measurement.The first hypothesis in the current study is that there will be a notable difference between the CBCT and panoramic radiography measurements, and the second null hypothesis is that there will be a difference between the measurements of an experienced and an inexperienced oral surgeon in these measurements.The study protocol was approved by the Near East University Scientific Research Ethics Committee (project number NEU/2019/89-1304).A retrospective study was conducted in the Near East University Faculty of Dentistry Department of Oral and Maxillofacial Surgery with 50 patients . The mean age of the patients was 62: the minimum age was 22 and the maximum age was 92, with upper edentulous jaws suitable for the all-on-4 technique, who had preoperative panoramic radiography and CBCT assessment for dental implant surgery between September 2016 and August 2021.Subsumption criteria were panoramic radiography and CBCT images showing the maxillary edentulous region clearly. Images showing artifacts, geometric distortion, and indeterminate anatomical structures were excluded from the study data .CBCT images were captured using Sirona Orthophos SL\u00ae 3D  with 85\u2009kV voltage, 6\u2009mA current, 16 \u00d7 5\u2009cm scan area, and 14\u2009sec. Scan time also panoramic radiographs were taken using the Orthophos XG\u00ae  using 1.2 magnification, 60\u2009kV voltage, 4\u2009mA current, and exposure time of 14\u2009sec. All measurements were performed using the same software program Sirona Sidexis\u00ae v.4 .Distance between the right and left nasal floors Distance between the right/left lateral nasal wall and right/left maxillary sinus Distance between the bottom of the left and/or right nasal floor and the alveolar crest Distance between the right/left maxillary lateral incisor tooth region and the right/left maxillary first molar tooth region Distance between the right and left maxillary lateral incisor tooth region Panoramic images at 1\u2009:\u20091 magnification and on CBCT scans were viewed in the coronal and sagittal planes. The vertical distances between anatomical structures were measured as follows:The measurements were made, at the Near East University Faculty of Dentistry Department of Oral and Maxillofacial Radiology, independently on the same monitor and under equal examining conditions by two oral and maxillofacial surgeons, one experienced (more than 10 years in the field) and the other inexperienced (less than 5 years in the field). Measurements obtained from each patient were recorded in mm. All examinations and measurements were performed on a 60.5\u2009cm, 1920 \u00d7 1080 resolution, 23.8-inch color LCD  monitor , under subdued room lighting.t-test was used to compare normally distributed data according to paired groups. Analysis results were presented as mean \u00b1 standard deviation and median (minimum-maximum) for quantitative data. The significance level was taken as p < 0.050.Data were analyzed with IBM SPSS V23. Relevance to normal distribution was evaluated by the Kolmogorov-Smirnov test. One-way analysis of variance was used in the comparison of normally distributed data according to groups of three or more, and multiple comparisons were performed with the Tukey HSD test. An independent two-sample p = 0.045). The mean for women was 5.5, while the mean for men was 6.0. A statistically significant difference was found between the mean values of the lateral nasal wall edge\u2014maxillary sinus (left) according to gender (p = 0.003). The mean for women was 5.3, while the mean for men was 6.1. A statistically significant difference was found between the mean values of the lateral incisor region (left) and lateral incisor region (right) according to gender (p = 0.03). The mean for women was 16.7, while the mean for men was 17.7. A statistically significant difference was found between the mean values of the nasal floor and the alveolar crest (left) by gender (p = 0.021). While the average for women was 12.9, the average for men was 14.1. There was no statistically significant difference between the mean values of other variables by gender (p > 0.050). The mean values of the distances by gender in panoramic radiography and CBCT images are shown in After evaluating the eligibility criteria, the final sample consisted of 29 men (58%) and 21 women (42%) aged between 22 and 92 (mean 62 years). A statistically significant difference was found between the mean values of the lateral nasal wall and maxillary sinus (right) according to gender (p < 0.001). However, there was a difference between all measurement regions in panoramic radiography and CBCT measurements. The mean values of the measurements are presented in When the measurements made by the experienced surgeon and the inexperienced surgeon were compared, there was no significant difference between panoramic radiography and CBCT (p < 0.05) , it was found that panoramic radiography gives significantly incorrect results compared to CBCT ( < 0.05) .In the CBCT, according to the experienced surgeon, 12 out of a total of 50 cases were found to have a nasal floor\u2014alveolar crest (left) distance value of <10. So, 24% of all-on-4 cases should be done with M-4 and V-4 configurations. When looking at the anterior-posterior (A\u2013P) distance, 10% of all-on-4 cases were found to be suitable for the M-4 configuration and 14% for the V-4 configuration. In the panoramic radiography group evaluated by the experienced surgeon, only 3 cases were found <10. In other words, 9 cases were incorrectly measured.In the CBCT group evaluated by the inexperienced surgeon, 11 of a total of 50 cases were found to have a nasal floor\u2014alveolar crest (left) distance value of <10. Accordingly, 22% of all-on-4 cases should be done with the M-4 and V-4 configurations. When looking at the A\u2013P distance, 2% of all-on-4 cases were found to be suitable for the M-4 configuration, and 20% for the V-4 configuration. In the panoramic radiography group evaluated by the inexperienced surgeon only 3 cases were found <10. In other words, 8 cases were incorrectly measured.p > 0.050) in No statistically significant difference was found between the distributions of lateral incisor region\u2014first molar region (right) according to the nasal floor\u2014alveolar crest (right) condition in each group  distance value was <10. So, 26% of all-on-4 cases should be done with the M-4 and V-4 configurations. When looking at the A\u2013P distance, it was seen that 6% of all-on-4 cases were suitable for the M-4 configuration, and 20% for the V-4 configuration. In the experienced surgeon panoramic group, only 3 cases were found. In other words, 10 cases were incorrectly measured .In the inexperienced surgeon CBCT group, of the total 50 cases, 10 had a nasal floor\u2014alveolar crest (Right) distance value of <10. That is, 20% of all-on-4 cases should be done with the M-4 and V-4 configurations. When looking at the A\u2013P distance, 4% of all-on-4 cases were suitable for the M-4 configuration, and 16% for the V-4 configuration. In the inexperienced surgeon panoramic group, only 3 cases were found. In other words, 7 cases were incorrectly measured .Different imaging techniques are available in maxillofacial radiology. Intraoral radiographs, panoramic radiographs, and CBCT are the most favored techniques in dental implant surgeries.The 2-dimensional (2D) nature of intraoral radiographs can cause anatomical proposition and dimensional distortion . Isidor Panoramic radiographs enable a detailed 2D inspection of the jaws. The most important benefits of panoramic radiographs are a low radiation dose, comparatively lesser time of exposure, and clarity of examination . On the CBCT, which is another imaging method and provides 3-dimensional (3D) examination has been the choice of use in dental implant surgeries . CBCT isThe results of implant treatment have become quite expectable in recent years . HoweverOn the other hand, one of the main problems associated with dental implant surgeries is the recovery of patients with extremely atrophic maxilla . ClinicaJensen and Adams in their research stated that compared to the standard all-on-4 configuration, the M-4 method could have more mechanical benefits and it is the choice for clinicians to achieve implant durability without exposing patients to further surgeries . He suggWhen primary stability is of concern, all-on-4 different variations with tilted anterior implants can be considered beneficial because of the insertion of longer implants. Therefore, in this study, the standard all-on-4 treatment concept and its variations, which are intended to be applied to patients with severely atrophic maxilla, were compared in terms of applicability with panoramic radiography and CBCT between the inexperienced surgeon and the experienced surgeon. In the CBCT, according to the experienced surgeon, 12 out of a total of 50 cases were found to have a nasal floor\u2014alveolar crest (left) distance value of <10. So, 24% of all-on-4 cases should be done with M-4 and V-4 configurations. When looking at the A\u2013P distance, 10% of all-on-4 cases were found to be suitable for the M-4 configuration and 14% for the V-4 configuration. In the panoramic radiography group evaluated by the experienced surgeon, only 3 cases were found <10. In other words, 9 cases were incorrectly measured. In the experienced CBCT group, in 13 out of 50 cases, the nasal floor\u2014alveolar crest (right) distance value was <10. So, 26% of all-on-4 cases should be done with the M-4 and V-4 configurations. When looking at the A\u2013P distance, it was seen that 6% of all-on-4 cases were suitable for the M-4 configuration, and 20% for the V-4 configuration. In the experienced surgeon panoramic group, only 3 cases were found. In other words, 10 cases were incorrectly measured. In the CBCT group evaluated by the inexperienced surgeon, 11 of a total of 50 cases were found to have a nasal floor\u2014alveolar crest (left) distance value of <10. Accordingly, 22% of all-on-4 cases should be done with the M-4 and V-4 configurations. When looking at the A\u2013P distance, 2% of all-on-4 cases were found to be suitable for the M-4 configuration, and 20% for the V-4 configuration. In the panoramic radiography group evaluated by the inexperienced surgeon only 3 cases were found <10. In other words, 8 cases were incorrectly measured. In the inexperienced surgeon CBCT group, of the total 50 cases, 10 had a nasal floor\u2014alveolar crest (right) distance value of <10. That is, 20% of all-on-4 cases should be done with the M-4 and V-4 configurations. When looking at the A\u2013P distance, 4% of all-on-4 cases were suitable for the M-4 configuration, and 16% for the V-4 configuration. In the inexperienced surgeon panoramic group, only 3 cases were found. In other words, 7 cases were incorrectly measured.This shows that in the majority of cases, it was determined that it was more suitable for the V-4 concept due to the short A\u2013P distance. At the same time, it was seen that the measurements made by the inexperienced surgeon and the experienced surgeon on panoramic radiography were similar and the margin of error was higher than that of CBCT. That is, CBCT has a lower margin of error to measure the A\u2013P distance while planning all-on-4.In different research that supports our study, Tang et al. compared the magnification rate of CBCT and panoramic radiography in the assessment of various maxillofacial loci and stated that the distances calculated by panoramic radiography were closely correlated with those assessed by CBCT . In suchUsing of various imaging modalities for preimplant assessment has been analyzed in numerous researches. Kopecka et al. compared the use of CBCT and panoramic radiography in the evaluation of the bone height present for dental implant insertion surgeries . In postThis study's primary limitation was the number of the cases selected for the study. This limitation was due to the limited number of maxillary edentulous population in our university and we tried to present all scans using the same software. The cases were selected between September 2016 and August 2021. In addition, each measurement has been done once, and by making more measurements, we can obtain a detailed average for each region.Within the study's limitations, we can conclude that CBCT is more reliable than panoramic radiography and eliminates the margin of error in the planning of all-on-4 or its variations (M-4 and V-4) to be made by either an experienced or an inexperienced oral surgeon. Further studies should review the availability of computer-aided implant surgery with a surgical guideline based on CBCT."}
+{"text": "Overall, only the GLP-1RA add-ons demonstrated a comprehensive beneficial effect on all outcomes. Furthermore, our results corroborated intraclass differences among therapies. Given the limited evidence, we could not reach a conclusion about the optimal therapies regarding mortality and vascular outcomes. Conclusion: The results suggested a potential treatment hierarchy for clinicians and patients, with the GLP-1RA add-ons being most preferred based on their favorable efficacy and safety profiles; and provided a unified hierarchy of evidence for conducting country-specific cost-effectiveness analyses. Background: Multiple glucose-lowering drugs are available as add-ons to metformin for a second-line treatment for type 2 diabetes. However, no systematic and comparative data are available for them in China. We aimed to compare the effects of glucose-lowering drugs added to metformin in China. Methods: PubMed, Embase, Web of Science, CNKI, WanFang Data, and Chongqing VIP from 1 January 2000 until 31 December 2020 were systematically searched for randomized controlled trials comparing a glucose-lowering drug added to metformin with metformin in Chinese type 2 diabetes patients. Drug classes included sulfonylureas (SUs), glinides (NIDEs), thiazolidinediones (TZDs), \u03b1-glucosidase inhibitors (AGIs), dipeptidyl peptidase-4 inhibitors (DPP-4is), sodium-glucose cotransporter-2 inhibitors (SGLT2is), glucagon-like peptide-1 receptor agonists (GLP-1RAs), and insulins (INSs). Two reviewers independently screened studies, extracted data, and appraised the risk of bias. Results: 315 trials were included. In patients receiving metformin alone, the addition of NIDEs produced the greatest additional HbA1c reductions ; while INSs yielded both the largest additional FPG reductions  and 2 hPG reductions . INS add-ons also conferred the largest additional HDL-C increases , whereas AGI add-ons generated the greatest TC reductions . The greatest incremental SBP reductions  were evident with SGLT2i add-ons. GLP-1RA add-ons had the greatest BMI reductions (1.96 kg/m Type 2 diabetes imposes serious threats to national health and economy . China hIt is of value to choose rational drugs for individualized patients as add-ons to MET. The choice of which drug to add is based on drug-specific effects and patient factors ,3,4. TheEvidence-based therapeutic decision making requires comparisons of all relevant competing treatments . AlthougThis study followed the statement for Preferred Reporting Items for Systematic Reviews and Meta-Analysis  and was PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), WanFang Data, and Chongqing VIP were systematically searched for randomized controlled trials (RCTs) published between 1 January 2000 and 31 December 2020, which compared a glucose-lowering drug added to MET with MET in Chinese type 2 diabetes patients. Search strategies are detailed in 1) participants were Chinese adults with type 2 diabetes; (2) intervention was a glucose-lowering drug added to MET, whose usage and dosage met the recommendation of Chinese clinical guideline [3) comparison was a MET monotherapy or placebo added to MET, whose usage and dosage were the same as that in intervention; (4) background therapy was limited to lifestyle intervention; (5) primary outcome was hemoglobin Alc (HbA1c), and secondary outcomes were fasting plasma glucose (FPG), 2 h postprandial plasma glucose (2 hPG), body mass index (BMI), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), systolic blood pressure (SBP), hypoglycemia, renal function, lactic acidosis, mortality, or vascular outcomes; (6) study design was RCT; (7) study duration was \u226512 weeks; and (8) study was published in Chinese or English. Eligibility criteria are detailed in Studies were eligible if the  independently screened the titles and abstracts of retrieved records, and examined the full texts of potentially eligible records. The results were cross-checked by two reviewers (Y.G. and M.H.) with any discrepancies resolved by consensus.Two reviewers (S.G. and X.S.) independently extracted data using a structured form, and appraised the risk of bias of the trials using the Cochrane Collaboration\u2019s risk of bias assessment tool . The extI2 statistic to assess heterogeneity and considered it important when it was greater than 50% [Initially, a series of meta-analyses were conducted to assess the treatment effects of MET-based dual therapies vs. MET on each outcome. Then, using MET as a common comparator, the adjusted indirect treatment comparisons based on the Bucher method were used to compare the treatment effects of MET-based dual therapies with each other ,17. We cthan 50% . The subThe initial searches retrieved 35,245 records, of which 1735 full texts were assessed for eligibility. Finally, 315 trials were included ,334,335,A total of 315 trials  provided data on HbA1c. In patients receiving MET alone, the addition of a glucose-lowering drug generated significant additional HbA1c reductions. The greatest reductions were