New cultivars adapted to major durum wheat growing environments are essential for the cultivationof this crop. The development of new cultivars has required the availability of diverse genetic material and theirextensive field trials. In this work, a collection of tetraploid wheat consisting of 85 accessions was tested in thefield conditions of Almaty region during 2018 and 2019. The accessions were ranged according to nine agronomictraits studied, and accessions with the highest yield performance for Almaty region of Kazakhstan were revealed.The ANOVA suggested that the performance of agronomic traits were influenced both by Environment and Genotype.Also, the collection was analyzed using seven SSR (simple sequence repeats) markers. From 3 to 6 alleles perlocus were revealed, with an average of 4.6, while the effective number of alleles was 2.8. Nei’s genetic diversitywas in the range of 0.45–0.69. The results showed high values of polymorphism index content (PIC) in the rangeof 0.46–0.70, with an average of 0.62, suggesting that 6 out of 7 SSRs were highly informative (PIC > 0.5). Phylogeneticanalysis of the collection has allowed the separation of accessions into six clusters. The local accessions werepresented in all six clusters with the majority of them grouped in the first three clusters designated as A, B, and C,respectively. The relations between SSR markers and agronomic traits in the collection were studied. The resultscan be efficiently used for the enhancement of local breeding projects for the improvement of yield productivityin durum wheat. Othertetraploid wheat species Triticum turgidum L. ssp. turanicum(Jakubz.) Á. Löve & D. Löve, Triticum turgidum L. ssp. polonicum(L.) Thell., Triticum turgidum L. ssp. carthlicum(Nevski) Á. Löve & D. Löve, Triticum turgidum ssp. dicoc-cum(Shrank ex Schübler) Thell. are used as food and feedcrops in different world regions. Wild species Triticum turgidumssp. dicoccoides (Korn. ex Asch. & Graebn.) Thell. isalso often included in crossing schemes as a source for resistanceto abiotic and biotic stresses .Durum wheat (Triticum turgidum L. ssp. turgidum convar. durum(Desf.) MacKey) is a tetraploid species of wheat and is themain crop to producers of pasta and cereals. The growing areaunder durum wheat is about 17 million hectares in the worldand production is 37 million tons . In 2019, durum wheat production in Kazakhstanamounted to 560 thousand tons . Hence, the comprehensive study ofthe diverse germplasm is a very important prerequisite forthe successful conservation and rational use of plant geneticresources, including both wild and cultivated tetraploid wheatspecies . Theappropriate assessment of the genetic diversity in these collectionsdepends on the application of informative and efficienttypes of DNA markers. In many centers of the world,research is underway to find and use different types of DNAmarkers with the aim of using them to study genetic diversity,inventory, genotyping, mapping, and identifying genesassociated with useful traits of cultivated plant varieties andlines . Various types of DNA markershave been developed and are successfully used to study thegenetic diversity of accessions of the genus Triticum L. . PCR-basedmarkers,such as RAPD, AFLP, and SSR, are widely used toolsfor studying genetic diversity and discrimination both durumand common wheat .The wheat genome contains a class of specific nucleotide sequencescalled microsatellites, also known as SSRs or simplesequences repeats . SSR markers havemany advantages, being highly polymorphic, codominant,informative, reliable, and the availability of information onchromosomal localization . Microsatellites are hypervariable, they often havedozensof alleles at one locus, differing from each other inthe number of repeats. They are widely used to study geneticdiversity, as well as for the analysis of paternity and mappingof quantitative trait loci (QTLs), kinship, belonging to aspecific population, for studying hybridization, evolutionaryprocesses, and for searching for paralogs .Durum wheat polymorphism studies are currently underwayworldwide. The survey of reports demonstrated the successfuluse of SSR markers for assessment of the genetic diversityin different collections of Europe , Africa , China , Russia, Turkey ,Syria , etc. Microsatellites are also highlyeffective in tagging specific genes that play an important rolein variation for yield components and biotic stress resistance.A number of studies reported relations between SSR lociand wheat traits, such as yield, etc. For instance, Zhang etal. (2013) showed that the Xgwm11-1B locus is significant( p < 0.001) for plant height. In the study reported by Li etal. (2015) it was shown that the marker Xgwm148-2B is associatedwith the manifestations of the traits “thousand grainweight”, “spike yield index” and “weight of kernels perspike”. Xgwm251 was associated with lipoxygenase (LOX)activity, which is an important factor determining the colorof flour and end-use products of wheat .Vinod et al. (2014) have identified the significant associationbetween Xgwm234 and the resistance of T. turgidum to leafrust. Golabadi et al. (2011) showed that the Xcfa2114-6Amarker was responsible for 20 % of the phenotypic variationin the yield index and thousand grain weights (TGW) underdifferent environmental conditions. SSR marker Xgwm219was also shown to be associated with TGW . These examples suggest that the assessment of thegenetic diversity of the varietal gene pool of durum wheatmay provide not only proper genetic documentation of theaccessions but also hinting the identification of a valuablesource of genes associated with agronomic traits.The purpose of this work was the study the genetic diversityusing seven SSR markers and phenotypic variation in yieldcomponents in the collection of tetraploid species harvestedin the conditions of South-East Kazakhstan.1. Seeds were provided bythe Research Center for Grain and Industrial Crops , University of Bologna , Aktobe andKarabalyk Agricultural Experimental Stations (Kazakhstan).The collection included 21 cultivars and 15 promising linesof durum wheat from Kazakhstan (see Suppl. Table 1).Plant material and experimental site conditions. The plantmaterial consisted of 85 accessions of tetraploid wheat (2 Triticumturgidum ssp. dicoccoides (Korn. ex Asch. & Graebn.)Thell., 2 Triticum turgidum ssp. dicoccum (Shrank ex Schübler)Thell., 65 Triticum turgidum L. ssp. turgidum convar. durum(Desf.) MacKey, 10 Triticum turgidum L. ssp. turanicum(Jakubz.) Á. Löve & D. Löve, 4 Triticum turgidum L. ssp. polonicum(L.) Thell., and 2 Triticum turgidum L. ssp. carthlicum(Nevski) Á. Löve & D. Löve from different geographicalorigins (Supplementary Table 1)1 Supplementary Tables 1 & 2 are available in the online version of the paper:http://www.bionet.nsc.ru/vogis/download/pict-2020-24/appx9.pdfThe studied collection of tetraploid wheat was evaluated intwo randomized replicates in the field conditions of Almatyregion (Table 1).Each accession was planted in two rows with a row spacingof 15 cm, 25 seeds per row. In total, nine agronomic traits connected with the vegetation period, plant morphology, and yieldcomponents were studied. The list of traits included the headingtime , flowering time , seed maturationtime , plant height , spike length , number of fertile spikes , number of kernelsper spike , thousand kernel weight , andyield per plant  .DNA extraction and SSR genotyping. Genomic DNAwas isolated from individual 4-day-old wheat seedlings, accordingto Dellaporta et al. (1983). The quality and quantityof isolated DNA were evaluated using a NanoDrop 2000 and agarose electrophoresisin 1 % gel. The list of markers used for SSR analysis wasthe following: Xgwm11, Xgwm148, Xgwm251, Xgwm234,Xcfa2114, Xgwm169, and Xgwm219 (Supplementary Table 2).Polymerase chain reaction (PCR) was conducted in a Veriti™Thermal Cycler . The PCRreaction mixture (10 μl) contained from 2.5 mM of 10× Taqbuffer; 0.2 mM of each dNTP; 1.5 mM MgCl2; 250 μM ofeach primer; 1 unit Taq polymerase  and50 ng of genomic DNA.The amplification program included the following cycles:94 °C – 3 min; 40 cycles: 94 °C – 1 min; annealing temperature(55 or 60 °C depending on the primer) – 1 min; 72 °C –2 min; and 72 °C – 10 min. PCR products were separated on6 % polyacrylamide gels  run in 0.5×TBE buffer pH 8.0 at 250 V for 1.5 h. Gels were stained withethidium bromide, and the images were recorded with a Bio-Rad Image System . Allele sizes wereestimated in comparison with 100 bp DNA ladder .http://software.dell.com/products/statistica).Statistical analyses of field data were estimated usingSPSS 22.0 and STATISTIKA 13.2 software . Thevalues of the PIC index (polymorphism information content)suggested the effectiveness of the markers used, giventhat markers with a value of PIC > 0.5 considered as highlyinformative; 0.5 > PIC > 0.25 as informative; and PIC ≤ 0.25as marginally informative . Variationamong populations was studied using Principal CoordinateAnalysis (PCoA) in the software GenAlex, ver.6.5 . The resulting similarity matrix was furtheranalyzed using the neighbor-joining clustering algorithm forthe construction of the dendrogram. The phylogenetic treewas constructed using PAST v.3.25 software . Analyses of marker-trait associations were conductedusing a simple t-test .Phenotypic variation in the studied collectionField trials for two years revealed a sharp difference inthe vegetationperiod between species of tetraploid wheat(Table 2).All accessions reached the ripening stage, with an exceptfor the wild accession PI346783 .The shortest HT was observed in genotypes of T. dicoccoides(56.5 ± 3.5 days), the longest – in T. polonicum(60.7 ± 3.9 days) (see Table 2).Plant height is one of the important morphological traitsof the crops. According to the species, the highest ones werethe samples from T. carthlicum (117.9 ± 5.4 cm), while theaccessions from T. dicoccum were the lowest (97.4 ± 7.4 cm).On the other hand for T. durum genotypes the PH rangedfrom 58.0 ± 3.7 cm  to 137.6 ± 3.0 cm for cultivarKargala 66 (see Suppl. Table 1). As for the SL, the lowestvalue (5.0 ± 0.2 cm) had the cultivar PI 184526 , while the highest value (17.5 ± 1.7 cm) wasin accession PI 210845 (T. polonicum from Iran).The value of a cultivar is determined by its productivity,which consists of several components, including TKW whichis significantly affected by weather conditions, violation ofmoisture supply, and mineral nutrition of plants during theformation and maturation of grain. The highest averagedTKW values were revealed for three T. turanicum accessions and T. polonicumfrom Iraq (PI 208911 – 61.8 ± 4.5 g). The lowestTKW value was in accessions of T. carthlicum (29.9 ± 1.1 g).The NFS ranged from 3.9 ± 0.6 pcs/plant in the accessionPI 343446 (T. dicoccoides) to 2.0 ± 0.5 pcs/plant in genotypesPI 210845 and PI 266846 of T. polonicum.As for NKS and YPP the highest value were on accessionsof T. durum and the lowest to T. dicoccoides (see Table 2). Themin value of NKS (24.8 ± 3.8 pcs) under both conditions wasobtained in PI 343446 , the max – inKazakh cultivar Gordeiforme 254 (67.7 ± 7.1 pcs) and Canadiancultivar Strongfield (62.2 ± 1.2). Overall 31 T. durumaccessions prevailed the local check cultivar Gordeiforme 254(4.4 ± 1.6 g/plant) by YPP. Top twenty accessions by yieldcontained cultivars from Canada (Strongfield – 7.6 ± 1.9 g/plant), Spain (Granizo – 7.0 ± 1.9 g/plant), Italy (Capeiti-8and Ancomarzio), Syria (Sharm5), Russia , Ukraine (Har’kovskaya 90 andHar’kovskaya 9), USA (LO92), as well as 5 cultivars and4 breeding lines (e. g. G 2607 – 7.2 ± 1.4 g/plant), from Kazakhstan(see Suppl. Table 1).The Pearson index analysis revealed a significant positivecorrelation ( p < 0.01) between yield components and phenotypictraits. The ANOVA test based on two-years field trialssuggested that Genotype significantly influenced the SMT,NFS, SL, and all yield components with p < 0.001 (Table 3).Microsatellite analysis of the tetraploid wheat collectionThe lines and cultivars of the studied tetraploid wheat collectionwere analyzed using 7 polymorphic microsatellite markers(see Suppl. Table 2) localized on 6 wheat chromosomes – 1B,2B, 4B, 5B, 6A, 6B. The results based on using 7 SSR markershave allowed identifying a total of 32 alleles, with average4.57 alleles per marker (Table 4).The effective number of alleles ranged from 1.82 to 3.27,with a mean value of 2.77. Nei’s genetic diversity index averaged0.62 (see Table 4). The average value of polymorphisminformation content (PIC) was 0.62, ranging from 0.46 forXgwm219 to 0.7 for Xgwm148, Xgwm251, and Xgwm11,respectively.The PCoA was conducted based on SSR genotyping of85 tetraploid wheat accessions using 7 SSR markers. Accessionsof the studied collection were divided into groupsdepending on their attribution to species and place of origin,respectively .The first principal component in the PCoA (46.31 %) clearlyseparated T. polonicum and T. turanicum from other species. The most genetically distant from other specieswas T. carthlicum. PCoA using origin data revealed that localgenotypes were genetically closer to the North American accessions. The accessions from Russia and NorthAfrica were genetically distant from other groups of origin.Based on the genetic diversity results using 7 polymorphicSSR markers, a phylogenetic tree of 85 accessions of tetraploidwheat was constructed .The analysis revealed a division into two large clusters. Thefirst cluster consisted mostly of cultivars of tetraploid wheatfrom Kazakhstan and North America. The second cluster wasdivided into three sub-clusters. Although the European accessionswere dominated in all three subclusters of cluster 2, allthree sub-clusters included cultivars and lines of Kazakhstan.The t-test was performed to confirm the significance of theSSR markers for the studied traits. The results identified themost informative SSR markers related to major agronomictraits (Table 5). Xgwm251 showed a significant relationshipto HT and FT. Four markers were related to variance in PH.DiscussionInitially, the studied collection was separated according totheir species classification and origin (see Suppl. Table 1).The average yield analysis in the collection of tetraploid accessionsover two years (2018 and 2019) suggested that it ishighly correlated with all studied phenotypic traits ( p < 0.01), confirming the importance of selected characters in the trials.The two-way ANOVA showed that Environment greatly