observed with NIDEs , followed by SUs  and GLP-1RAs ; while TZDs  conferred the smallest reductions. There were large differences in HbA1c reductions found within SUs, ranging from 0.86%  with glipizide to 1.70%  with gliclazide, and within DPP-4is, ranging from 0.76%  with vildagliptin to 1.35%  with alogliptin .Across all dual therapies, NIDEs+MET, SUs+MET, and GLP-1RAs+MET had significantly greater HbA1c reductions than TZDs+MET, ranging from 0.35%  with GLP-1RAs+MET to 0.49%  with NIDEs+MET. No significant difference was noted between other comparisons .A total of 311 trials  provided data on FPG. When used as add-ons to MET, all drug classes produced significantly incremental FPG reductions, among which INSs  yielded the largest reductions, followed by NIDEs  and GLP-1RAs . As a class, SGLT2is  conferred the smallest reductions, which were inferior to AGIs ; but there were large differences within AGIs, with a 1.33 mmol/L  reduction with voglibose vs. no significant reduction with miglitol. Considerable differences were also found within SUs, ranging from 1.01 mmol/L  with glipizide to 2.20 mmol/L  with gliquidone .Amongst all dual therapies, INSs+MET and GLP-1RAs+MET demonstrated significantly greater FPG reductions than DPP-4is+MET, TZDs+MET, AGIs+MET, and SGLT2is+MET, with reductions ranging from 0.23 mmol/L  with GLP-1RAs+MET vs. DPP-4is+MET to 0.71 mmol/L  with INSs+MET vs. SGLT2is+MET. In addition, NIDEs+MET were superior to AGIs+MET and SGLT2is+MET, whilst SUs+MET were superior to SGLT2is+MET .A total of 282 trials  provided data on 2 hPG. The addition of a glucose-lowering drug to MET reported significant additional 2 hPG reductions. The largest reductions were 2.52 mmol/L  evident with INSs, followed by 1.90 mmol/L  with NIDEs and 1.90 mmol/L  with SUs; while the smallest reductions were 1.44 mmol/L  induced by SGLT2is. There were considerable differences in 2 hPG reductions within AGIs, ranging from 1.27 mmol/L  with miglitol to 3.29 mmol/L  with voglibose; within SUs, ranging from 1.70 mmol/L  with gliclazide to 2.97 mmol/L  with gliquidone; and within DPP-4is, ranging from 1.20 mmol/L  with vildagliptin to 2.03 mmol/L  with sitagliptin .Across all dual therapies, the only significant differences noted were between INSs+MET and DPP-4is+MET, TZDs+MET, GLP-1RAs+MET, AGIs+MET, or SGLT2is+MET. INSs+MET were favored over these therapies, with 2 hPG reductions ranging from 0.76 mmol/L  vs. DPP-4is+MET to 1.08 mmol/L  vs. SGLT2is+MET .2  with DPP-4is to 1.96 kg/m2  with GLP-1RAs; while no significant changes were observed with other add-ons. Across individual drugs, all GLP-1RAs and SGLT2is significantly reduced BMI, with reductions ranging from 1.39 kg/m2  with dapagliflozin to 2.11 kg/m2  with exenatide. By contrast, large differences existed within five DPP-4is, as only sitagliptin and vildagliptin yielded significant BMI decreases, in the order of 1.66 kg/m2  and 0.82 kg/m2   provided data on BMI. As add-ons to MET, GLP-1RAs, AGIs, SGLT2is, and DPP-4is generated significantly additional BMI reductions, by a range of 1.19 kg/m6, 1.38) .2  with SGLT2is+MET vs. INSs+MET to 1.90 kg/m2  with GLP-1RAs+MET vs. TZDs+MET. Moreover, GLP-1RAs+MET were also favored over DPP-4is+MET, while DPP-4is+MET were favored over INSs+MET and SUs+MET  provided data on TC. With the exception of NIDEs and SUs (no data available), the addition of other drug classes to MET all yielded significantly incremental TC reductions. The greatest reductions were detected with AGIs , followed by GLP-1RAs  and DPP-4is . TZDs  conferred the smallest reductions, although there were big intraclass differences, with a 0.61 mmol/L  reduction with pioglitazone vs. no significant reduction with rosiglitazone. Large differences were also found within SGLT2is, with a 0.54 mmol/L  reduction with dapagliflozin vs. no significant reduction with empagliflozin. Notably, all GLP-1RAs significantly reduced TC with relatively small intraclass difference, in the order of 0.90 mmol/L  with liraglutide and 1.11 mmol/L  with exenatide .Among all dual therapies, AGIs+MET and GLP-1RAs+MET lowered TC to a significantly greater extent than SGLT2is+MET, INSs+MET, and TZDs+MET, with reductions ranging from 0.44 mmol/L  with GLP-1RAs+MET vs. SGLT2is+MET to 0.70 mmol/L  with AGIs+MET vs. TZDs+MET. Additionally, AGIs+MET were more efficacious than DPP-4is+MET, while DPP-4is+MET were more efficacious than TZDs+MET .A total of 54 trials (5272 patients) provided data on HDL-C. The addition of INSs, AGIs, DPP-4is, or GLP-1RAs to MET generated significantly incremental HDL-C increases, while other add-ons demonstrated no significant changes. The greatest increases were observed with INSs , followed by AGIs  and DPP-4is . The smallest increases were evident with GLP-1RAs , although big intraclass differences existed, with a 0.12 mmol/L  increase with liraglutide vs. no significant increase with exenatide. Within DPP-4is, there were also great differences, as only sitagliptin  and alogliptin  demonstrated significant HDL-C increases .Across all dual therapies, INSs+MET, AGIs+MET, and DPP-4is+MET had significantly greater HDL-C increases than GLP-1RAs+MET and SUs+MET, ranging from 0.21 mmol/L  with DPP-4is+MET vs. GLP-1RAs+MET to 0.55 mmol/L  with INSs+MET vs. SUs+MET. Besides, GLP-1RAs+MET and SGLT2is+MET worked better than SUs+MET .A total of 19 trials (1522 patients) provided data on SBP. As add-ons to MET, only SGLT2is  and GLP-1RAs  produced significantly additional SBP reductions, while no significant changes were observed with other add-ons. SGLT2is produced relatively larger reductions, in the order of 6.46 mmHg  with dapagliflozin and 6.61 mmHg  with empagliflozin. GLP-1RAs yielded smaller reductions, with a 5.77 mmHg  reduction with liraglutide vs. no significant reduction with exenatide. Of note, as a DPP-4i, sitagliptin also conferred a large SBP reduction of 12.12 mmHg  .Across all dual therapies, no significant difference on SBP effect between all comparisons was detected .A total of 204 trials  provided data on hypoglycemia. GLP-1RAs  and DPP-4is  as add-ons to MET reported significantly lower hypoglycemia risks than MET alone, while no significant changes were noted in other add-ons . AmongstA total of 27 trials (2621 patients) provided the data of DPP-4is , TZDs , AGIs , SUs , SGLT2is , INSs , and GLP-1RAs  as add-ons to MET on renal function. A total of 26 of them reported that no renal function impairment occurred during the trials, with the remaining one reporting that glipizide added to MET did not have a significant impact on renal function compared with MET alone. In addition, three trials provided data of sitagliptin, vildagliptin, and liraglutide added to MET on lactic acidosis, all of which reported that no lactic acidosis occurred during the trials.Only two trials provided the mortality data of sitagliptin, liraglutide, and insulin glargine as add-ons to MET, reporting that no death occurred during the trials. Seven trials reported vascular outcomes, but without subdividing and detailing the events. One trial on miglitol and one trial on gliclazide reported that no cardiovascular events occurred during the trials. By contrast, one trial found that the addition of acarbose to MET demonstrated a 0.18  times lower risk of vascular events than MET alone. Two trials reported that vildagliptin added to MET conferred a 0.16  times lower risk of vascular events than MET alone. Whereas, two trials demonstrated that the addition of rosiglitazone to MET did not have significant impact on vascular events .Sensitivity analyses did not significantly change the overall results .Ideally, the choice of a drug to add to MET requires a multi-factorial consideration that goes beyond glucose control, and encompasses a control of other risk factors and a beneficial effect on long-term outcomes. However, despite the wide use of MET-based dual therapies in China, conclusive evidence on mortality and vascular benefits is lacking. Given the limited data on long-term outcomes, we provided detailed comparative evidence on intermediate outcomes and common adverse events, which are cardiovascular risk factors and routinely monitored throughout diabetes care. Moreover, there are eight classes of add-on drugs with varying treatment effects, not only between drug classes but also between drugs of the same class. Thus, we reported efficacy and safety results across drug classes and individual drugs, and compared them against each other, to aid decision-makers in selecting therapy.Evidences suggest that intensive glycemic control can reduce the risks of diabetes-related vascular events among patients in the early stage of diabetes ,337. ThiObesity or overweight is common in type 2 diabetes patients, and weight is an important treatment-related outcome to patients ,342. RedDyslipidemia and hypertension are also common conditions coexisting with type 2 diabetes, which are routinely monitored and frequently addressed during treatment. A total of 72% of Chinese type 2 diabetes patients reported either dyslipidemia, hypertension, or both, and were 6 times more likely to develop cardiovascular diseases than those with diabetes alone . It is oA patient-centered treatment approach should not only optimize efficacy, but also minimize adverse events and have beneficial effect on long-term outcomes . HypoglyTo our knowledge, only a few systematic reviews were found to compare several drug classes, but they were not targeted at Chinese patients, did not cover all available drugs, and only included English-language studies ,349,351.Certain limitations should be acknowledged. First, systematic review and meta-analysis inevitably have inherent limitations, such as risk of the bias of trials, potential publication bias and clinical heterogeneity. Although methodological controversies of indirect comparison remained, the adjusted indirect treatment comparison based on Bucher method can partially take into account the baseline risk and other prognostic factors of participants in different trials, and maintain certain strengths of randomized allocation in the original trials when estimating effect size ,352. SecThe choice of a glucose-lowering drug added to MET should be tailored, depend on drug-specific effects and individualized patient characteristics and preferences. Our results provided a unified hierarchy of evidence for caring for type 2 diabetes and suggested a potential treatment hierarchy for clinicians and patients in China, with GLP-1RA add-ons being the most preferred based on their favorable combined efficacy and safety profiles; NIDEs or SUs suggested for patients whose HbA1c is far from the target; INSs or NIDEs suggested for patients whose FPG or 2 hPG is out of control; GLP-1RAs or AGIs suggested for patients for whom BMI or TC management is a priority; INSs or AGIs suggested for patients for whom HDL-C control is an emphasis; SGLT2is or GLP-1RAs suggested for patients having a need to lower SBP; and GLP-1RAs or DPP-4is suggested for patients wishing to minimize hypoglycemia. Additionally, our results corroborated the intraclass differences among therapies. Of course, these results may incorporate recent findings in cardiovascular outcomes in foreign populations.From an overall point of view, long-term treatment costs across therapies should be considered alongside their clinical outcomes, by means of country-specific cost-effectiveness analyses. This study also provided systematic and comparative clinical data for conducting cost-effectiveness studies, which will add cost evidence for the rational choice of therapies and improving the allocation of healthcare resources. In a future study, we intend to use these data to conduct a cost-effectiveness study of these therapies."}
+{"text": "Their chemical structures were elucidated by detailed analysis of their NMR spectra, HRESIMS and X-ray crystallographic data, and by comparison with literature data as well. The antibacterial and DPPH scavenging activities of compounds 1\u20136 were evaluated. Compounds 1\u20133 showed inhibitory activity against some of the tested bacteria whereas compounds 2 and 5 showed potent DPPH radical scavenging activities, which were better than that of the positive control butylated hydroxytoluene (BHT). This work is likely the first report on marine natural products of mussel-derived fungus living in cold seep environments.Five new aromatic polyketides, including a unique benzofuran derivative, talarominine A ( Gigantidas platifrons (deep-sea mussel) belongs to the subfamily Bathymodiolinae . It is the most typical macroinvertebrate in the global deep-sea cold seep environments \u2212 . Compound DMSO-d6,  displayeDMSO-d6,  revealed1 was further identified by detailed analysis of 1H-1H COSY and HMBC data  to C-6, C-7, and C-8, whereas a hydroxy group at C-9 was established by the HMBC correlations from the proton of OH-9 to C-8  to C-1\u2032, C-2\u2032, and C-3\u2032 and from CH3-4\u2032 (\u03b4H 2.04) to C-3\u2032, C-4\u2032, and C-5\u2032 (\u03b4C 153.7 (C-3\u2032), the locations of two hydroxy groups were designated at C-5\u2032 and C-3\u2032, respectively. The side chain was determined by the HMBC cross peaks from the proton of a methoxy group at \u03b4H 3.55  to C-1, from H2-3 and H2-2 to C-1, and by the COSY correlations from H2-3 to H2-2 (The structure of compound MBC data . HMBC co9 to C-8 . In addiand C-5\u2032 . Support to H2-2 . \u03b4C 154.7 (C-11), 149.8 (C-4), 116.9 (C-5), and 119.5 (C-6) and the HMBC cross peaks from H-2 to C-4 and from H-6\u2032 and H-3 to C-5 determined that the 1,2,3,5-tetrasubstituted benzene ring and the side chain were connected to a furan ring through C-5 and C-4, respectively  to C-5, C-7, C-8, and C-10 led to the designation of this proton as H-6  to C-2, C-3, and C-4 designated the location of the oxygenated methylene group at C-3. In addition, the chemical shifts of the carbon atoms of 4 at benzene ring have changed greatly when compared with that of compound 2, which was speculated to be caused by the different substitution patterns of the methoxy and hydroxy groups on the benzene ring. HMBC correlations from the proton of OCH3-8 (\u03b4H 3.74) to C-8 and the chemical shifts of \u03b4C 159.7 (C-7) verified the locations of the hydroxy and methoxyl groups. After the NMR data collection, we set 4 to single-crystal X-ray diffraction experiment and confirmed our deduction  to C-2 and C-10 designated this proton as H-3. In addition, chemical shifts of the carbonyl and several aromatic carbon atoms changed significantly, which indicated that the substituent on benzene ring has changed. HMBC correlations from OCH3-7 (\u03b4H 3.92) to C-7 and from OCH3-5 (\u03b4H 3.79) to C-5 verified the locations of the methoxy groups. The single-crystal X-ray diffraction experiment , Micrococcus luteus, Pseudomonas aeruginosa, Vibrio harveyi, and Vibrio vulnificus, with MIC values ranging from 32 to 64 \u03bcg/mL. Compound 2 showed certain activity against V. vulnificus with an MIC value of 32 \u03bcg/mL, whereas compound 3 exhibited inhibitory activities against MRSA and V. vulnificus. For both, the MIC values were 64 \u03bcg/mL. Compounds 4\u20136 showed no or weak activity to the tested strains (MIC > 64 \u03bcg/mL). All of the isolated compounds were tested for antibacterial activities against three humanic and 9 aquatic pathogenic bacteria. As shown in 1\u20136 were further evaluated for their 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The results . A structure activity relationship analysis of compounds 2\u20136 indicated that the hydroxy group at C-8 of compounds 2 and 5 was essential for their antioxidant property.Compounds  results  showed tOne-dimensional and 2D NMR date was acquired on a Bruker Avance 500 spectrometer . UV spectra were read from a PuXi TU-1810 UV-visible spectrophotometer . Mass spectra were recorded on an API QSTAR Pulsar 1 mass spectrometer . Analytical HPLC was performed using a SHIMADZU prominence HPLC system equipped with LC-20AT pump, SIL-20A automated sample injector, CTO-20AC colomn oven, and SPD-M20A diode array detector controlled by LCSolution software.Column chromatography (CC) was used with silica gel , Lobar LiChroprep RP-18 , and Sephadex LH-20 . Thin Layer Chromatography (TLC) was performed with silica gel GF254 precoated plates . A separation and purification experiment was carried out with distilled organic solvents.Talaromyces minioluteus CS-138 was isolated from the inner fresh tissue of the Gigantidas platifrons, which is a deep-sea mussel collected from the cold seep area of the south Sea of China in July 2018. After strain identification with the morphological character and ITS region sequence \u2212 .Talarominine A (2): yellow crystals; mp 207\u2013209 \u00b0C; UV (MeOH) \u03bbmax (log \u03b5) 261 (4.36), 348 (3.59); 1H and 13C NMR data, see m/z 237.0762 [M + H]+ .Talamin A (3): yellow powder; UV (MeOH) \u03bbmax (log \u03b5) 252 (4.22), 332 (3.55); 1H and 13C NMR data, see m/z 251.0912 [M + H]+ .Talamin B (4): yellow crystals; mp > 350 \u00b0C; UV (MeOH) \u03bbmax (log \u03b5) 228 (4.44), 260 (4.60), 326 (3.88); 1H and 13C NMR data, see m/z 275.0520 [M + Na]+ .Talamin C (5): light yellow crystals; mp 227\u2013229 \u00b0C; UV (MeOH) \u03bbmax (log \u03b5) 225 (3.37), 260 (3.55), 333 (2.86); 1H and 13C NMR data, see m/z 235.0614 [M \u2212 H]\u2212 .Talamin D (4 were collected on a Bruker Smart-1000 diffractometer equipped with a graphite-monochromatic Cu K\u03b1 radiation (\u03bb = 1.54178) \u00c5 at 296 (2) K, whereas those of compounds 2 and 5 were collected with a graphite-monochromatic Mo K\u03b1 radiation (\u03bb = 0.71073) \u00c5 at 293 (2) K and 298 (2) K, respectively. The data were corrected for absorption by using the program SADABS .4:Crystal data for compound  C12H12O6, F.W. = 252.22, Triclinic, space group P-1, unit cell dimensions a = 7.0563(10) \u00c5, b = 9.0035(13) \u00c5, c = 11.8983(17) \u00c5, V = 722.36(18) \u00c53, \u03b1 = 81.350(4)\u00b0, \u03b2 = 75.651(4)\u00b0, \u03b3 = 84.160(4)\u00b0, Z = 2, dcalcd = 1.160 mg/m3, crystal dimensions 0.180 \u00d7 0.160 \u00d7 0.150 mm, \u03bc = 0.806 mm\u20131, F(000) = 264. The 12,637 measurements yielded 2616 independent reflections after equivalent data were averaged. The final refinement gave R1 = 0.0872 and wR2 = 0.2001 [I > 2\u03c3(I)].5:Crystal data for compound  C12H12O5, F.W. = 236.22, monoclinic, space group C2/c, unit cell dimensions a = 21.326(2) \u00c5, b = 6.1455(8) \u00c5, c = 16.7254(19) \u00c5, V = 2182.7(5) \u00c53, \u03b1 = \u03b3 = 90.00\u00b0, \u03b2 = 95.304(2)\u00b0, Z = 8, dcalcd = 1.438 mg/m3, crystal dimensions 0.35 \u00d7 0.17 \u00d7 0.10 mm, \u03bc = 0.113 mm\u20131, F(000) = 992. The 4926 measurements yielded 1896 independent reflections after equivalent data were averaged. The final refinement gave R1 = 0.0934 and wR2 = 0.2083 [I > 2\u03c3(I)].Escherichia coli EMBLC-1, methicillin-resistant Staphylococcus aureus EMBLC-2, and Micrococcus luteus QDIO-3) and 9 aquatic bacteria  were carried out by the microplate assay with a microplate assay with three repetitions [Antibacterial activities against three human pathogens . Butylated hydroxytoluene (BHT) was used as positive control. All the measurements were performed in triplicate and each value was presented as the mean \u00b1 standard deviation. The ability to scavenge the DPPH was calculated according to the equation:The scavenging activity against DPPH radicals was carried out according to the method of Sharma with some modifications ,19. Certsample \u2212 Asample blank) \u00d7 100/(Acontrol \u2212 Ablank)Scavenging effect (%) = 100 \u2212 , were isolated and identified. Among them, compounds 2, 4, and 5 were further confirmed by single-crystal X-ray diffraction analysis. Compounds 1, 2, and 3 exhibited inhibitory activities against some human pathogenic and aquatic bacteria, with MIC values ranging from 32 to 64 \u03bcg/mL. Moreover, compounds 1, 2, and 5 exhibited potent DPPH radical scavenging activities, significantly better than that of the positive control BHT, possessing the potential to be developed as antioxidants.In summary, the secondary metabolites of"}