influencedHT and SMT. In addition, it was found that SMT isalso influenced by Genotype, showing the prospects of possibilityto adjust maturation time in the breeding process, asearly seed maturation is vital to avoid abiotic stresses duringthe important stages of plant growth. Particularly, it was shownthat in T. polonicum the seeds are ripening nearly five daysearlier than in T. durum (see Table 2). The field trials haveallowed the identification of accessions with outstanding fieldperformances. For instance, the cultivar Strongfield (Canada)showed 7.6 ± 1.9 g/plant, which was the highest yield valueamong 31 T. durum accessions that prevailed local standardGordeiforme 254 (4.4 ± 1.6 g/plant). In general, two-wayANOVA indicated the great influence of the environmentalfactors, as they were affected both adaptation-related traits,such as HT and SMT, and yield components, such as SL andNKS (see Table 3).The entire collection was studied using seven SSR markersthat were located on six different chromosomes (see Suppl.Table 2). According to the previous works, a list of markers inthis study was most useful to evaluation of genetic diversityand associations with agronomic traits of durum wheat . The average PIC value was higher than 0.6,suggesting that the level of polymorphism was very high.The high level of variation in the collection has effectivelyallowed the separation of accessions according to their speciesclassification . Notably, the PC1 (46.3 %)separated T. polonicum and T. turanicum from the remainingspecies, and the PC1 (34.1 %) distinguished T. carthlicum andT. durum from T. dicoccum and T. dicoccoides. Interestingly,the accessions originated in Kazakhstan were geneticallyclose to North American samples , and it is tosome extent confirm the phylogeny of hexaploid bread wheatstudies using SNP (single nucleotide polymorphism) markers. The PC plot is suggesting that sixaccessions of durum wheat from the Russian Federation aredistinctly different from accessions with other origins . The Neighbor-joining phylogenetic tree suggestedthat all accessions can be divided into two clusters, wherecluster 1 was mostly populated by accessions from Kazakhstan.The significance of each SSR marker for studied traits wasassessed using a two-tailed t-test . The results of the test suggested that five out ofseven SSRs were significant at least for one studied trait (seeTable 5). The PH was the trait where four SSR markers, twowith negative and two with positive values, were significantlycorrelated. In addition, the test showed that Xgwm234 is significantlycorrelated with TKW and Xgwm219 and Xgwm169with YPP (see Table 5). Thus, the application of SSR markersin the analysis of tetraploid wheat collection consistingof 85 accessions was used for (1) genetic documentation ofsamples, (2) for phylogenetic clusterization based on the speciesclassification and geographic origin, and (3) associationsbetween DNA markers and studied phylogenetic traits. Hence,the results can be efficiently used for the enhancement of localbreeding projects for the improvement of yield productivityin durum wheat.The phenotypic analysis of the tetraploid wheat collectionconsistingof 85 accessions showed a high correlation ofYPP with all 8 phenotypic traits in conditions of South-EastKazakhstan. The ANOVA suggested that the environmentalconditionssignificantly affected the variation in HT and SMT,while Genotype has contributed significantly to main yieldcomponents, including TKW. Overall, 31 accessions of T. durumshowed higher average yield values in comparison withlocal check cultivar Gordeiforme 254 (4.4 ± 1.6 g/plant), andCanadian cultivar Strongfield was with the highest yield value(7.6 ± 1.9 g/plant). The application of seven SSR markerssuggested that local accessions were distinctly different fromdurum accession from other parts of the world. Particularly,the Principal Coordinate plot showed that local durum sampleswere most close to North American samples. The Neighborjoiningphylogenetic tree separated 85 samples to two mainclusters, where the cluster 1 was mainly represented by Kazakhaccessions and cluster 2 mostly by European accessions. Theapplication of the t-test indicated that five out of seven SSRswere significant at least with one agronomic trait. 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Caseinolytic protease P (ClpP), which is located on the inner mitochondrial membrane, degrades mitochondrial proteins damaged by oxidative stress. The role of ClpP varies among tumor types. However, the expression pattern and biological functions of ClpP in breast cancer (BC) have not yet been investigated.The Cancer Genome Atlas (TCGA) and Kaplan Meier-plotter database were used to analyze the expression level of ClpP in BC tissues, relationships with clinicopathological characteristics, and the influence on the prognosis of BC. Protein and mRNA expression levels of ClpP in BC cell lines and tissues were detected by quantitative real-time PCR, western blot and immunohistochemical (IHC) analyses. The colony formation assay, transwell assay and flow cytometric analysis were performed to assess various functions of ClpP. Western blot analysis was also conducted to determine the mechanism of ClpP.ClpP expression was markedly increased in BC cells and tissues. High expression of ClpP was significantly correlated with the T stage, estrogen receptor (ER) expression, and poor recurrence-free survival (RFS) in TCGA and Kaplan Meier-plotter database. ClpP silencing significantly inhibited proliferation, migration, invasion, and promoted apoptosis of BC cells, which resulted in suppression of the Src/PI3K/Akt signaling pathway. The gain-of-function assay confirmed partial these results. Breast cancer (BC) is the most common malignant tumor among women worldwide . At presThe mitochondria have been a focus of cancer research since the 1950s, when it was discovered that cancer cells produce adenosine triphosphate (ATP) and employ different mechanisms to support cell growth than the normal surrounding tissues. Therefore, it was suggested that a defect in the mitochondrial mechanism will not only lead to increased glycolysis, but also to the transformation of normal cells into cancer cells . The maiin vivo, and correlated with shortened survival. However, the ClpP and ClpX subunits may not have completely overlapping function(s) in the tumor mitochondria (The ClpXP protease complex is composed of two proteins: hexamers of a AAA+ ATPase (ClpX) and the tetradecameric peptidase caseinolytic protease P (ClpP) , which cchondria .ClpP is encoded by nuclear genes in mammalian cells and plays a central role in the quality control of mitochondrial proteins via the degradation of misfolded proteins. ClpP was first identified in bacteria and has since aroused interest as a potential anti-microbial therapeutic target . StudiesThe expression level of ClpP is greater in acute myeloid leukemia (AML) cells than in normal hematopoietic cells . FurtherHuman BC tissues and corresponding adjacent normal tissues were collected from patients who underwent surgery at the First Affiliated Hospital of Chongqing Medical University from 2014 to 2018, snap-frozen in liquid nitrogen, and then stored at −80 °C. This study