+{"text": "Autism spectrum disorder (ASD) is diagnosed based on socio-communicative difficulties, which are believed to result from deficits in mentalizing, mainly evidenced by alterations in recognizing and responding to the mental states of others. In recent years, efforts have been made to develop mentalization-based treatment (MBT) models for this population. These models focus on enhancing individuals\u2019 ability to understand and reflect on their own mental states, as well as those of others. However, MBT approaches for people with ASD are limited by their existing theoretical background, which lacks a strong foundation grounded in neuroscience-based evidence properly integrated with attachment, and mentalizing. These are crucial aspects for understanding psychological processes in autism, and as such, they play a pivotal role in shaping the development of tailored and effective therapeutic strategies for this specific population. In this paper we review evidence related to the neurobiological, interpersonal, and psychological dimensions of autism and their implications for mentalizing processes. We also review previous mentalization-based frameworks on the psychosis continuum to provide a comprehensive understanding of attachment, neurobiology, and mentalization domains in therapeutic approaches for autism. After presenting a synthesis of the literature, we offer a set of clinical strategies for the work with children with autism. Finally, we provide recommendations to advance the field towards more robust models that can serve as a basis for evidence-based therapeutic strategies. AutisIndividuals with ASD are often described as having difficulties in mentalizing - the ability to understand and think about other people\u2019s thoughts, feelings, and intentions \u201313. SpecTherapeutic approaches to address these difficulties have arisen from mentalization-based treatment (MBT). While MBT was initially developed for individuals with borderline personality disorder (BPD), its application has broadened significantly over time. Today, it is used to treat a variety of psychological conditions in different populations, demonstrating its versatility and effectiveness as a therapeutic approach .One of the MBT approach applications that might be interesting to look at when thinking about ASD is the development of MBT for individuals with psychosis , 20. AltConsidering this, we argue that interventions targeting mentalizing deficits could help alleviate psychological symptoms in individuals with ASD. MBT, with its focus on essential social cognitive processes that support social functioning, may particularly benefit individuals with ASD who struggle to identify and differentiate between different mental states in themselves and others. Furthermore, due to its emphasis on interpersonal relationships, MBT may assist individuals in managing socio-relational challenges.In the following sections, we will review the evidence of social functioning difficulties in ASD from both symptomatological and neurobiological perspectives to provide a comprehensive understanding of the dimensions of ASD and their implications for mentalizing processes. We will also examine evidence related to MBTs and how insights from neuroscience research can inform the development of targeted interventions. Finally, we will propose clinical strategies that can be incorporated into existing mentalization-based models to better address the needs of children with ASD and present potential directions for advancing research in this field.2From a clinical and neurocognitive perspective, behavioral difficulties in social functioning have been associated with autism from an early stage of development , 14, 24.joint attention   \u201313. Mentnd (ToM) \u201331. Thisnd (ToM) , 30, 32.nd (ToM) . AlteratStudies have identified reduced neural responses in key regions of the mentalization brain network, notably the temporoparietal junction (TPJ) and the medial prefrontal cortex (mPFC), during mentalization tasks \u201313. HoweThe social nature of the mentalizing ability underscores its relational component, prompting questions about the levels of complexity where this ability is required or involved. For instance, understanding scenarios involving false beliefs, as mentioned earlier, necessitates the capacity to think from another person\u2019s perspective in the context in which the observer (oneself) is involved. Notably, studies have described a specific phenomenon known as \u2018camouflage,\u2019 which is observed in both men and women with ASD who present high cognitive abilities. This phenomenon plays a significant role in compensating for communication difficulties and may contribute to the challenges of diagnosing ASD in womenU \u201353. IndeTaken together, this evidence highlights the relational dimension of social functioning, in terms of a diverse knitting of human relations that could entail emotions, satisfactions (or not) of needs, contexts, etc. Importantly, these relations can be experienced as reciprocal, synchronized, stable, i.e., trustworthy and secure, or unpredictable, ambivalent, non-reciprocal, and even threatening, i.e., unreliable and insecure, from a very early age. Therefore, if the social phenomenon is closely associated with how those interactions are mutually experienced, and autism is described as having social interferences, the question that naturally arises should be does autism impact the relationship between infants and their caregivers? If it does, how does autism impact those relationships?In the following section, we will delve into the topic of attachment development in autism. We will also examine the neuroscience perspective on attachment in autism to gain a more comprehensive understanding of the condition and its potential treatment with MBT.3Attachment theory has made significant contributions to the fields of psychology, psychopathology, education, and health in recent years. However, studying attachment patterns in relation to autism can be a complex undertaking.Historically, attachment has been assessed using the Strange Situation Test . This meThe literature on the topic of attachment in children with autism has produced mixed results. Some studies have found that these children have higher levels of insecure attachment compared to other groups. However, there have been inconsistencies in the analysis and evaluation methods used . Recent During the dyadic interaction between children and their attachment figures, a complex process of accommodation and coordination occurs at multiple levels, resulting in the emergence of self-reflecting awareness and socioemotional skills \u201374. ThisThe neuropeptide oxytocin is one of the biomarkers of attachment. According to research, oxytocin contributes to interpersonal bonding, parental care, trust establishment and social attachment in typical development , 87. In For example, an experimental trial on multiple-dose oxytocin treatment found a beneficial effect on repetitive behaviors and feelings of avoidance . It has The above reflects the complex nature of the oxytocin influence on social cognition. Further research is needed to fully understand oxytocin\u2019s mechanisms and potential therapeutic applications in autism.Based on the reviewed findings, it is important for an integrative psychotherapeutic model for individuals with autism to consider the child\u2019s characteristics, their impact on the bond with their caregiver, and the effects of having a diagnosis. How a caregiver perceives the mental state behind a child\u2019s behavior can affect how they respond, ultimately affecting the quality of their relationship.This highlights the vital link between attachment and mentalizing, which will be further examined in the following section, particularly concerning autism.4Studies indicate that a diagnosis of autism can have positive effects on the parent\u2013child relationship in certain cases. Parents report decreased negative evaluations of their child\u2019s behavior following diagnosis and individuals with autism report gaining valuable insights into their past experiences and being able to reframe their sense of self after receiving a diagnosis. This newfound understanding can serve as a protective factor in the relationship between parents and their child with autism .When it comes to self-identity and mentalizing, it\u2019s important to note that the camouflage mechanism can play a role in self-awareness. While many individuals may model their behavior based on societal expectations or influence from others, individuals with autism may use camouflage as a way to compensate and mask their true selves. This can require a significant amount of cognitive effort, leading to heightened stress responses, social overload, anxiety, depression, and even a negative impact on self-identity development [as noted by Hull et al. ].The evidence presented supports the significance of addressing the ability to understand and interpret the thoughts, feelings, and intentions of oneself and others, aligning with the core principles of mentalization-based therapy. In the following sections, we aim to incorporate these elements to propose a therapeutic model capable of helping transform one\u2019s and others\u2019 sense of self.5MBT is an evidence-based psychotherapy approach with the primary goal of improving individuals\u2019 capacity to comprehend their own thoughts and emotions, as well as those of others. This approach combines concepts from psychoanalysis with attachment theory and research on social cognition. MBT has demonstrated effectiveness in reducing symptoms, enhancing interpersonal skills, and ultimately, elevating overall quality of life. Originally, MBT was developed to foster mentalizing in individuals diagnosed with borderline personality disorder . Over tiThe framework of MBT is built on two key assumptions. First, it believes that the ability to understand mental states is developed through early attachment relationships and is closely intertwined with the development of the self. Second, MBT recognizes that disruptions in these early attachment relationships can hinder the growth of an individual\u2019s capacity for mentalization and the development of their self-structure.MBT mainly proposes a developmental model of the self, drawing from concepts in developmental psychology, attachment theory and psychoanalysis. However, as previously mentioned, when adapting MBT for individuals with ASD, it\u2019s crucial to establish a strong foundation rooted in neuroscience-based evidence while coherently incorporating attachment theory and mentalization processes. To address this challenge, the following sections will explore the therapeutic principles and applications of MBT, with a focus on the model for psychosis as a starting point. Additionally, we will delve into the current state of MBT in the context of autism.5.1The primary objective of MBT is to establish an intersubjective narrative construction space that fosters the development of mentalizing capacity, enabling individuals to effectively process emerging thoughts and feelings. This is achieved by establishing a patient-therapist relationship that evokes affective states and engages the patient in a reflective process. An essential component of MBT is the repair of mentalizing ruptures that may occur during therapy sessions.In psychosis, Debban\u00e9 et al.  have proBased upon these considerations about the MBT model in psychosis, and considering the difficulties described in mentalizing ability in autism, may the question arise as to whether it is possible to develop an autism MBT model that integrates neuroscience, attachment, and mentalizing evidence?5.2While there are distinct neurodevelopmental characteristics between autism and psychosis, social functioning challenges have also been observed in individuals with psychosis, particularly those diagnosed with Schizophrenia Spectrum Disorders (SSD) as a proxy for psychosis , 96, 97.Social cognition entails various cognitive processes that integrate different brain structures and networks , 34 and Unlike psychosis and schizophrenia, the distinction of ASD as a neurodevelopmental condition presents a significant opportunity to explore the challenges in understanding mental states. This difference can be crucial for developing a specialized MBT model tailored for autism. It is possible to consider that the pervasive feature of autism encompasses an early and primary interference with the ability to mentalize from the beginning. Thus, mentalization-based interventions may be beneficial for individuals on the autism spectrum who struggle to identify and distinguish between different mental states in themselves and others. This is because, through MBT, individuals can develop a more nuanced awareness of their thoughts, emotions, and motivations, leading to increased self-reflection and self-understanding. Moreover, considering that MBT recognizes the interactive nature of mentalizing and that mentalization deficits are associated with social dysfunction , 7, 99, 5.3Current interventions to improve social and mentalizing abilities in individuals with ASD have been mainly based on Cognitive Behavioral Therapy (CBT) , 101. HoStudies have shown the benefits of working alongside parents and focusing on relational aspects.It has been argued elsewhere the potential of the MBT-C model to increase the child\u2019s capacity for emotional regulation and improve general psychosocial functioning . This isAdditionally, there are interventions that may not specifically focus on mentalizing, but they still work towards improving related capacities. These interventions can offer valuable information on the potential effectiveness of this model for the targeted population. In this regard, there is evidence that children with autism improve their social communication skills by increasing their role as initiators of social interactions such as improvements in social and emotional behavior, communication, eye contact, joint attention, and imitative play .It has been also found that working with children with ASD through interactions between children and parents using a Developmental, Individual Difference, Relationship-Based model of intervention (DIR) may enhance important aspects of mentalizing such as joint attention and regulation, engagement across a wide range of emotions, two-way communication, and complex social problem solving .The evidence examined thus far suggests that treatments centred around mentalizing and related skills have the potential to significantly improve social functioning, psychological adjustment, and emotional regulation in children with autism. However, there is currently no research supporting the effectiveness of a MBT model specifically for children with ASD.In this regard, we argue that in order to develop therapeutic approaches in autism suitable to be empirically tested, there is a need for a more robust underlying theory. We propose that such a theory should integrate attachment, neuroscience, and mentalizing. In this article, we have presented some of the current advancements in autism research, specifically in the areas of attachment and mentalizing, from a neuroscience perspective.In the subsequent section, we outline possible avenues for progress, considering both clinical and research possibilities.6After reviewing the available evidence, we propose a set of elements that should be considered within current mentalization-based models in order to better meet the needs of children with ASD.By focusing on a core capacity that may promote resilience in a wide range of children with various presenting problems, MBT aims to be a transdiagnostic therapy that can be adapted to the particular needs of a range of difficulties.Regarding ASD, MBT approaches, and especially those that work with children  may be helpful as they offer an alternative model of the relationship between the caregiver and the child, which fits both the child\u2019s and caregiver\u2019s capacities to reflect on their own and other\u2019s mental states and generalize a new way of regulating, connecting, and communicating with themselves and others. MBT with children may support developmental experiences through a secure, predictable, yet flexible therapeutic framework to improve psycho-social functioning and increase emotional regulation skills.Considering these elements, we propose a body of therapeutic actions that are focused on the complex interplay among the therapist, the child, and the caregiver in the context of ASD. In this respect, it is important to note that ASD exhibits a widely heterogeneous range of social and cognitive symptoms, which has challenged comprehension and therapeutic approaches. It has been argued that this enormous phenotypic heterogeneity is closely related to a complex multifactorial etiology \u2013116, maka) Knowing the features of autism while keeping the not-knowing stance: a comprehensive understanding of relevant information and scientific knowledge will contribute to debunking myths and misconceptions regarding autism and individuals with ASD. This knowledge holds significant potential in developing self-identity and fostering a deeper sense of self-understanding by:Acknowledging the child\u2019s unique experience and co-constructing the diagnosis together\u2014not taking the child\u2019s experience for granted.Being curious about their states of mind that arises in situations linked with difficulties in social communication and/or related with their pattern of interests and behaviors.Considering the presence of restrictive and repetitive behaviors and patterns, it is crucial to assist the child in balancing an \u201coverinterpretation\u201d and an \u201coverlook\u201d of both their experiences and their own characteristics. The exercise of weighing each individual\u2019s role in a situation (whichever may be) is challenging. In addition, social interactions are highly complex situations that require predictive abilities, and mentalization is a critical skill needed to interpret, comprehend, and attribute both one\u2019s own and other\u2019s behaviors , 118\u2013120b) Creating mentalizing narratives about events related to social communication difficulties: it may be helpful for children to have someone join them in reflecting on and understanding their thoughts and feelings about their own unique  characteristics of personality, interests or thoughts in a curious rather than critical approach. Exploring autism\u2019s meanings constructed from non-mentalizing interactions with others plays a crucial role in self-identity. For example, health professionals, teachers, classmates, relatives, etc. may give misleading labels, leading to children feeling confused, misunderstood and invalidated about themselves and their experiences.c) Working with the autobiographical narrative: creating a life story that incorporates the child\u2019s own characteristics, interests and experiences, can strengthen self-esteem, emotional regulation and self-identity. By exploring the impact of cultural perspective regarding autism  in the autobiographical narrative of the child, self-awareness, re-routing and mentalization are reinforced.d) Working with process rather than content: learn and understand in the here-and-now how to cope with and regulate the emotions associated with rigid thinking and/or restrictive interests and behaviors, the presence of comorbidities or symptoms, sensorial interferences, among others.e) Working with parents (caregivers) in mentalizing autism: it may be helpful to visit specific episodes where the child-caregiver dyad faces autism-related issues and to mentalize what happened. The parent-therapist work should reflect on the caregiver\u2019s thoughts and feelings related to the child\u2019s characteristics and behaviors in order to revise the role of the diagnostic on their relationship with their child. For example, a diagnosis may be overused when it is used excessively to explain a child\u2019s behavior, disregarding other possible causes and hindering a complete comprehension of the child\u2019s inner thoughts and feelings. Also, mentalizing the diagnosis can promote an empathic awareness of when the caregiver\u2019s own anxieties lead them to intrusive and controlling behaviors in the relationship with the child. On this point, it is also important to consider that relatives of children with autism might display subclinical symptoms , so the f) Collaborative work with educational and health systems: therapeutic strategies should include collaborative work with the child\u2019s educational system. Communication with the school regarding the child\u2019s coping at school and how to support them is crucial to reinforce their self-identity development and facilitate the inclusion of the child in the social context. Additionally, and considering the eventual presence of comorbidities, therapeutic strategies should also include working together with the health system, such as a psychiatrist, neurologist, occupational therapist, and speech therapist, in order to strengthen the interventions and to understand the entire network of meanings in which the child is immersed.7As we mentioned earlier, no research currently supports the effectiveness of a MBT model specifically for children with ASD. We have argued that a therapeutic approach worthy of empirical testing needs a more robust underlying theory. Here, we have presented some of the current advancements in research in ASD, specifically in the areas of attachment and mentalizing, from a neuroscience perspective. Although there have been some developments in this area, more information is needed to understand how therapeutic strategies aiming to enhance mentalizing might work better for individuals with ASD.Therefore, to move forward in the research field, it is crucial to conduct further research exploring the neurobiological mechanisms underlying attachment and mentalizing, particularly in the context of ASD.Once equipped with this knowledge, the subsequent step may involve the design of therapeutic approaches that seamlessly integrate attachment theory, neuroscience, and mentalizing. Such integration has the potential to give rise to tailored therapeutic approaches that take into account the distinctive neurobiological characteristics of individuals with autism, potentially resulting in enhanced care and the facilitation of empirically validated interventions.Following this, the research should shift its focus to the conduct of empirical studies aimed at evaluating the effectiveness of these therapeutic approaches. These studies may encompass a range of methodologies, including randomized controlled trials, feasibility studies, and secondary analyses. Therapeutic outcomes should include improving social functioning, psychological adjustment, and emotional regulation . Additionally, achieving these improvements may reduce caregivers\u2019 distress, which can be seen as a secondary therapeutic outcome.To assess these therapeutic outcomes, studies might benefit from the incorporation of measures of general social functioning, social interaction functioning, communication abilities, and the presence of restricted, repetitive, and stereotyped behavior. For example, studies can use validated scales such as The Social Communication Questionnaire (SCQ) \u2013123. ForFinally, it is crucial to acknowledge the heterogeneous nature of autism, which is reflected in important individual differences among individuals with ASD. This includes considering factors such as age, gender, language abilities, cognitive performance and cultural backgrounds. It is equally important to consider potential cultural differences that may impact outcomes in different populations and situations when examining the effectiveness of MBT for individuals with autism.8In this paper we have shown that comprehensive models to understand the psychological processes of children with ASD have prospects for advancement. Integrating attachment, neuroscience, and mentalizing can help in achieving this goal. While there is room for improvement, there is already a path to follow. The current evidence supports the importance of utilizing neuroscience within psychological therapeutic models like MBT. Our literature review suggests that MBT models are suitable for integrating the latest neuroscientific findings into therapeutic considerations. This approach will provide a secure environment for individuals with autism to address any psychological issues they may be experiencing\u2014particularly those related to mentalizing.MBT has the potential to be a valuable therapeutic approach for children with ASD. By fostering mentalization, it can help children develop a more nuanced awareness of their thoughts, emotions, and motivations, ultimately leading to increased self-reflection and self-understanding. Furthermore, MBT promotes the development of more flexible thinking patterns, aiding individuals in coping with and regulating their emotions effectively.We also highlighted the importance of considering the caregiver\u2019s role in the therapeutic process. Collaborative work with parents or caregivers can enhance their understanding of the child\u2019s unique experiences and challenges, ultimately strengthening the parent\u2013child relationship. This approach extends to educational and health systems, emphasizing the need for cooperation to support the child\u2019s social inclusion and overall well-being.We additionally emphasize the importance of recognizing the diverse needs of pre-verbal children and those with language or cognitive difficulties as a crucial aspect to consider when tailoring effective clinical strategies. It is important to acknowledge individual characteristics and adapt interventions accordingly, regardless of age. Proper therapeutic strategies must take into account the stage of development at which each child is and should be adapted to address the challenges specific to that developmental level. We argue that an appropriate therapeutic approach should not only foster a supportive environment for the child\u2019s development but also a caregiver-child relationship.However, the next step is to conduct studies that evaluate the outcomes of MBT interventions, considering factors such as social functioning, psychological adjustment, emotional regulation, and mentalizing abilities. These studies should be mindful of the heterogeneity within the autism spectrum, accounting for variations in age, gender, language abilities, cognitive performance, and cultural backgrounds.In summary, we offer a promising avenue for the development of effective therapeutic approaches for children with ASD. By incorporating attachment theory, neuroscience, and mentalization processes, we can work toward providing tailored interventions that address the unique needs of each child. Through empirical research and a collaborative effort involving caregivers, educational systems, and healthcare professionals, we can advance our understanding and application of MBT, ultimately improving the well-being and social integration of children with autism.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.SC-C: Conceptualization, Writing \u2013 original draft, Writing \u2013 review & editing. PS-I: Conceptualization, Writing \u2013 original draft, Writing \u2013 review & editing. KB: Conceptualization, Writing \u2013 original draft. AG-C: Writing \u2013 original draft. NM: Conceptualization, Writing \u2013 review & editing."}
+{"text": "Photodynamic therapy is a photochemistry-based approach, approved for the treatment of several malignant and non-malignant pathologies. It relies on the use of a non-toxic, light activatable chemical, photosensitizer, which preferentially accumulates in tissues/cells and, upon irradiation with the appropriate wavelength of light, confers cytotoxicity by generation of reactive molecular species. The preferential accumulation however is not universal and, depending on the anatomical site, the ratio of tumor to normal tissue may be reversed in favor of normal tissue. Under such circumstances, control of the volume of light illumination provides a second handle of selectivity. Singlet oxygen is the putative favorite reactive molecular species although other entities such as nitric oxide have been credibly implicated. Typically, most photosensitizers in current clinical use have a finite quantum yield of fluorescence which is exploited for surgery guidance and can also be incorporated for monitoring and treatment design. In addition, the photodynamic process alters the cellular, stromal, and/or vascular microenvironment transiently in a process termed photodynamic priming, making it more receptive to subsequent additional therapies including chemo- and immunotherapy. Thus, photodynamic priming may be considered as an enabling technology for the more commonly used frontline treatments. Recently, there has been an increase in the exploitation of the theranostic potential of photodynamic therapy in different preclinical and clinical settings with the use of new photosensitizer formulations and combinatorial therapeutic options. The emergence of nanomedicine has further added to the repertoire of photodynamic therapy\u2019s potential and the convergence and co-evolution of these two exciting tools is expected to push the barriers of smart therapies, where such optical approaches might have a special niche. This review provides a perspective on current status of photodynamic therapy in anti-cancer and anti-microbial therapies and it suggests how evolving technologies combined with photochemically-initiated molecular processes may be exploited to become co-conspirators in optimization of treatment outcomes. We also project, at least for the short term, the direction that this modality may be taking in the near future. Photodynamic therapy (PDT) is a United States Food and Drug Administration (FDA) and European Medicines Agency (EMEA) approved modality for the treatment of a number of cancer and non-cancer pathologies , 2. WhilParamecia incubated with acridine orange and accidental exposure to sunlight, resulting in cytotoxicity to the organism, was possibly one of the key early observations that led to the current version of PDT applications , 8584, 8via two main initiating routes involving either the activation of death receptors (the extrinsic pathway) or the mitochondrial release of cytochrome c (the intrinsic pathway). Alternatively, generation of tBid through the extrinsic pathway can trigger the intrinsic pathway and amplify the apoptotic response. Ultimately, these pathways converge at the caspase activation step and lead to cellular changes such as poly nucleosomal DNA fragmentation, plasma membrane blebbing, and nuclear shrinking, which could be morphologically and biochemically detected in dying cells  was used in photosens-mediated PDT, highlighting the occurrence of ferroptosis mediated cell death fluoro-D-glucose (FDG) with PET was used to monitor tumor metabolism following PDT treatment. The authors found significant reduction in FDG signal in Pf-PDT treated tumors early after PDT, whereas AlPsC-PDT treated tumors demonstrated a slower, more gradual decrease in FDG, reflecting slower decrease in tumor activity, with fewer vascular effects and rather more direct tumor cell inactivation. Alternatively, the initial decrease in FDG seen in Pf-PDT treated tumors indicates stronger indirect vascular effects initially following treatment \u2013266.et al. in 2001 . T. Tet al.et al. fabricated iRGD  modified nanoparticles loaded with ICG and TPZ (Tirapazamine) for enhancing penetration and anti-tumor efficacy  , 344 proefficacy  346]. D. Det al.In contrast to PDT being used as an activator of prodrugs for enhancing their therapeutic potential, the combination of photothermal therapy with PDT has been used to enhance the therapeutic effects of PDT. Theoretically, the photothermal effect results in a local temperature increase \u2013349, theP process is the elicitation of immunogenicity in different tumors via the activation of ICD  to to367] tet al. conju gated verteporfin (BPD) to the anti-EGFR antibody; Cet, showing the targeting specificity of the conjug ate to EGFR expressing human cancer cells [NCT02422979 and NCT03769506) for head and neck cancers [et al. demonstrated a nanoengineering approach of immobilizing PICs onto nanocarriers successfully directing their internalization to the target cells, and enhancing PS delivery [Owing to their specificity for antigens, monoclonal antibody (mAb) mediated active targeting approaches have been the gold standard for over the past two decades. Moreover, as most of the preferred mAbs bind to overexpressed receptors on tumor cells, they also provide therapeutic benefits by suppressing signaling pathways involved in uncontrolled cellular proliferation and migration . Many mAer cells . The con cancers , 375, wi cancers . As an edelivery .in vitro, and doxorubicin-loaded anti-HER2 immunoliposomes exhibited marked therapeutic effects in HER2-overexpressing xenograft models [Another molecular target that has been widely exploited for PDT is the human epidermal growth factor receptor 2 (HER2). Several studies with HER2 targeted PDT have established the efficacy of this target in enhancing selectivity in various tumor types including preclinical models of disseminated ovarian and gastric tumors , 377. Ant models . A wide t models \u2013382 funct models , folate t models , peptidet models , 386, cyt models ), aptamet models  and mannt models  have beet models , 391.et al. showed an efficient strategy for the photodestruction of desmoplasia in heterotypic pancreatic tumors with the molecular precision of antibody-based therapeutics. This study established a platform for the delivery of chemi cally tuned NIR activatable targeted photoimmuno-nanoconstructs (PINs), demonstrating an effective binding and penetration into the tumor, with respect to the non-targeted nanoconstructs leading to significant photomodulation of tumor collagen, which is considered to be a major barrier to drug delivery, drug distribution, and immune-cell infiltration in the TME [Apart from targeting moieties, nanoconstructs and their surfaces can be extensively modified to endow them with multiple functionalities, including modifications for increasing cellular internalization and accumulation at the target tissue, long systemic circulation or organelle-specific drug delivery , with th the TME \u2013394.in situ PDT in the nucleus damages nuclear DNA and induces apoptosis, enhancing PDT efficiencies both in vitro and in vivo [While targeting surface markers has been extensively reported, targeting nuclear molecules has not been achieved with the same amount of success. The major barrier to this is the endosomal degradation of the targeting moiety and the nanoconstruct. Following the success of several anti-cancer drugs that have their targets in the nucleus, nanoagents targeted to nuclear molecules including DNA are also being considered , 396. In in vivo . In this in vivo , utilizi in vivo .et al. demonstrated an exciting approach to target and photo-inactivate the nuclear proliferation protein Ki-67 [et al. demonstrated the phototherapeutic cellular destruction by a two-step light triggering strategy [As the selective photodamage and inhibition of intracellular and nuclear proteins is a promising strategy to improve PDT efficiency Rahmanzadeh in Ki-67 . PIC-encin Ki-67 , 401. Bastrategy . This instrategy . HoweveraPDT), is briefly discussed below. In this section, selected reports and potential future applications of aPDT are summarized. The section by no means claims to be a comprehensive review of the field and for thorough discussions on aPDT, the reader is referred to other reviews [Although this review primarily focuses on PDT of cancer, non-cancerous pathologies also benefit from the photodynamic processes. Of these, the best known is the treatment of AMD, mentioned earlier, which became a clinical and commercial success , 405. An reviews \u2013409 and, reviews  and Wain reviews , 411.aPDT has been studied primarily for extracellular bacteria but there have been clinical studies against intracellular pathogens discussed later in the section. The emergence of drug-resistant pathogens and the agnosticism of aPDT to this resistance open up new opportunities for antimicrobial photomedicine [Staphylococcus aureus, Enterococcus faecalis and Streptococcus pyogenes) and Gram (\u2212)  strains. The modality has also been successful against intracellular, Gram (+) (Listeria monocytogenes) and Gram (\u2212)  bacteria as well as the acid-fast Mycobacteria species [e.g. Candida species) and viruses (e.g. human papillomavirus) [medicine , 412. Phmedicine . This ma species \u2013418. Themavirus) \u2013424.aPDT of bacteria reported killing of E. coli with neutral red [aPDT effect against Gram (+) bacteria [aPDT efficacy. Different conjugation approaches have also been applied in an effort to enhance the antimicrobial effects of PDT. For example, in a study, the anionic PS (chlorin e6) was conjugated with cationic-charge-bearing polymers which showed successful increase in PS-conjugate penetration through the outer membrane of Gram (\u2212) bacteria as shown in e.g. TMPyP) [in vitro anti-bacterial efficacy of PDT but there were only a few studies that demonstrated selective killing of pathogens compared to host cells [et al. in 2001, to kill skin microorganisms  using PDT, was one of the initial investigations that showed selective damage of the bacteria compared to the host keratinocytes. Currently, a wide variety of other PSs such as methylene blue [in vitro studies of aPDT. More recently, the relatively old concept of dual-action antibiotics [Some of the earliest studies of tral red  and acritral red . Studiesbacteria , due to bacteria , 428. Thbacteria . The teibacteria . Contrarbacteria . Studiesbacteria , 431, whbacteria , to tran. TMPyP) , where t. TMPyP) . Until tst cells . To thisene blue , rose beene blue , porphyrene blue  and indoene blue  have shoibiotics  such as ibiotics \u2013444 and in vitro and in vivo models of bacterial infection, aPDT has been explored as a potential clinical treatment in randomized clinical trials, with most studies targeting periodontal disease [et al. (Jordan University of Science and Technology), involving a conventional treatment  for periodontal disease combined with aPDT suggested significant improvement in all evaluated clinical parameters for at least 1-year post-treatment. An interesting application to note, although not yet studied in clinical trials, is a 2018 publication by the Hamblin group, in which methylene blue (MB) and potassium iodide (KI) mediated aPDT was utilized to treat urinary tract infections (UTIs) in a rat model [aPDT, followed by delivery of intravesicular illumination with a diffusing fiber connected to a 660 nm laser. Significantly shorter infection duration was found in the aPDT treated rats compared to controls, and a smart solution to monitor treatment efficacy was shown via bioluminescent imaging of the bacteria following treatment up to 6 days post-aPDT. Further work needs to be performed for full translation into clinic, but light-based methods for the treatment of antibiotic-resistant bacteria is an exciting new approach with potentially simple clinical adaptations. In particular, aPDT against biofilm growth, which is characterized by unique capabilities to thwart traditional antibiotic treatments and cause recurrent infection, will likely continue to develop [Other than success with  disease \u2013447. In at model . Drug-re develop , 448.et al. showed bactericidal efficacy of PDT, in vitro, against intracellular pathogens including Salmonella and Listeria using a range of PSs  [aPDT, include Mycobacterium spp., Cutaneous Leishmaniasis (CL), and malarial parasites. An interesting approach has been aimed to control vectors that act as host reservoirs to complete different parts of the parasite life cycle e.g. mosquitoes, snails, and flies. Abdel-Kader and coworkers used naturally derived porphyric PSs such as chlorophyll and hematoporphyrin derivates as photopesticides [Leishmania is the most studied pathogen in the field of antimicrobial photomedicine. PDT has shown excellent results against this otherwise tedious organism and is worth discussing. Leishmania parasites are engulfed by the macrophages and proliferate inside them while escaping the host immune response by producing reducing agents against oxidative stress [Leishmania parasitic load, as shown in preclinical and clinical studies  and nitric oxide synthase , which together with RMS generated during phagocytosis, kill the parasites [L. major, 28 days post-PDT, with absence of amastigotes. However, other studies [et al. report an increase in IFN-\u03b3 in response to ALA-PDT against L. braziliensis and conclude that the reduction in parasite load was through both direct and indirect mechanisms [Leishmania which may vary among different species. Recently, a clinical study in Israel done by self-administering topical methyl aminolevulinate followed by daylight PDT has reported much success to treat cutaneous leishmaniasis [Intracellular pathogens have also been shown as reasonable targets for PDT. The mechanisms are naturally complex as the PS now has to traverse the cell to find organelles harboring the pathogens. Brovko e green) . Other msticides \u2013452. Thee stress . Generale stress , 454. Ev studies , there a killing . On the arasites , 457. Inarasites , ALA-PpI studies , 460 indchanisms . Howevermaniasis , which dmaniasis .Among all viral infections antiviral PDT has been most successful against human papilloma virus manifestations. Another area that has earned much success over the past years for photodynamic inactivation of viruses is decontamination of blood products . Of parte.g. methylene blue) and fluorophores (e.g. BODIPY FL)  or \u03b2-lactamase activated fluorophore (\u03b2-LEAF) and can be used for treatment and diagnosis respectively. The design of the system and probe contains a cephalosporin core including the \u03b2-lactamase cleavable lactam ring conjugated to two chromophore moieties , 471\u2013476DIPY FL) . This ap 24\u201348 h . The app 24\u201348 h . Contraret al. have shown selective targeting of Plasmodium falciparum, the causative organism of malaria [Plasmodium. Interestingly, when red blood cells (RBC) get infected by parasites, they hijack the machinery of RBCs and develop new nutrient acquisition pathways. As a result, this new permeability machinery enhances selective uptake of ALA into the infected RBCs as compared to the uninfected RBCs. Feeding parasites with exogenous ALA results in an increase in the production of parasite\u2019s PpIX, one of the precursors of heme. In this process, the infected RBCs become photoreactive and upon irradiation generate RMS which in turn kills the parasites, as shown in e.g. ticks and mites) and internal (e.g. babesia and theileria) parasites have unique nutrient acquisition pathways that can be manipulated by leveraging photodynamic mechanisms.Like other organisms, parasites also deploy unique host\u2013pathogen interactions for their survival. More recently, exploiting such mechanisms, Sigala  malaria . The autaPDT has also provided many options for the treatment of infectious diseases. The main advantage of aPDT is that it is effective against both drug-susceptible and resistant pathogens [aPDT has the ability to alter lipopolysaccharides, (a potent immune stimulant) which can induce secretion of pro-inflammatory cytokines by host cells [Even though PDT has a long-standing history of clinical use mainly in cancer, the phenomenon of athogens , 482. Unathogens \u2013487. Morathogens . In addist cells \u2013490. Thust cells . PDT hasst cells , 480.e.g. the upregulation of VEGF secretion or increase in tumor permeability [P, as it primes the tumor for secondary complementary treatments to enhance the therapeutic outcome and deter any unwanted side-effects such as increased metastasis observed with almost all treatments [P may be viewed as an enabler of other therapies and forms the rationale for PDT-based combinations, where one treatment mechanism reinforces the next. Both PDT and complementary treatments may be administered separately [P effect [P and combinatorial treatments in preclinical studies showing promising outcomes even at sites remote to the locally illuminated ones. So, one might look forward to a day when PDT is more than a local therapy using a deliberative, mechanism-based understanding and exploitation of PDP. The same is true for targeted treatments that provide a higher level of selectivity than is possible with small molecules [NCT02422979 and NCT03769506) with photo-immunotherapy, even though focused on local disease, is exciting and the community looks forward to the results of these trials [P enhanced immuno-oncology is a future focus in the field. PDT mediated destruction of tumor cells happens primarily via necrosis and other cellular death mechanisms including apoptosis. While inducing cell damage or death, PDT releases DAMPs, which activate the innate and adaptive immune system components and enhance ICD. Local inflammation due to PDT-mediated recruitment of immune cells to the site of irradiation and increase in the infiltration of cytotoxic T lymphocytes can kill the tumor cells. Thus, the photodynamic process primes the TME, which may be beneficial in treating less immunogenic \u201ccold\u201d tumors and turning them into more inflamed \u201chot\u201d ones compared to traditional treatments such as chemotherapy which can be immunosuppressive. Approaches for the use of PDT to treat non-cancerous diseases are also moving although, since the initial excitement and the success of the treatment of AMD, there have not been significant clinical approvals where PDT is used as a first-line treatment. From the end of 2019, with the COVID-19 pandemic, viral decontamination in patients and/or in devices such as ventilator inlets using PDT could prove to be an extremely useful technology. Preclinical and clinical studies suggest that aPDT, along with nanotechnology and photochemically primed immune system and combination treatments are likely to be the next areas of advanced development/application of PDT.The essential principles of PDT have been used in therapy since the days of the early Egyptians over 5000 years ago and have been developed significantly in recent years. Much has become known about mechanisms involved and the complexity of the PDT process so that the modality can now be investigated not only as a local disease control approach but also as a systemic therapeutic option in combinatorial regimens. The process of being subjected to the PDT transforms not only the cells targeted with light but also the microenvironment, both at the cellular and tissue level. The changes are often transient, eability \u2013496, andeatments , 497. PDparately  or simulparately . PDT basP effect , 498. Alolecules , 499. Tholecules . The onge trials , 375. The trials  with a Pe trials . PDP enh"}
+{"text": "IntroductionThe association of acute appendicitis with caecal or colorectal cancer is known. One of the proposed theories for acute appendicitis is luminal blockage by mass at the base of the appendix. There have been no national recommendations or guidelines for follow-up with patients aged 40 and older after an emergency appendicectomy. The purpose of this study was to evaluate the prevalence of caecal and colonic cancer or polyps in patients over the age of 40 who have undergone an appendicectomy. This shall enable us to develop the necessary strategies to investigate and diagnose associated caecal and colonic pathology in acute appendicitis to prevent delayed diagnosis of colon cancer.MethodsAll patients who underwent appendicectomy between October 2011 and October 31, 2021, and who were 40 years of age or older were included in this retrospective cohort study. Patients aged 40 to 54 years old and patients 55 years or older underwent subgroup analyses. We looked at any investigations of the colon (CT pneumocolon or colonoscopy) within three years before the appendicectomy or three years after an appendicectomy. All colorectal cancers diagnosed within five years of the index episode of appendicitis were included in the analysis.ResultsA total of 1076 appendicectomies were performed on patients aged 40 and older during the study period of 10 years. A total of 769 patients were confirmed to have appendicitis on histology. One hundred and fifty-seven patients had colonic investigations within three years of the diagnosis of acute appendicitis. In our study, 51 of the 769 patients (6.63%) were found to have colorectal neoplasms. Eight patients  were diagnosed with colorectal cancers, and the occurrence of caecal cancer was 0.26% (2/769). The mortality rate was 75% (6/8) in these patients diagnosed with colorectal cancer. Four out of six died due to advanced metastatic colonic cancer. In comparison to patients aged 40 to 54, patients over the age of 55 had a statistically significant increased risk of caecal pathology (polyp and cancer) (p = 0.07).ConclusionThere seems to be an increased risk of significant colorectal neoplasm in patients over the age of 55 who are admitted with acute appendicitis, and there appears to be an increased severity with a poor prognosis of cancer in these individuals. We recommend the use of routine colonoscopy or CT pneumocolon, particularly for those over the age of 55 who present with acute appendicitis or the histology of appendicular neoplasms. Appendicectomy for acute appendicitis is one of the most frequently performed surgical operations. One of the proposed theories for acute appendicitis is luminal blockage by faecolith, lymphoid hyperplasia, or mass at the base of the appendix . The assAlthough the occurrence of appendicitis with caecal cancer is uncommon, it should be considered in patients over the age of 40 . VariousAlthough there has been a proven association between acute appendicitis and right-sided colonic cancers and the incidence of colorectal cancer in patients presenting with acute appendicitis is higher than in the general population over 40 years of age, there have been no national recommendations or guidelines for the follow-up of these patients. This study aimed to assess the incidence of significant caecal and colonic pathologies in patients over 40 who have undergone appendicectomy. This will enable us to develop the necessary strategies to investigate and diagnose associated caecal and colonic