was approved by the Institutional Ethics Committees of the First Affiliated Hospital of Chongqing Medical University  and conducted in accordance with the tenets of the Declaration of Helsinki. Each participant signed an informed consent form prior to study inclusion.2/95% air.Seven human BC cell lines  and two normal mammary epithelial cell lines (MCF-10A and HBL-100) were obtained from the American Type Culture Collection . MCF-10A cells were cultured as described previously  and all ClpP-specific and the negative control siRNAs were synthesized by OriGene . The following siRNAs sequences were generated: SR305388A-rGrCrUrCrArArGrArArGrCrArGrCrUrCrUrArUrArArCrATC; SR305388B-rGrUrUrUrGrGrCrArUrCrUrUrArGrArCrArArGrGrUrUrCTG; and SR305388C-rGrGrCrCrArUrCrUrArCrGrArCrArCrGrArUrGrCrArGrUAC. The expression vector pCMV6-Myc-DDK-ClpP was produced by OriGene ; the empty pCMV6-Myc-DDK vector was used as a control. Lipofectamine 2000  was used for siRNAs and plasmids transfection, in accordance with the manufacturer’s protocols.Total RNA was extracted from cells and tissues with TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. RT-qPCR was performed using an ABI 7500 Real-Time PCR System  with the SYBR Green kit (Invitrogen). β-actin was used as an internal control. Each sample was tested in triplicate. The followig primers were used for RT-qPCR analysis: ClpP, forward primer: 5′-GCC AAG CAC ACC AAA CAG A-3′, reverse primer: 5′-GGA CCA GAA CCT TGT CTA AG-3′; β-actin, forward primer: 5′-CCT GTG GCA TCC ACG AAA CT-3′, reverse primer: 5′-GAA GCA TTT GCG GTG GAC GAT- 3′.Total proteins were extracted with radioimmunoprecipitation assay lysis buffer . Protein concentrations were determined using the bicinchoninic acid protein assay kit . Western blot analysis was conducted as previously described  with theAll specimens were formalin-fixed, paraffin-embedded and cut into 4 µm-thick sections, which were mounted onto glass slides. IHC analysis was conducted as described previously . BrieflyCell apoptosis was detected with the Annexin V-FITC Apoptosis Detection Kit  in accordance with the manufacturer’s protocol and a FACSCalibur flow cytometer (BD Biosciences).MDA-MB-231 and ZR-75-1 cells were seeded into triplicate wells of 6-well plates at 1,000 cells/well, and cultured for 7 days. The cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution .4 cells/well) were added into the upper transwell chamber, and 800 µL of medium containing 10% FBS were added to the lower chamber. After incubation at 37 °C under an atmosphere of 5% CO2/95% air for 24 h (MDA-MB-231 and ZR-75-1 cells), or 72 h (MCF-7 and T47D cells), cells that migrated through the membrane pores were fixed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet  for 15 min at room temperature. Matrigel™-coated transwell filters  were used to evaluate the invasion capability of the cells. The subsequent procedures were the same as those for the cell migration assay. Cells from six random fields were counted under a microscope. All experiments were repeated three times.Transwell chambers  were used to detect the migratory and invasive capabilities of BC cells. For the transwell migration assays, 200-µlL aliquots of transfected MDA-MB-231, ZR-75-1, MCF-7 and T47D cells (4 * 10https://tcga-data.nci.nih.gov/tcga/). The study cohort consisted of a total of 1010 BC patients. ClpP expression data of 112 normal breast samples were included to compare differences in ClpP expression levels in BC tissues. Overall survival (OS) and complete clinicopathological data of 990 BC patients, and RFS data of 792 BC patients were screened. ClpP expression levels were ranked from low to high based on median values. The first 50% of patients were considered as the low-expression group and the second 50% as the high-expression group.Gene expression data of BC tissues were downloaded from the TCGA database .Prognosis based on ClpP levels in BC patients was analyzed using the Kaplan Meier-plotter database  and GraphPad Prism 7.0 software . The two-tailed Student’s p < 0.001) (p < 0.0001) (p < 0.01) (p = 0.029) (p < 0.001) (p = 0.0154) and ER expression (p = 0.0164). Kaplan–Meier survival curves from TCGA indicated that ClpP was not associated with RFS (p = 0.506) or OS (p = 0.619) (p = 0.00071)  . Receive 0.0001) . To veri < 0.01) . The AUC= 0.029) . ClpP pr< 0.001) –1G. Thes< 0.001) . High Cl= 0.619)  and 2B. 0.00071)  and 2D. ClpP mRNA and protein expression levels in a panel of BC and normal breast epithelial cell lines were determined. The results showed that ClpP was increased in most malignant cell lines, as compared with normal cells  and 3B. p < 0.01 and <0.001, respectively) (The suppressive effect of ClpP silencing on cancer cell growth was confirmed by the colony formation assay. The colony formation capabilities of the BC cell lines MDA-MB-231 and ZR-75-1 were markedly inhibited by si-ClpP-B (ctively) –4E.The transwell migration and invasion assay was performed to quantitatively assess cell metastasis and invasiveness. The results showed that cell migration and invasion were significantly reduced in cells transfected with si-ClpP-B, as compared with control cells, due to the down-regulation of MMP7 and vimentin, and the up-regulation of E-cadherin –5T. Convp < 0.01 and <0.001, respectively), accompanied by increased levels of cleaved caspase-9, cleaved caspase-8 and cleaved poly (ADP-ribose) polymerase (PARP) (Flow cytometry was carried out to detect cell apoptosis. The percentages of apoptotic MDA-MB-231 and ZR-75-1 cells were increased by si-ClpP-B (e (PARP) –5N.To confirm the specificity of ClpP siRNAs, the same functional experiments were performed with a second siRNA (si-ClpP-A). The results of the loss-of-function studies were consistent .These data revealed the anti-proliferative, anti-migration, anti-invasion and pro-apoptotic roles of silencing ClpP in BC cells.in vivo by increasing phosphorylation of the key cellular kinases Akt and Src  of 14 analyzed datasets. Importantly, high ClpP expression was correlated with shorter metastasis-free survival in BC patients and reduced RFS in those with lung adenocarcinoma . ConsistClpP is a subunit of the ClpXP complex. The Akt signaling pathway is important in the regulation of various cellular functions, including metabolism, growth, proliferation, survival, transcription, protein synthesis and tumorigenesis . MutatioIn summary, the present study provides the first evidence that ClpP is frequently up-regulated in BC and that ClpP has high diagnostic value.ClpP expression is markedly increased in BC and significantly correlated with the T stage, ER expression, and poor RFS. Silencing of ClpP significantly inhibited proliferation, migration and invasion, and promoted apoptosis of BC cells, resulting in suppression of the Src/PI3K/Akt signaling pathway. These data indicate that ClpP is an oncogene, and may be a promising diagnostic biomarker and therapeutic target in BC.10.7717/peerj.8754/supp-1Figure S1p < 0.01, ***p < 0.001.(A) The overexpressing efficiency in ClpP (MCF-7 and T47D cells) was evaluated by RT-qPCR.  The effects of overexpressed-ClpP on BC cell migration were evaluated using transwell assays.  