pathology in acute appendicitis to prevent delayed diagnosis of colon cancer.MethodsThis retrospective cohort study collected data for all patients aged 40 years or older who underwent appendicectomy during the period of 10 years between October 2011 and October 31, 2021, at Colchester Hospital, East Suffolk and North Essex Foundation NHS Trust. Patients were identified using the hospital\u2019s electronic database. Only emergency appendicectomies were included in this retrospective study, and those patients who had undergone appendicectomy as a part of a more extended procedure were excluded . All patients undergoing appendicectomy had their histology results examined. This study included patients who had undergone appendicectomy as the index procedure and had been diagnosed with acute appendicitis based on histology for additional analysis.Those aged 40 years or older were chosen since patients in this age range are frequently mentioned in published data. Patients between 40 and 54 years old and patients 55 years of age or older underwent subgroup analyses. Comparing \"younger\" and \"older\" patients allowed for a better understanding of any differences that might exist in colorectal cancer development in both groups. Patients with normal appendicular histology (excluding appendicitis) were excluded from the main analysis. The hospital's audit department approved this project .DataThe data gathered encompassed demographic information, the date of appendicectomy, preoperative CT imaging at the initial admission, and histological diagnosis. We also looked at any investigations of the colon (CT pneumocolon or colonoscopy) within three years before the appendicectomy or within three years after the appendicectomy. A comparison of the rates of colorectal pathologies (polyps and cancer) was made between patients aged 40 to 54 years and 55 years and older. Patients with colorectal pathologies were identified by reviewing electronic reports for imaging and endoscopic procedures. We also cross-referenced the hospital\u2019s colorectal cancer database to identify: any colorectal cancers in patients who did not have any colonic investigations three years before or after their index admission with histologically proven appendicitis; any colorectal cancers in patients who were found to have colorectal polyps in their colonic investigations. All colorectal cancers diagnosed within five years of the index episode of appendicitis were included in the analysis as related to the episode with appendicitis, especially as most colorectal neoplasms will take about five years to evolve .Statistical analysisDescriptive analytics were employed to characterize the data. Odds ratios were calculated to compare age groups 40 to 54 years and 55 years and older for any significant difference between them when they were found to have caecal pathologies associated with appendicitis. Based on previous studies, the association of colorectal cancer with acute appendicitis in patients aged 40 and older is 2.4%, and the incidence of colorectal cancer in patients aged 40 and older in the UK in the general population is 0.27%. Considering the probability of a type I error of 0.05 and the power of the study, 90% of the sample size necessary was 196 .A total of 1076 appendicectomies were performed in the study population of 40 and older during the study period of 10 years. A total of 78% of operations were performed for acute presentations of abdominal pain. Around 91.43% (769/841) were confirmed cases of acute appendicitis on histopathological reports  within three years of the diagnosis of acute appendicitis , and the occurrence of caecal cancer was 0.26% (2/769). The mortality rate was 75% (6/8) in patients diagnosed with colorectal cancer. Four out of six died due to advanced metastatic colonic cancer. The demographic characteristics of the patients who had cancer are depicted in Table 9, and thFindings in participants less than 55 years of ageIn patients younger than 55 years, the incidence of caecal pathology (polyps and cancer) was 0.75% (3/399). One patient was diagnosed with caecal cancer  and two with caecal polyps . Overall, in the colon and rectum, six polyps were noted  with two patients having caecal, two patients having sigmoid cancer, one patient having a rectal polyp, and a single patient having multiple polyps at different sites  was 2.43% (9/370), and the incidence of caecal cancer was 0.27% (1/370). The latter patient had caecal adenocarcinoma. Eight patients had benign caecal polyps. None of the patients with polyps develop cancer later on. Overall, seven colorectal cancers  were noted in this age group: one in the caecum and two cancers in the transverse colon, sigmoid, and rectum, respectively.Patients aged 55 years or older were found to be significantly more likely to possess caecal pathology (polyps and cancer) than those aged 40 to 54 (p = 0.07). The odds ratio of developing caecal pathology (polyps and cancer) was 3.3 times higher (95% CI 0.88-12.18) in individuals aged 55 years and above compared to people between the ages of 40 and 54 years. Patients over 55 were also found to be more likely to have associated colorectal cancer compared to those aged between 40 and 54, with an odds ratio of 4.35 (p = 0.19).In our study, 51 of the 769 patients (6.63%) were found to have colorectal neoplasms after confirmed appendicitis in patients over the age of 40, and eight patients  were diagnosed with colorectal cancers. In the United Kingdom, the age-wise incidence of colorectal cancer in the same age group (40 and over) is 0.25% .Lai et al. specifically examined individuals aged 40 and over, discovering that 0.85% of them had colorectal cancer at the time of appendicectomy or within 40 months of the initial operation . They alIn our study of patients aged more than 55 years, the reported incidence of caecal pathology (polyps and cancer) was 2.43% 9/370) and the incidence of caecal cancer was 0.27% (1/370). The association of appendicitis with caecal cancer has been documented in recent studies. A study of patients aged 55 years or older undergoing appendicectomy for acute appendicitis by Mohamed et al. , Bizer e0 and theIn the present study, cancers were evenly distributed across the colon, with two patients having caecal, transverse, sigmoid, and rectal cancers each. In the literature, some studies have noticed an uneven distribution of cancers. In a study by Pedersen et al. , seven oThe prognosis of tumours occurring in the caecum or proximal colon presenting as acute appendicitis is reported as poor, with the reason partly being delayed diagnosis. In our study, six of eight patients died. Four of them died within three years of the diagnosis of colon cancer due to advanced metastatic disease, thus indicating a poor prognosis for patients presenting with acute appendicitis and having underlying colonic cancer.Although appendicular cancers are rare, synchronous colorectal neoplasia has been reported in 3% to 5% of patients with appendicular neoplasia . ConsequOlder patients presenting with symptoms of acute appendicitis are often investigated with CT scans. Although patients can be reassured of no pathology on a negative initial CT scan, it has been shown in various studies that the accuracy of picking up caecal pathology in acute appendicitis remains low. Hence, post-appendicectomy colonoscopy is recommended in the elderly population. In our study, 468 (61%) patients were investigated with a CT scan. Only three CT scans raised the possibility of caecal cancer, of which none were found to have colorectal cancers. However, three patients presenting with acute appendicitis who were diagnosed with colon cancers on colonoscopies later were not picked up by the initial CT scan. In a study by Khan et al., of the 80 patients presenting with acute appendicitis investigated radiologically, a CT scan identified only 1 patient (1.25%) with suspicion of a caecal tumour . In anotThere are certain limitations to our study. The retrospective design of the study itself is an important limitation. Only 157 patients had a colonoscopy within three years of surgery for acute appendicitis, thus indicating a selection bias in requesting colonic investigations. With the set limit for the observation period at three years, those patients admitted in late 2019 and 2020 have not yet had a three-year observation period at the time of the study, and any findings in these patients after 30 March 2022 have not been registered. We did not include patients who were managed conservatively. The study could not explain the association of colorectal carcinoma with appendicitis, although the relation is explicable in caecal pathology. The study population has not undergone colonoscopy; hence, the actual incidence of colorectal pathology cannot be assessed. Finally, we may have missed those patients who were investigated elsewhere.There seems to be an increased risk of significant colorectal neoplasm in patients over the age of 55 admitted with acute appendicitis, and there seems to be an increased severity and poor prognosis of cancer in these individuals. The sensitivity of CT scans to detect colorectal cancer in the setting of acute appendicitis is low. We recommend the use of routine colonoscopy or CT pneumocolon, particularly in those over the age of 55 years presenting with acute appendicitis or with histology of appendicular neoplasms. We also recommend that clinicians err on the side of caution in patients between the age of 45 and 54 years\u00a0with appendicitis. Further randomized trials are necessary to increase the level of evidence."}
+{"text": "The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway.Human serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation This study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology.in vitro\u2013generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood.The plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma.Plasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated These data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs. Microtiter plate wells were blocked with blocking buffer  for 1 hour before performing chromogenic assays. Activation of FXII (100 nM) was monitored in 100 \u03bcL standard HEPES-buffered saline (HBS) buffer  PTT-a was used as a positive control. FXII-/FXIIa-dependent FXIa generation was determined by incubating 2 nM FXII with 30 nM FXI \u00b1 30 nM HK and agonists for 3 hours, before the addition of 2 mM S2288; 1% (v/v) PTT-a or kaolin were used as positive controls. In some cases, 300 pM FXIIa was added instead of 2 nM FXII, and the incubation time was omitted. For the 2 discontinuous assays, a standard curve of PKa (0.39\u201330 nM) or FXIa (0.39\u201330 nM) activity was generated on each microtiter plate.2.1.3 |\u22121).FXIa on S2288 was determined by mixing 3 nM FXIa with serial dilutions of S2288 following the addition of HSA-AGE. FIXa on FX was determined by mixing 100 nM FIXa with dilutions of FX (3.9\u20131000 \u03bcM), 100 \u03bcM phospholipid micelles, and 700 \u03bcM S2765 after adding HSA-AGE . Some clotting assays were measured turbidometrically and prepared in the same way as aPTT assays in 96-well clear microtiter plates. Assays were performed by measuring plasma turbidity at 340 nm to monitor fibrin polymerization using a Bio Tek PowerWave HT Microplate Spectrophotometer. Clotting times were determined by measuring the time of 50% maximum clot formation.aPTTs, prothrombin times (PTs), and reptilase assays were performed using a STart benchtop coagulometer (Stago). For aPTTs, 47.5 \u03bcL pooled normal plasma (PNP) was added to 2.5 \u03bcL HBS or HSA-AGE. Fifty microliter PTT-a was added to the PNP mixture, then pre-warmed 50 \u03bcL 25 mM CaCl2.1.5 |\u22121 fibrillar type I collagen (Labmedics)  and incubated for 1 hour in a humid atmosphere at 37 \u00b0C. Slides were then blocked with an HBS-bovine serum albumin advanced (BSA) buffer  BSA, pH 7.4) for 30 minutes at 37 \u00b0C before experiments. Final concentrations of both 0.5 \u03bcg.mL\u22121 3,3\u2019-dihexyloxacarbocyanine iodide, [DiOC6(3)] (ThermoFisher Scientific) and 40 \u03bcg.mL\u22121 Alexa Fluor 647 conjugated human fibrinogen (AF647-fibrinogen) were added to freshly collected sodium citrate\u2013anticoagulated blood samples and incubated at room temperature for 10 minutes. Two 1-mL syringes were filled with whole blood or recalcification buffer , and loaded onto an AL1800\u2013220 Aladdin programmable 8-channel multi-syringe pump. Both syringes were connected to silicone tubing with a 0.8-mm internal diameter (Rubber BV). The 2 pieces of tubing were connected via a Y-style splitter (Thistle Scientific), which in turn connected to the channel slide via a Luer elbow connector (Thistle Scientific). Whole blood and recalcification buffer were perfused into the channel at equal flow rates, diluting whole blood by half and resulting in a final shear rate of 150s\u22121. The blood/recalcification buffer perfusion was terminated after a time allowing for sufficient thrombus formation, depending on the donor sample, usually between 20 to 40 minutes. Fluorescent images were taken using a Zeiss Axio Observer 7 microscope equipped with a Hamamatsu Flash 4 LT sCMOS camera and Definite Focus2 (Zeiss). Two-dimensional images of the total area covered by collagen for each channel were scanned at 1-minute intervals using a 10x/0.3 M27 EC Plan-Neofluar objective (Zeiss) and automated scanning stage (Zeiss). Z-stacks were scanned for both DiOC6 labeled platelets and AF-647 fibrin(ogen) using a 40\u00d7/0.95 Plan-Apochromat air objective (Zeiss). Images were processed using ZEN 3.0 with deconvolution (Zeiss).Channel slides  were half-filled with 1.3 \u03bcL 50 \u03bcg.mL3 |3.1 |p < .05; medical center\u2019s normal range, 26\u201338 seconds) (p = .0055) in diabetic samples compared with IGT and ND samples  (p < .05) or absence of PTT-a (p < .01) , 2 individuals with impaired glucose tolerance IGT), and 10 nondiabetic (ND) volunteers, which were matched for age and comorbidities . There wseconds) . First, seconds) , B. Anal samples , D, reflp < .05) , D. In cp < .05) , B, E. Ap < .01) , G. The T, and 10p < .01) .p < .01)  or a selective FXIIa (corn trypsin inhibitor [CTI]) inhibitor was added to plasma before the PTT-a agonist, dose-response inhibition of both the inhibitors was observed using the S2302 substrate. However, when CTI or SBTI were added following previous incubation of PTT-a with plasma S2302 (which would generate FXIIa and PKa before the addition of the inhibitors), dose-dependent proteolysis was inhibited by CTI but not SBTI, which demonstrates that PKa rather than FXIIa was responsible for S2302 proteolysis . Constitp < .01) . A fluorp < .01, .3.2 |\u22121 kaolin, or 2.5 \u03bcg.mL\u22121 HSA-AGE (\u22121) failed to trigger FXII-dependent PKa generation  PTT-a (\u22121 HSA-AGE or 1% (v/v) PTT-a \u00b1 40 \u03bcg.mL\u22121 protamine sulfate and analyzed by immunoblotting (HSA-AGE was added to PNP and dose-dependently triggered PKa-like activity . Protamiv) PTT-a . PNP or blotting . Moreove3.4 |\u22121 kaolin, or 2.5 \u03bcg.mL\u22121 HSA-AGE (\u22121) used to trigger optimal FXII-dependent PKa generation (\u22121) or kaolin (1.56\u2013100 \u03bcg.mL\u22121) was then titrated into the FXII-dependent FXIa generation assay. Although kaolin dose-dependently increased FXIa generation with maximal FXIa generation reaching 1.40 \u00b1 0.09 nM at 50 \u03bcg.mL\u22121, HSA-AGE generated a maximum of 0.263 \u00b1 0.019 nM using 5 \u03bcg.mL\u22121  but was dose-dependently inhibited by HSA-AGE (0.78\u201350 \u03bcg.mL\u22121) , 50 \u03bcg.mL HSA-AGE . FXIa waneration . HSA-AGE \u03bcg.mL\u22121 . These a\u03bcg.mL\u22121) .2, a phospholipid mixture of 20% phosphatidylserine:20% phosphatidylethanolamine:60% phosphatidylcholine to mimic an activated platelet surface, and either no surface (HBS vehicle), PTT-a, or kaolin gave clotting times of 608.9 \u00b1 8.3 seconds, 212.0 \u00b1 4.0 seconds, and 240 \u00b1 6.9 seconds, respectively (\u22121) were added to plasma in procoagulant conditions; clotting times began to shorten from baseline readings , with the shortest clotting time of 499.7 \u00b1 6.5 seconds noted with the 15.25 \u03bcg.mL\u22121 HSA-AGE concentration but began to get prolonged beyond baseline readings (cat) of FXIa with minimal effects on the Michaelis constant (Km) this is a characteristic of noncompetitive allosteric enzyme inhibition.The kinetics of FXIa toward S2288 with HSA-AGE  was studectively . HSA-AGEreadings . A summa3.5 |\u22121 HSA-AGE prolonged the aPTT by 50%  showed no effect on the aPTT (\u22121) did not affect the PNP reptilase time, suggesting fibrinopeptide-A cleavage or fibrin oligomerization is unaltered (\u22121) prolonged the aPTT of PNP, FXI-, and PK-but not FIX-deficient plasma. These data suggest that HSA-AGE inhibits the function or activation of FIXa. Reconstituting FIX-deficient plasma with 5% PNP, restored the inhibitory effect of HSA-AGE on aPTT assays. Determinations of aPTTs (in the absence of other reagents) for PNP, FXII-, PK-, FXI-, and FIX-deficient plasmas are shown in aPTT assays were performed with HSA-AGE using PNP to investigate any anticoagulant effects. HSA-AGE dose-dependently prolonged the aPTT and 400 \u03bcg.mLthe aPTT . HSA-AGEnaltered . aPTT-trnaltered . HSA-AGE3.6 |m 263.5 \u00b1 39 \u03bcM and kcat 3.8 \u00b1 0.3 (\u00d710\u22124) s\u22121 and HSA-AGE increased the Km of FIXa-mediated FX activation with minimal effects toward the kcat, indicative of competitive inhibition , rather than procoagulant because of the inhibitory effect of HSA-AGE at various points of the coagulation cascade. Interestingly, HSA-AGE