The effects of overexpressed-ClpP on BC cell invasion were evaluated using transwell assays. The data are presented as mean ± SD, **Click here for additional data file.10.7717/peerj.8754/supp-2Figure S2p < 0.01, ***p < 0.001. The effects of si-ClpP-A on BC cell proliferation were analyzed using the colony formation assay.  The effects of si-ClpP-A on BC cell migration were evaluated using the transwell assay.  The effects of si-ClpP-A on BC cell invasion were evaluated using the transwell assay.  The effect of si-ClpP-A on MDA-MB-231 cells apoptosis was analyzed using flow cytometry. The data are presented as the mean ± SD, **Click here for additional data file.10.7717/peerj.8754/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.8754/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.8754/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.8754/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.8754/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.8754/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.8754/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.8754/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.8754/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.8754/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.8754/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.8754/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.8754/supp-15Supplemental Information 15Click here for additional data file.10.7717/peerj.8754/supp-16Supplemental Information 16Click here for additional data file.10.7717/peerj.8754/supp-17Supplemental Information 17Click here for additional data file.10.7717/peerj.8754/supp-18Supplemental Information 18Click here for additional data file.10.7717/peerj.8754/supp-19Supplemental Information 19Click here for additional data file.10.7717/peerj.8754/supp-20Supplemental Information 20Click here for additional data file.10.7717/peerj.8754/supp-21Supplemental Information 21Click here for additional data file.10.7717/peerj.8754/supp-22Supplemental Information 22Click here for additional data file.10.7717/peerj.8754/supp-23Supplemental Information 23Click here for additional data file.10.7717/peerj.8754/supp-24Supplemental Information 24Click here for additional data file.10.7717/peerj.8754/supp-25Supplemental Information 25Click here for additional data file.10.7717/peerj.8754/supp-26Supplemental Information 26Click here for additional data file.10.7717/peerj.8754/supp-27Supplemental Information 27Click here for additional data file.10.7717/peerj.8754/supp-28Supplemental Information 28Click here for additional data file.10.7717/peerj.8754/supp-29Supplemental Information 29Click here for additional data file.10.7717/peerj.8754/supp-30Supplemental Information 30Click here for additional data file.10.7717/peerj.8754/supp-31Supplemental Information 31Click here for additional data file.10.7717/peerj.8754/supp-32Supplemental Information 32Click here for additional data file.10.7717/peerj.8754/supp-33Supplemental Information 33Click here for additional data file.10.7717/peerj.8754/supp-34Supplemental Information 34Click here for additional data file.10.7717/peerj.8754/supp-35Supplemental Information 35Click here for additional data file.10.7717/peerj.8754/supp-36Supplemental Information 36Click here for additional data file.10.7717/peerj.8754/supp-37Supplemental Information 37Click here for additional data file.10.7717/peerj.8754/supp-38Supplemental Information 38Click here for additional data file.10.7717/peerj.8754/supp-39Supplemental Information 39Click here for additional data file.10.7717/peerj.8754/supp-40Supplemental Information 40Click here for additional data file.10.7717/peerj.8754/supp-41Supplemental Information 41Click here for additional data file.10.7717/peerj.8754/supp-42Supplemental Information 42Click here for additional data file.10.7717/peerj.8754/supp-43Supplemental Information 43Click here for additional data file.10.7717/peerj.8754/supp-44Supplemental Information 44Click here for additional data file.10.7717/peerj.8754/supp-45Supplemental Information 45Click here for additional data file.10.7717/peerj.8754/supp-46Supplemental Information 46Click here for additional data file.10.7717/peerj.8754/supp-47Supplemental Information 47Click here for additional data file.10.7717/peerj.8754/supp-48Supplemental Information 48Click here for additional data file.


Acanthosaura have been recorded so far, including Acanthosauraarmata from the southern region, A.cardamomensis from the eastern region, A.crucigera from the western region, A.lepidogaster from the northern region and A.phuketensis from the Phuket Island and south-western region. However, comprehensive studies of diversity patterns and distribution of Acanthosaura are still lacking in some areas and need further information for designating areas of special conservation importance and nature protection planning in Thailand.In Thailand, five species of Acanthosauraaurantiacrista is a new species of long-horned lizard of the genus Acanthosaura from northern Thailand. It is distinguished from all other species of Acanthosaura by a dagger-like nuchal spine with yellowish-orange colouration in females, bright yellow colouration in males and a combination of other morphological characters: a greater tail length to snout-vent length ratio; a larger postorbital spine, nuchal spine, dorsal spine and occipital spine compared to its head length; a smaller diastema to snout-vent length ratio; a greater number of subdigital lamellae on the fourth finger and fourth toe; and a larger gular pouch than other Acanthosaura species. Analysis of mitochondrial ND2 gene sequences revealed a sister clade between the A.aurantiacrista lineage and the A.crucigera lineage with a 100% probability of divergence, according to Bayesian analysis and strong support value for Maximum Likelihood analysis. The pairwise distance ranged from 13.8-15.0% between A.aurantiacrista and A.cardamomensis, 10.9-14.5% between A.aurantiacrista and A.crucigera and 0-1.2% amongst A.aurantiacrista populations. The discovery of this lizard increases the known endemic herpetological diversity and underscores the importance of conservation in the mountain rainforest region of northern Thailand. Acanthosaura Gray, 1831 are recognised, ranging from Myanmar, east through Thailand, Cambodia, Laos, Vietnam and southern China and southwards through the Malaysian Peninsula and archipelagoes , A.capra , A.cardamomensis Wood, Grismer, Grismer, Neang, Chav & Holden, 2010, A.murphyi Nguyen, Do, Hoang, Nguyen, McCormack, Nguyen, Orlov, Nguyen & Nguyen, 2018, A.nataliae Orlov, Truong, & Sang, 2006 and A.phuketensis Pauwels, Sumontha, Kunya, Nitikul, Samphanthamit, Wood & Grismer, 2015 are classified as a long-horned group exhibiting the largest nuchal and dorsal spines with lengths greater than 10 mm; A.bintangensis Wood, Grismer, Grismer, Northayati, Chan & Bauer, 2009, A.brachypoda Ananjeva, Orlov, Nguyen & Ryabov, 2011, A.crucigera Boulenger, 1885, A.lepidogaster , A.phongdienensis Nguyen, Jin, Vo, Nguyen, Zhou, Che, Murphy, & Zhang, 2019, A.titiwangsaensis Wood, Grismer, Grismer, Northayati, Chan & Bauer, 2009 and A.tongbiguanensis Liu & Rao, 2019 belong to a short-horned group exhibiting nuchal and dorsal spines that are shorter than 10 mm; and the nuchal crest is absent in A.coronata Günther, 1861  or bright yellow  nuchal crest on the dorsum. To clarify the taxonomic status of this Acanthosaura population, we describe the new species on the basis of its distinctive morphological and colouration characteristics and a genetic analysis. Its name is provided to be included in conservation planning for agamid species in wildlife sanctuaries and national parks in the Thanon Thong Chai Mountain Range.A recent field sampling of Specimens were collected by hand from two locations in Thanon Thong Chai Mountain Range, Thailand: one specimen from Mae Sariang District, Mae Hong Son Province on 27 April 2018; seven specimens from Sop Khong Subdistrict, Omkoi District, Chiang Mai Province on 14 November 2018; and one specimen from Nang Lae Subdistrict, Mueang District, Chiang Rai Province on 24 January 2019. For all specimens, photographs were taken to document their colour patterns prior to euthanisation. Liver samples were taken and stored in absolute ethanol. Specimens were fixed in 10% formalin before being transferred to 70% ethanol for permanent storage.Acanthosaura in Thailand were examined from the Natural History Museum, National Science Museum, Technopolis, Pathum Thani Province (THNHM) and the Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok Province, Thailand (QSMI): Acanthosauraarmata from Hala-Bala, Narathiwat Province (THNHM15209), Sungai Kolok District, Narathiwat Province (THNHM18884); Acanthosauracardamomensis from Koh Kut, Trat Province , Khao Yai National Park, Nakhon Ratchasrima Province ; Acanthosauracrucigera from Na Yong District, Trang Province , Taksin Maharat National Park, Muang District, Tak Province , Thong Pha Phum District, Kanchanaburi Province (THNHM22658), Huai Kha Khaeng, Lan Sak District, Uthai Thani Province (THNHM18594); Acanthosauralepidogaster from Phu Luang District, Loei Province , Phu Kieo District, Chaiyaphum Province (THNHM19619), Ban Sun Phae Kae, Chiang Dao District, Chiang Mai Province (THNHM20537), Roi Praputabath, Umphang District, Tak Province (THNHM20647), Huai Na Tee, Pua District, Nan Province (THNHM10080), Doi Khun Tan National Park, Lam Phun Province ; Acanthosauraphuketensis from Ton Sai Waterfall, Thalang District, Phuket Province (THNHM08865), Khao Sok, Ban Ta Khun, Surat Thani Province (THNHM22663); and Acanthosauranataliae from Xe Sap, Samoy, Lao .Comparative materials of all currently recognised species of Acanthosaura were also taken from original descriptions and subsequent studies  obtained from GenBank  was prepared using an EP0402 TAQ DNA POLYMERASE. Two primers, METF6  and ACANTHND2.833. R1 (5’-AGGGAGGTTATTGTTGCTAG-3’), were used to amplify a 698 bp fragment of the NADH dehydrogenase subunit 2 (ND2) gene . PCR proThe ND2 sequences were aligned using ClustalW v.1.83  integratBayesian Interference was performed in Mr.Bayes v.3.1.2 , based o212B9A56-ACDD-536A-958C-C3115F384365urn:lsid:zoobank.org:act:E0D80F05-8884-4426-B2D6-6C939310D2A4Type status:Holotype. Location: country: Thailand (northern region); stateProvince: Mae Hong Son Province; county: Mae Sariang District; verbatimElevation: 728 m; verbatimLatitude: 18°09'02.8"N; verbatimLongitude: 97°58'50.2"E; verbatimCoordinateSystem: degrees minutes seconds; verbatimSRS: WGS84; Event: year: 2018; month: April; day: 27; habitat: evergreen forests on hills up to at least 600 m elevation; fieldNotes: collector = Poramad Trivalaira; Record Level: institutionCode: THNHM; collectionCode: 28064; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = adult femaleType status:Paratype. Location: country: Thailand (northern region); stateProvince: Chiang Mai Province; county: Omkoi District; locality: Sop Khong Subdistrict; verbatimElevation: 935; verbatimLatitude: 17°39'45.4"N; verbatimLongitude: 98°11'53.6"E; verbatimCoordinateSystem: degrees minutes seconds; verbatimSRS: WGS84; Event: year: 2018; month: November; day: 14; habitat: evergreen forests on hills up to at least 600 m elevation; fieldNotes: collector = Kirati Kunya; Record Level: institutionCode: THNHM; collectionCode: 28521; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = adult femaleType status:Paratype. Record Level: institutionCode: THNHM; collectionCode: 28522; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = adult female, same collection date, collector and location as paratype THNHM28521Type status:Paratype. Record Level: institutionCode: THNHM; collectionCode: 28523; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = subadult male, same collection date, collector and location as the THNHM28521Type status:Paratype. Record Level: institutionCode: THNHM; collectionCode: 28524; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = subadult male, same collection date, collector and location as the THNHM28521Type status:Paratype. Record Level: institutionCode: QSMI; collectionCode: 1446; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = adult female, same collection date, collector and location as the THNHM28521Type status:Paratype. Record Level: institutionCode: QSMI; collectionCode: 1447; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = adult female, same collection date, collector and location as the THNHM28521Type status:Paratype. Record Level: institutionCode: QSMI; collectionCode: 1448; basisOfRecord: PreservedSpecimen; dynamicProperties: sex = adult male, same collection date, collector and location as the THNHM28521Acanthosauraaurantiacrista sp. n.  ; differences of 13.8-15.0% compared to two specimens of A.cardamomensis (GenBank GU817397 and GU817400); differences of 16.2-16.3% compared to two specimens of A.armata (GenBank AB266452 and NC014175); differences of 18.0-19.6% compared to two specimens of A.lepidogaster (GenBank AF128499 and KR092427); and differences of 19.1-19.8% compared to a specimen of A.capra (GenBank AF128498) (Table 4). The phylogenetic relationships within the genus Acanthosaura revealed through Maximum-Likelihood trees and Bayesian Inference tree of the ND2 gene showed high posterior probabilities and high bootstrap support values , more FI (17-23 vs. 13-17), more TO (25-29 vs. 19-26), fewer NS (5-6 vs. 6-10), fewer NCS (11-13 vs. 10-17) and the presence of a BEP and more GP (1-4 vs. 1) , greater PS/HL ratio (0.24-0.84 vs. 0.07-0.19), greater DS/HL ratio (0.15-0.38 vs. 0.08-0.09), fewer DIASN (8-9 vs. 11-15), more VENT (63-66 vs. 51-55), fewer NSSOS (5 vs. 6-7), fewer NS (5-6 vs. 8) and the presence of a LKP.Acanthosauraaurantiacrista sp. n. differs from A.brachypoda in presenting greater PS/HL ratio (0.24-0.84 vs. 0.11), greater DS/HL ratio (0.15-0.38 vs. 0.06), fewer RS (4-6 vs. 5-9), fewer NS (5-6 vs. 9) and more GP (1-4 vs. 0).Acanthosauraaurantiacrista sp. n. differs from A.capra in presenting fewer INFRAL (9-11 vs. 12-13), more FI (17-23 vs. 16-17), more TO (25-29 vs. 22-24), fewer NS (5-6 vs. 9) and the presence of an occipital spine and scales surrounding the occipital spine.Acanthosauraaurantiacrista sp. n. differs from A.cardamomensis in presenting fewer RS (4-6 vs. 7-9), fewer NS (5-6 vs. 7-10) and fewer NSSLC (9-13 vs. 10-19).Acanthosauraaurantiacrista sp. n. differs from A.coronata in presenting greater TaL/SVL ratio (1.40-1.70 vs. 0.60-1.00), more FI (17-23 vs. 13-14), more TO (25-29 vs. 17-19), fewer RS (4-6 vs. 9), fewer NS (5-6 vs. 7-9), fewer NR (1-2 vs. 3-4) and the presence of a postorbital spine, nuchal spine, dorsal spine, diastema, occipital spine, YAS, ND, BEP and more GP (1-4 vs. 0).Acanthosauraaurantiacrista sp. n. differs from A.crucigera in presenting fewer DIASN (8-9 vs. 9-25), more VENT (63-66 vs. 55-63), more FI (17-23 vs. 16-18), more TO (25-29 vs. 21-26), fewer RS (4-6 vs. 7-8), fewer NS (5-6 vs. 7-9) and more GP (1-4 vs. 1-2).Acanthosauraaurantiacrista sp. n. differs from A.lepidogaster in presenting greater TaL/SVL ratio (1.40-1.70 vs. 1.00-1.50), greater PS/HL ratio (0.24-0.84 vs. 0.06-0.17), greater NSL/HL ratio (0.35-0.95 vs. 0.12-0.15), greater DS/HL ratio (0.15-0.38 vs. 0.06-0.15), fewer DIASN (8-9 vs. 10-14), more VENT (63-66 vs. 52-61), more TO (25-29 vs. 22-23), greater OS/HL ratio (0.19-0.44 vs. 0.14-0.15), fewer NS (5-6 vs. 7-8), fewer PM (4 vs. 5), absence of scale on tympanum and more GP (1-4 vs. 0-1).Acanthosauraaurantiacrista sp. n. differs from A.murphyi in presenting greater PS/HL ratio (0.24-0.84 vs. 0.16-0.34), greater NSL/HL ratio (0.35-0.95 vs. 0.24-0.43), fewer INFRAL (9-11 vs. 12-14), more FI (17-23 vs. 15-18), more TO (25-29 vs. 21-23), fewer RS (4-6 vs. 8-9), fewer NS (5-6 vs. 7-8), fewer NR (1-2 vs. 3-4) and the absence of a tympanum scale.Acanthosauraaurantiacrista sp. n. differs from A.nataliae in presenting fewer VENT (63-66 vs. 64-71), fewer RS (4-6 vs. 7), fewer NSSLC (9-13 vs. 13-16) and the presence of an occipital spine, scales surrounding the occipital spine, ND and LKP.Acanthosauraaurantiacrista sp. n. differs from A.phongdienensis in presenting greater PS/HL ratio (0.24-0.84 vs. 0.06-0.09), greater NSL/HL ratio (0.35-0.95 vs. 0.07-0.18), larger DS/HL ratio (0.15-0.38 vs. 0.03-0.07), more FI (17-23 vs. 14-17), more TO (25-29 vs. 19-23) and the presence of a diastema.Acanthosauraaurantiacrista sp. n. differs from A.phuketensis in presenting greater NSL/HL ratio (0.35-0.95 vs. 0.21-0.39), fewer DIASN (8-9 vs. 12-17), more FI (17-23 vs. 15-17), more TO (25-29 vs. 21-24), fewer RS (4-6 vs. 5-9), fewer NS (5-6 vs. 7-8) and more GP (1-4 vs. 1-2).Acanthosauraaurantiacrista sp. n. differs from A.titiwangsaensis in presenting greater PS/HL ratio (0.24-0.84 vs. 0.14-0.18), greater NSL/HL ratio (0.35-0.95 vs. 0.11-0.18), greater DS/HL ratio (0.15-0.38 vs. 0.07-0.09), fewer DIASN (8-9 vs. 10-13), more VENT (63-66 vs. 47-57), fewer NS (5-6 vs. 8) and the presence of a LKP.Acanthosauraaurantiacrista sp. n. differs from A.tongbiguanensis in presenting greater PS/HL ratio (0.24-0.84 vs. 0.13-0.19), greater NSL/HL ratio (0.35-0.95 vs. 0.15-0.21), greater OS/HL ratio (0.19-0.44 vs. 0.16-0.23), fewer RS (4-6 vs. 6-9), fewer NS (5-6 vs. 8-9) and more GP (1-4 vs. 1-2).Acanthosauraaurantiacristasp. n. is differentiated from all other congeners by this combination of characters: A large size  and a single long conical spine above the posterior margin of the eye; a large spine on the occiput between the tympanum and nuchal crest; tympanum naked, large, roundish; large developed gular pouch; scales on flanks randomly intermixed with small keeled and small tubercle scales; large nuchal crest with 8 large dagger-like and pointed spines; narrow diastema with 8-9 scales between the nuchal and vertebral crests; vertebral crest composed of large dagger-like, pointed spines beginning at the shoulder region and decreasing in size until the base of the tail; nuchal and dorsal crests are orange in females and yellow in males; tail 1.40-1.70 times the SVL; and black collar and black eye patch present, extending posteriorly until reaching the nuchal crest.Description of the holotype: Adult female. SVL 105.7 mm; TaL 151.8 mm (1.44 times SVL), tail complete; HL (23.3 mm) slightly longer than HW (18.9 mm); HL one-fifth SVL (0.22 times SVL), HW narrow (0.179 times SVL) and HD tall (0.64 times HL); head triangular in dorsal and lateral views; SL moderately long (0.46 times HL); RW wide (2.31 times RH); steeply sloping anteriorly; CS prominent, forming a large projecting shelf extending above eye, composed of 14/13 large scales; shelf terminates with a notch anterior to postorbital spine; rostrum moderate in size, rectangular, bordered laterally by first SUPRALs and posteriorly by five smaller scales; nostrils roundish, surrounded by one prenasal anteriorly, four postnasals posteriorly and two subnasals; six NS; oval supranasals; large scales above orbit weakly keeled; three rows of moderately-keeled scales below orbit extending from the anterior margin of the eye to posterior; large EYE (0.28 times HL) and ORBIT (0.44 times HL); interorbital, prefrontal and frontal scales slightly keeled and smaller than scales below orbit; seven large, keeled, azygous prefrontal scales arranged in a Y-shaped pattern; parietal eyespot surrounded by a larger row of scales; large conical PS above posterior margin of the eye surrounded by five small lanceolate scales; single row of seven large keeled scales extending from suborbital below posterior margin of eye to above tympanic margin, increasing in size posteriorly; elongated conical OS on lateral margin of nape surrounded by a rosette of five small lanceolate NSSOS; tympanum exposed, roundish, with a size two-thirds that of EYE (0.69 times EYE), surrounded by tiny conical scales; thirteen rectangular SUPRALs similar in size; mental pentagonal, larger than adjacent INFRALs; two postmentals similar in size, four scales contacting PM; chin shields large, extending posteriorly to angle of jaw, separated from infralabials by one scale row anteriorly and three at angle of jaw; eleven rectangular INFRALs of similar size; gular scales sharply keeled and spinose with a larger midventral row; extensible dewlap present; nuchal crest composed of eight elongated, dagger-like scales, bordered on each side by two rows of large, flat, keeled, triangular scales; nuchal crest followed by a diastema of nine DIASN at base of nape; dorsal body crest extending from posterior margin of diastema to base of tail; dorsal crest composed of small laterally compressed, triangular epidermal scales, bordered by a row of smaller paravertebral triangular scales; DS slightly decreasing to sacrum, then fading progressively; and nuchal and dorsal crests present as orange in live specimen  and OS (5.3-7.5 vs. 4.5) and greater NSL (9.6-12.7 vs. 8.3), wider WNC (0.9-1.6 vs. 0.6), more SUPRAL (10-11/10 vs. 13/13) and fewer number of FI (18-20/17-20 vs. 23/21), higher number of NSSLC (10 vs. 13) in QSMI1446, QSMI1447, THNHM28521 and THNHM28522, and larger GP (3 vs. 1) in QSMI1446 and THNHM28522. The adult male paratype (QSMI1448) differs from the adult female holotype in presenting a longer SVL (130.1 vs. 105.7), greater TaL (202.2 vs. 151.8) and SL (22.3 vs. 10.7), wider TBW (19.2 vs. 10.3), greater HD (21.7 vs. 14.9), larger ORBIT (8.5 vs. 6.4), longer PS (19.1 vs. 5.7), OS (10 vs. 4.5), greater NSL (21.6 vs. 8.3), FOREL (54.2 vs. 49.8), HINDL (71.4 vs. 59.7) and GP (4 vs. 1) size, wider WNC (2.9 vs. 0.6), narrower DIAS (3.5 vs. 5.4) and fewer number of SUPRAL (10 vs. 13) and FI (19/18 vs. 23/21). Two subadult male paratypes are smaller and present fewer differences in morphological