appears to be a physiologic equivalent to the in vitro effect of the synthetic polyanion dextran sulfate, which also triggers FXII and PK generation but inhibits thrombin generation [A key objective of this study was to determine how misfolded protein aggregates can trigger contact activation without associated intrinsic coagulation. We chose to investigate this phenomenon using neration .in vivo\u2013acquired material, which was beyond the scope of this study.We first investigated the activity of CAS in diabetes. The DM group in our study had a mean glycated hemoglobin level of 7.58% (\u22656.5% threshold for DM). The results show that DM is associated with elevated levels of FXIIa and cHK antigen, which we determined by immunoblotting. Plasma of patients with DM also showed significantly higher constitutive PKa activity via a chromogenic approach when compared with ND controls, which positively correlated with glycated hemoglobin %. The AGE-specific fluorescence emitted by plasma of patients with DM was approximately 6.22-fold higher than ND controls, which agrees with previous findings \u201330. Previn vivo [The current study\u2019s data agree with previous findings that patients with DM have slightly shorter aPTTs but not PTs, compared with euglycemic controls , althougin vivo .in vitro\u2013generated HSA-AGE added to PNP, which is devoid of the complex metabolic milieu or other glycated plasma proteins. Future studies will be required using advanced purification methods to obtain AGE-modified proteins from the plasma of patients with DM to fully delineate the effects of isolated AGE-modified proteins toward contact activation and intrinsic coagulation.Our studies with HSA-AGE agree with previous findings by Maas et al. , who desin vitro by long-term incubation with glucose, which is characterized by autofluorescence, inter- and intramolecular crosslinking to form both soluble and nonsoluble amorphous, amyloid-like anionic high-molecular-weight aggregates with decreased proteolytic susceptibility and protein clearance in vivo [in vitro preparations of BSA-AGE. Experiments using 6D12 found common epitopes are shared with other AGE-modified proteins and peptides [in vitro AGE formation have physiologic relevance. The epitope shared among AGE-proteins was later found to be N\u03b5-(carboxymethyl)lysine, which is a modified lysine adduct formed on the protein surface in the advanced stages of the Maillard reaction, which increases the net negative charge of AGE-proteins [AGEs are generated  in vivo ,19,40. Ipeptides \u201344. Thisproteins \u201317,45.Our results showed HSA-AGE triggered purified FXII autoactivation and FXII-dependent PKa activity in both purified and plasma-based assays. The anionic charge dependency of these reactions was seen following the addition of the cationic peptide protamine sulfate to reactions triggered by HSA-AGE. Heat denaturation of albumin is a simple method used to induce fibril formation in albumin rich in \u03b1-helices, as opposed to the high \u03b2-sheet content of HSA-AGE . ThT andTo investigate the mechanism underlying the inhibitory effect of HSA-AGE on intrinsic coagulation, mixing studies were performed using factor-deficient plasmas. PTT-a\u2013triggered clotting in FIX-deficient plasma was not affected by HSA-AGE compared with baseline FIX-deficient plasma. However, mixing FIX-deficient plasma with 5% PNP restored the HSA-AGE prolongation of clotting times, suggesting that HSA-AGE is additionally capable of inhibiting at the axis of FIXa. We pursued this finding and exposed FIX-deficient plasma to exogenous FIXa or FXa before adding HSA-AGE. Therefore, we confirmed that HSA-AGE inhibited clotting when FIXa was supplemented under these conditions but did not interfere with clotting when exogenous FXa was employed in the presence of PTT-a-triggered coagulation. In purified chromogenic assays, HSA-AGE inhibited FIXa-dependent FX activation; analysis of the kinetics of these reactions suggested that HSA-AGE was a competitive inhibitor of FX activation by FIXa.There are limitations to the current study. The purified FIX-dependent FXa generation chromogenic assays were performed without cofactors FVIIIa or FVa, but we believe these cofactors may not be so critical because we demonstrate the anticoagulant effect of HSA-AGE in normal plasma. Therefore, we believe that the effects of FVIIIa or FVa may not be critical to the inhibition mechanism exerted by HSA-AGE to FIXa-dependent FX activation. Future studies should specifically address whether FIX or FX binding to misfolded protein was responsible for the phenotype of bleeding disorders previously reported in systemic amyloidosis .in vitro flow-based model but was also dose-dependently prolonged. Therefore, providing direct evidence that the presence of procoagulant platelets does not override the inhibitory effect of HSA-AGE toward fibrin formation.The HSA-AGE inhibition toward the intrinsic pathway was not only limited to static PTT-a\u2013triggered plasma assays because platelet-mediated fibrin formation using an FXII is a protein that has critical roles in disease pathology, such as the acute sterile inflammation seen in hereditary angioedema and sepsis, but also chronic conditions, such as Alzheimer disease, systemic amyloidosis, and thrombosis ,7,9,10. Supplement"}
+{"text": "The Scandinavian countries make interesting samples for the study of shared parenting as they are characterized by some of the highest levels of father involvement and gender equality globally. Despite numerous studies, data from Denmark is noticeably absent in the international debate, partly due to a researcher preference for publishing in Danish. Here, I present an overview of the increase in father involvement in Denmark since the 1960s and on the increase in shared parenting across recent decades. I further examine Danish law, ministerial guidelines and guidelines from major Danish public and private institutions/organizations involved in deciding or advising on parenting practices post-divorce. I relate these to international research findings as well as to findings from Danish research. Overall, I find that Danish guidelines/practice have several reservations against shared parenting and substantial father involvement, which are not considered warranted by a substantial number of scientists and which are not supported by the majority of the available evidence. It thus appears that societal transition toward increased shared parenting has happened on a largely voluntary basis in spite of official law/practice. Updated law and/or ministerial guidelines are likely necessary if politicians desire that children experience the same high degree of father involvement post-divorce that they experience in society in general. Numerous studies are published internationally from particularly Sweden but also Norway. However, data from the third Scandinavian country, Denmark, is remarkably absent in the international literature. This is not because studies are not conducted, but rather that they have been published in Danish, and thus are not easily accessible to readers outside of Scandinavia. A main purpose of this article is to remedy this situation by conducting a detailed analysis of the development of shared parenting in Denmark over recent decades. Specifically, I review the literature on the development in custody and parenting time in Denmark in the context of local and international research on the topic, and I discuss this research in relation to Danish law, official guidelines and legal practice. The review examines whether the historical increase in shared parenting has happened on a voluntary basis or whether it has been facilitated by law/professional guidelines, and it raises the question of whether joint physical custody should be a legal presumption.The scientific literature on the topic discusses parenting time using a set of terms that are to some extent bound to specific societies/laws. For example, the English-language literature often uses terms from the US legal system: joint custody (JC) and sole custody (SC). These can be further elaborated to specify if the custody is physical or legal: sole/joint physical custody (SPC/JPC) and sole/joint legal custody (SLC/JLC). JPC and SPC are defined on the basis of how much time the child spends with each parent. In some older studies, JPC is defined as children spending at least 25% of the time with each parent   whereas The terms and definitions in Danish law and practice are in many ways comparable to those in the US system, but also differs in some aspects. It is legally split into three separate domains: custody, residence and visitation. In Danish law (\u201cFor\u00e6ldreansvarsloven\u201d), custody refers to legal custody exclusively, and it is estimated to be shared in over 90% of the cases , p. 37. In this article, I first provide an overview of the developments in parental caregiving time across recent decades for Danish men and women. Next, I compare men\u2019s share of caregiving after divorce2.As in many other countries, the division of labor and parental roles in Denmark has changed dramatically over the past two generations, and equal divisions are closer than ever historically. I first examine this change and subsequently compare it to changes in children\u2019s residence and to time spent with each parent post-divorce.Over the past 60\u2009years in Denmark, a dramatic change is evident both in terms of how time is spent overall and how women and men spend their time, respectively. cf.The children seem to be a significant part of the answer. In 2008, fathers and mothers both spent approximately 30\u201340\u2009min more each day on primary caregiving of children than fathers/mothers did in 1987  on active days but an overall contribution of 38% for men due to the fewer active days , the center has published a report on the welfare and wellbeing of children \u2013 including children of divorce \u2013 in Denmark, and they have occasionally published research reports fully dedicated to shared parenting (in 2011 and 2012). One such publication reports a longitudinal study that followed children born in 1995. Here, The 2012 report provides relatively precise definitions of the various visitation groups with the last measurement taken in 2011 when the children were 15\u2009years old (and the earliest in 1996 when the children were 4\u20135\u2009months). Knowing the probability of residence with the mother/father, respectively, for each age group, the proportion of children in each visitation group for each age group and how many nights the child spent with each parent, a fairly precise estimate can be made of how much time the children spent with their father and mother after a divorce.Corresponding calculations based on the 2014, 2018, and 2022 reports are somewhat less precise, as the visitation groups were defined more loosely and subjectively. In the latter reports, for example, it is divided into \u201cno contact,\u201d \u201cshared parenting,\u201d  and \u201cother,\u201d where the latter thus covers the three intermediate arrangements of the 2012 report. The 2022 report contains the numbers for all years so estimations can be made based on this report alone. If we assume an equal division of children reported as having the \u201cother\u201d arrangement into the three visitation groups from the 2012 report, the fathers\u2019 share childcare post-divorce is estimated to just below 30% in 2013 and around 30\u201335% in 2017 for children of most ages. However, the younger children are again the exception, where the fathers\u2019 share is down to 25% in 2017 after having been at nearly 30% in 2013. The assumption of an equal division appears relatively accurate as the more precise 2011 paternal caregiving estimates are generally \u22121.5% percent higher than the 2009 estimates and 3\u20138% lower than the 2013 estimates across the various age categories.Using data from the 2022 report , fathersIn principle, there can be many reasons why fathers\u2019 involvement in childcare after divorce lags decades behind general societal development such as the children\u2019s/parents\u2019 wishes, the children\u2019s needs (and parents\u2019 perception of this) and the practice of the family law system. Common to all perspectives appear to be a desire to act in the child\u2019s best interests, but there are different perceptions of what that is. Below, I examine the current recommendations of Danish institutions and authorities in the field and subsequently relate these to recent scientific research and consensus.4.With respect to residency and visitation, the Danish law (\u201cFor\u00e6ldreansvarsloven\u201d) is remarkably vague. \u00a74 establishes that decisions must be based on the child\u2019s best interests, without further specifying these except with respect to physical violence. \u00a717 establishes that the courts have the authority to decide the child\u2019s residence if the parents disagree. Importantly, \u00a718a establishes that shared residency can only be established voluntarily (and thus not decided by the authorities), and it can be revoked by one parent (whether or not the other agrees). In terms of visitation, \u00a719 establishes that the child  has a right to visitation with the non-residential parent, and \u00a721 establishes that the extent is defined to be set based on concrete assessment of the child\u2019s situation without specifying how. \u00a742 further establishes that the minister of social affairs can set rules/guidelines for these aspects, and this is indeed done .Much of these ministerial guidelines relate to procedures and to considerations for complex cases  while relatively little space is dedicated to specific guidelines for deciding residency and visitation in non-complex cases where the parents disagree. For residency, section 4.2 of the ministerial guidelines primarily establish that emphasis may be placed on the parent\u2013child attachment, the parents\u2019 personal characteristics, and on how the child will react to potentially moving as a consequence of the decision. Gatekeeping behavior (primarily obstruction of visitation according to section 2.2 of the guidelines) may also be considered as well as the risk of violence or witnessing violence. For visitation, Section 5 of the guidelines lists that the decision may be based on the age and development of the child, the child\u2019s own opinion, their everyday life and activities, prior contact, interparental collaboration, the personal characteristics of the parents, the distance between homes, contact to siblings and other practical matters. It lists that any arrangement can be set, but for equal time it is usually required that it should not affect the child\u2019s school or social life, and it is a decisive requirement that parents can collaborate to create continuity between the two homes and allow for flexibility with respect to the child\u2019s need for contact. Specific guidelines are also set for children under the age of 3. It is, for example, mentioned that within the first 5\u2009months of the child\u2019s life, frequent but brief visitation of less than an hour may be set, and that these can be increased with age. At around 9\u201312\u2009months, overnights can be initiated. Together, the law and ministerial guidelines primarily establish that unless both parents agree to shared residence, a single parent holds the residence and the courts can decide who. Apart from the decision of equal visitation time, no explicit rules guide the verdicts, but some factors are listed that may (or may not) be considered, leaving a lot of power of decision to the courts and The Agency of Family Law (\u201cFamilieretshuset\u201d), which is described below.The Agency of Family Law is the first and, in many cases, only institution that parents encounter during divorce. The agency \u2013 for example \u2013 handles divorce applications, provides mediation between parties, provides advice on custody/visitation arrangements, conducts interviews with children, and they can assign residence/visitation temporarily and refer a case to court. The majority of families set a visitation scheme without any official involvement, but for the substantial minority \u2013 23-30% , p. 59 \u2013The Agency of Family Law have recently updated their visitation guidelines (November 2022) , but theThe previous guide listed some very specific recommendations for visitation and residence. It was stated that young children need a primary caregiver with whom the child resides. Initially overnight stays with the other parents are discouraged, but contact may be gradually extended so that overnights can be attempted between the ages of 1 and 3 . For children between ages 3 and 6, contact and the number of overnights can be increased, and if it works well for the child, shared time can be approached. For 6-12-year-olds, it is stated that nothing can be said about specific needs in relation to visitation schemes, apart from the fact that it can be important to listen to the child\u2019s wishes. From the age of 12 and up, it is mentioned that children themselves typically do not desire shared parenting. Furthermore, it is mentioned that when there is a high level of conflict between parents, children should live primarily with one parent.Overall, the previous guidelines focus on the relationship with the primary caregiver , and then you may or may not gradually develop a relationship with the other parent, who would typically be the father, if circumstances allow for it. A very cautious attitude is expressed toward shared parenting both for young children, older children and in divorces with conflict or where parental cooperation is less than ideal. According to In the updated guidelines from November 2022 , it is sIn the recent guidelines , researcOverall, The Agency of Family Law appears to have changed their view from emphasizing the importance of a primary caregiver to the importance of caring relationships with both parents. The statements about young children\u2019s need for unequal care have been replaced by statements that the research is unclear or does not indicate that children are harmed by shared parenting. Indeed, the recent guidelines indicate a greater openness to shared parenting overall , but the specific examples of parenting plans are still based on young children having a single home and gradually seeing the other parent more until shared parenting can be approached around school age and likely abandoned again for teenagers. Statements about parental cooperation and low conflict are also toned down in relation to shared parenting and now appear to be viewed more as independently important factors, but there are still some cautious statements that might indicate reluctance toward shared parenting in case of conflict. Despite the less negative or reluctant attitude toward shared parenting in general, however, it is not presented as a general recommendation for most families, and research is only mentioned once as positively supporting it, followed by a sentence urging not to generalize.In addition to state entities, at least two other major Danish organisations with significant funding provide support and advice in relation to divorce: M\u00f8drehj\u00e6lpen (meaning \u201cMothers\u2019 help\u201d) and B\u00f8rns Vilk\u00e5r (meaning \u201cChildren\u2019s conditions\u201d). These express similar opinions. For example, B\u00f8rns Vilk\u00e5r writes that \u201cit is your cooperation, level of conflict and responsiveness to the child that are most important for your child\u2019s well-being \u2013 not where your child lives and sleeps,\u201d and it is emphasized, that shared parenting requires extensive cooperation. Nevertheless, they do mention shared parenting in relation to relatively young children, and they mention in one example that it is something a four-year-old might suddenly need . InteresTaken together, Danish law and ministerial guidelines are relatively vague and mostly provide a list of aspects that may be taken into account. Critically, however, the law establishes that when parents disagree, residency can be listed with one parent only, effectively establishing this parent as the primary caregiver. This parent has additional rights, and the child has preferential access to them as they must live at least half of the time with this parent. This means that shared parenting can only be practiced through the rules on visitation in case of disagreement. In that context, the ministerial guidelines place a hard requirement of interparental collaboration on equal time, meaning that this becomes difficult to establish outside of a mutual decision by the parents. Other organizations advising and taking part in the initial decisions on residence/visitation are relatively conservative and generally refrain from endorsing shared parenting as a default solution. Below, I review the latest research and relate it to Danish law/guidelines. The main focus is placed on The Agency of Family Law\u2019s guides as these are both the most detailed/explicit in terms of recommendations and as the latest guide provides a list of specific references.5.frequency of contact is not a meaningful measure of the amount of contact, and that the \u201cquality\u201d measures in reality reflect quantity  showed that frequency of contact was generally not related to child outcome , and thiquantity . For exaquantity .When the amount of contact has been examined directly, the results are quite different. Children in JC typically do substantially better than children in SC as evidenced in two meta-analyses (neither of which are referenced by The Agency of Family Law). In an early meta-analysis of 33 studies, Most studies contrast SC and JC as dichotomous categories, but graded increases toward equal time have also been examined. A meta-analysis of 16 studies found a large number of benefits related to JPC, and the effects were greater for children who spent at least 40% of their time in each home compared to those who spent only 30\u201339% in one of the homes . SimilarIn Denmark, The literature thus consistently finds a positive relationship between equal parenting time and well-being of the children on a wide range of parameters, and equal parenting time is related to optimal parent\u2013child relationships mirroring those found in intact families. Given the consistency of the findings, subsequent skepticism has focused on whether JC has a causal effect or whether the effect is due to other factors such as wealthy, educated, resourceful parents with low mutual conflict and older children self-selecting JC. The research has therefore tried to separate these factors in increasingly sophisticated designs to examine if equal time in itself has causal, positive effects. In the sections below, I review the literature on the proposed confounding factors.6.Interparental conflict in the context of divorce is particularly interesting as it has not only been proposed as a confounding factor (the claim that lower conflict is the cause of benefits of JPC), but also as one that interacts with the type of custody . It is specifically mentioned in all the guidelines presented above, but at the same time, the level of conflict post-divorce frequently changes, and conflict is rarely ongoing for years. For example, the level of conflict is relatively high up to and immediately following divorce \u2013 when custody/residence is determined \u2013 but it declines afterwards . In OttoInterestingly, Bauserman investigated conflict already in 2002 and did not find that it moderated the positive effects of JC, but he also noted that the data at the time was sparse . A more The Agency of Family Law does not refer to the reviews of Nielsen or Mahrer and colleagues, but instead to two others by Taken together, the reviews of 7.Other factors such as parental income and the existing parent\u2013child relationship have also been investigated. As can be seen in 8.Another topic mentioned in all of the above Danish recommendations is the age of the children. Here, there have been theoretical reasons as well as early research indicating a lack of benefits of JPC. However, recent studies are generally more positive. In a study of 3,656 children aged 3\u20135\u2009years (including 287 children of divorce), One of the very recent studies mentioned by After this overview article, another Swedish study was published. In her review article, Of the four reviews presented above, The Agency of Family Law refers only to the latter two, of which at least one is very limited and both take a relatively conservative perspective on JPC and neglect to mention individual studies with positive outcomes. The Agency of Family Law does, however, additionally list one study by 9.Establishing the causal effects of different visitation arrangements is notoriously difficult as random, controlled trials obviously cannot be done. Instead, researchers have used a range of different methodologies to make inferences about causal effects. For example, parental relocation often causes abrupt, drastic changes in the amount of contact with one parent, and one can therefore examine the effect of moving on parent\u2013child relationships. Self-selection is typically considered the alternative to a causal explanation so another line of research has examined the extent of self-selection and attempted to rule this out as an explanatory factor. For example, it can be examined whether the benefits of shared parenting disappear when the parents initially oppose it, i.e., whether they have self-selected or (possibly reluctantly or after a court decision) have accepted it. 10.The Danish-language research literature places a prominent focus on the perspective of the children, often in qualitative studies, and this is reflected in the guides of The Agency of Family Law. A large, qualitative study of 200+ pages examines the experiences of children in shared parenting arrangements through interviews with 28 non-randomly selected children along with 24 parents and 4 adult children . As in tA major quantitative Danish study has also been conducted on the topic. Overall, it thus appears that, both in Denmark and internationally, children\u2019s desires are taken significantly more into account when they want time with their mother than when they want time with their father, and at the same time, it is the children\u2019s impression that the mother has significantly more power in relation to determining custody/visitation than they themselves and their father have had. The issue is evident in Danish research, but it is not reported outside a table listing. Presumably for this reason, The Agency of Family Law does not refer to it, and it is not mentioned in ministerial guidelines.11.Several international groups of experts have published consensus reports or conclusions from panel discussions, but these do not appear on The Agency of Family Law\u2019s reference list. In the most recent consensus statement  from 110Regarding conflict, the report concludes that it should not rule out shared parenting, but that the focus should instead be on conflict reduction. This can be done, for example, through practical measures such as reducing the number of times the parents have to meet to hand over the children . The report highlights the danger of considering conflict as a valid reason for avoiding shared parenting as this can give one parent an incentive for creating and maintaining conflict, effectively exposing the children to a higher level of conflict than otherwise. It is also emphasized that shared parenting may actually shield the children from the effects of conflict instead of exposing them to it. This recommendation very much stands in contrast to most Danish guidelines.As mentioned above, shared parenting is still only rarely a legal presumption, but one such implementation has been evaluated by a range of professionals. Regarding Danish experts, researchers at the Danish Center for Social Science Research appears to have had a number of reservations regarding shared parenting around 2011 and 2012 whereas the stance appears more neutral in later publications. For example, The 2022 publication generally has a much less extensive review but presents a more neutral or positive view toward shared parenting, likely reflecting that more evidence has become available and that there is now less reason for caution. Nevertheless, it does report the finding from the 2018 publication that there was no positive effects of equal time when controlling for additional variables without mentioning the authors\u2019 previous caution not to draw causal conclusions , p. 228.12.Over the past 60\u2009years, the caregiving role of Danish fathers has transitioned from peripheral involvement to providing around 40% of the primary care, and at the same time spending more time with the children than mothers did one and two generations ago (see Section 2 of this article). Despite the increased role in caregiving in society in general, fathers\u2019 share of care after divorce has lagged decades after societal development (Section 3). While there is no clear scientific consensus on all aspects, the majority of studies report benefits associated with increased father involvement up to and including equal time (Sections 5\u20139). Similarly, a large number of experts recommend shared parenting in the vast majority of cases (Section 11), just as the children themselves report the greatest satisfaction in shared parenting and later in adulthood assess that this is the best for children in general (Section 10). Specifically in Denmark, a substantial proportion of children report that they wish to have more time with their fathers (Section 10).Although a causal link cannot be established, the slow transition toward shared parenting post-divorce in Denmark has coincided with law and guidelines that reflect a cautious stance toward it. Specifically, current law and guidelines are quite open to interpretation and set only a minimal framework for children\u2019s rights to contact with both parents, yet they impose special requirements on shared parenting. The law establishes that in case of disagreement, one parent is decided to hold residency, thus effectively establishing an unequal starting point by default. In ministerial guidelines, equal parenting time as a visitation scheme has some additional relatively strict and specific requirements (regarding collaboration) that do not align with recent consensus statements and which make it difficult to establish equal time outside of mutual agreement among the parents. Guidelines from the most important Danish institution, The Agency of Family Law, have until very recently recommended against shared parenting for the vast majority of children, meaning that societal transition toward shared parenting can be said to have happened on a voluntary basis in spite of official recommendations and with a legal framework against it. Researchers at the Danish Center for Social Science Research agree that the change is largely cultural and not facilitated by law or structural changes , p. 236,The most recent Agency of Family Law guidelines are less conservative, but nevertheless reference selectively the review articles that dedicate the least space to studies about young children and interparental conflict, yet express the least positive view on shared parenting. Reviews that argue that the evidence supports a positive stance on shared parenting for most families are not listed and neither are statements from leading international researchers and experts. Quantitative studies showing that children generally desire more time with their father are not mentioned either. Reference is made to studies reporting little to no impact of paternal contact frequency but not to studies reporting numerous positive effects of overall contact duration.Taken together, current Danish institutional guidelines/law/legal practice appear to reflect a more reluctant stance on shared parenting than research evidence, children\u2019s reports and societal practice warrants. This is not unique to Denmark but indeed appears more the rule than the exception internationally. The status is nevertheless particularly surprising given the high degree of father involvement in Danish society and Denmark\u2019s relatively high degree of gender equality in general. With a father involvement of 40\u201345% in society in general, it appears in fact that the reduction to 30\u201335% post-divorce is a main limiting factor in achieving near-complete equality overall.causally related, and the main alternative explanation is that the extent of contact does not matter when taking confounding factors related to the parents into account. It may thus be argued that a departure from the stance that sole maternal residence is best for the child unless both parents agree otherwise carries primarily a risk of not having an effect. In contrast, if the effect is causal, restraint in departing from current practice restricts tens of thousands of Danish children to parenting arrangements that negatively impact their parent\u2013child relationships, their development and their mental health, and which they themselves do not desire.The slow implementation of research and expert opinion into Danish practice may stem in part from a principle of caution to avoid departing from traditional practice without clear evidence. It may also have been influenced by a relatively cautious stance taken by leading Danish researchers. In this context, it is worth noting that the debate presently does not focus on whether shared parenting is related to the best outcome, but whether it is It may be mentioned in this context that divorce is not a traditional event with a traditional solution, but rather something that became common just 50\u201360\u2009years ago in Denmark. The solution of maternal residence and unequal parenting time in the vast majority of cases can in itself be described as a large-scale societal experiment, which was not based on empirical evidence, and which authorities should not be afraid to revise in light of such evidence. It is particularly interesting that shared parenting appears to allow children to benefit from a good father-child relationship and good parental cooperation while the benefits of these are reduced or lost entirely in other arrangements. In contrast, skewed arrangements do not appear to offer anything unique that is not possible in shared parenting. Particularly in a society with high pre-divorce father involvement, it is worth considering whether shared parenting as a legal presumption might not be the most effective way of preventing widespread, negative divorce-related changes in parent\u2013child relationships. An update of ministerial guidelines on visitation schemes may serve a similar function to establish equal parenting time . There also appears to be some room for Agency of Family Law staff to update their guidelines and decrease the gap in parenting time within the existing rules. Of course, such changes should not exclude that the parties involved can choose another arrangement if there is agreement that it is the best, or that the authorities can rule against it in a number of cases.The author confirms being the sole contributor of this work and has approved it for publication."
\ No newline at end of file