characters compared with the holotype, except for a longer PS (5.5-7.5 vs. 5.7) and OS (5.9 vs. 4.5) in THNHM28523, a greater WNC (0.7-0.8 vs. 0.6) in THNHM28523 and THNHM28524 and higher number of TO (29/29 vs. 27/26)  and crista (crest). The name refers to a distinctive characteristic of the first discovered female specimen, which exhibited nuchal and dorsal crests with an orange colour. We suggest the following common names: kingkakhaownaam seesom (Thai), orange crested horned lizard (English), orange-verzierter gehörnter Nackenstachler (German) and Acanthosaurus à crête orange (French).The specific epithet Acanthosauraaurantiacrista sp. n. occurs in the Thanon Thong Chai Mountain Range in northern Thailand: Mae Sariang District, Mae Hong Son Province  at 728 m a.s.l.; Sop Khong Subdistrict, Omkoi District, Chiang Mai Province  at 935 m a.s.l.; and Nang Lae Subdistrict, Mueang District, Chiang Rai Province  at 636 m a.s.l. This species usually lives in rainforests on mountains at elevations over 600 m a.s.l. . This herpetological discovery yields the sixth member of the genus Acanthosaura from Thailand and the taxa in this genus that have not yet been identified, will be described in the near future.The comparisons revealed morphological characters of a new long-horned lizard species, Thailand . In addiAcanthosauraaurantiacrista sp. n. is expected to distribute throughout the Thanon Thong Chai Mountain Range, which includes several important protected areas where the biological fauna is preserved in northern regions of Thailand, such as Doi Inthanon National Park; Doi Suthep-Pui National Park; Khun Khan National Park; Mae Ngao National Park; Mae Ping National Park; Mae Tho National Park; Mae Wang National Park; Namtok Mae Surin National Park; Op Luang National Park; Op Khan National Park; Chiang Dao Wildlife Sanctuary; Lum Nam Pai Wildlife Sanctuary; Mae Lao-Mae Sae Wildlife Sanctuary; Mae Tuen Wildlife Sanctuary; and Om Koi Wildlife Sanctuary. Moreover, our research team recommends this new long-horned species, whose nuchal and dorsal crests have an appearance similar to fire, as a representative animal for drawing the attention of the local people, scientific community and government towards addressing the current problem of forest burning in northern regions of Thailand.Many forest areas in northern regions of Thailand are currently faced with deforestation due to timber logging and forest fire for human use, which negatively influences the conservation of the herpetological fauna . However6CF57014-D7C4-5583-91CF-B24FDE50E47610.3897/BDJ.8.e48587.suppl1Supplementary material 1Acanthosaura and Acanthosauraaurantiacrista sp. n., “?” = data not available.Comparison of morphometric (in mm) and meristic data for all currently recognised species of Data typemorphologicalBrief descriptionAcanthosaura lizardThe comparison table of morphometric for File: oo_395107.docxhttps://binary.pensoft.net/file/395107Poramad Trivalairat


Callitris and Widdringtonia, the diterpene acids from Widdringtonia have never been described and no comparison to the Australian clade sister genus Callitris has been made. The critically endangered South African Clanwilliam cedar, Widdringtonia wallichii (syn. W. cedarbergensis), of the Cederberg Mountains was once prized for its enduring fragrant timbers and an essential oil that gives an aroma comparable to better known Mediterranean cedars, predominantly comprised by widdrol, cedrol, and thujopsene. In South Africa, two other ‘cedars’ are known, which are called W. nodiflora and W. schwarzii, but, until now, their chemical similarity to W. wallichii has not been investigated. Much like Widdringtonia, Callitris was once prized for its termite resistant timbers and an ‘earthy’ essential oil, but predominantly guaiol. The current study demonstrates that the essential oils were similar across all three species of Widdringtonia and two known non-volatile diterpene acids were identified in leaves: the pimaradiene sandaracopimaric acid (1) and the labdane Z-communic acid (2) with a lower yield of the E-isomer (3). Additionally, in the leaves of the three species, the structures of five new antimicrobial labdanes were assigned: 12-hydroxy-8R,17-epoxy-isocommunic acid (4), 8S-formyl-isocommunic acid (5), 8R,17-epoxy-isocommunic acid (6), 8R-17R-epoxy-E-communic acid (7), and 8R-17-epoxy-E-communic acid (8). Australian Callitris columellaris (syn. C. glaucophylla) also produced 1 and its isomer isopimaric acid, pisiferal (9), and pisiferic acid (10) from its leaves. Callitris endlicheri (Parl.) F.M.Bailey yielded isoozic acid (11) as the only major diterpene. Diterpenes 4–6, pisiferic acid (10), spathulenol, and guaiol (12) demonstrated antimicrobial and acaricidal activity.In spite of the evidence for antimicrobial and acaricidal effects in ethnobotanical reports of  Widdringtonia were once prized for their timbers and have been heavily exploited over the course of more than a century. The most pronounced impact has been on the now critically endangered W. wallichii Endl. ex Carrière (=W. cedarbergensis J.A.Marsh) + ; 8S-formyl-isocommunic acid (5), HRESIMS m/z 341.2094 [M + Na]+ , 8R,17-epoxy-isocommunic acid (6), HRESIMS m/z 319.2258 [M + H]+ , 8R-17-epoxy-E-communic acid (7), HRESIMS m/z 341.2081 [M + Na]+ , 8R-17-epoxy-Z-communic acid (8), HRESIMS m/z 341.2081 [M + Na]+ .Five of the compounds are undescribed. These are structures 4–8, which were all isolated as amorphous translucent white solids: 12-hydroxy-8Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 29213 & a methicillin resistant strain), Pseudomonas aeruginosa (ATCC 27703), Bacillus subtilis (University of New England strain), and Escherichia coli (ATCC 25922). The positive control used was tetracycline.The minimum inhibitory concentration (MIC) method described by Eloff , which im/z 30–400.Relative abundances of essential oil components and esterified diterpene acids were studied using gas chromatography with mass spectrometric detection (GC-MS). GC-MS analyses were performed using an Agilent Technologies 7890A GC-System coupled with an Agilent 5975C mass selective detector . An autosampler unit (Agilent Technologies 7693-100 positions) held samples. Separation of 1-μL injections used an HP-5MS Agilent column (30 m × 250 μm × 0.25 μm). Operating conditions were as follows: injector split ratio 25:1, temperature 250 °C, carrier gas helium, 1.0 mL/min, and constant flow. Column temperature was 50 °C (no hold) and 5 °C per minute. Then, at 280 °C, it was held at 5 min. Mass fragmentation patterns were acquired at −70 eV using a mass scan range of n-alkanes, when compared with values published in Adams [Primary identifications were performed by comparison of mass spectra with an electronic library database  and confin Adams  by visuain Adams . Semi-quHRESIMS spectra were recorded using an AB Sciex 5600 TripleTOF mass spectrometer in positive mode.


Acinetobacter baumannii (XDRAB), isolated in Thailand. These results revealed multiple antimicrobial-resistant genes, each involving two sequence type 16 (ST16) isolates, ST2, and a novel sequence type isolate, ST1479.Here, we report the complete genome sequences of four clinical isolates of extensively drug-resistant  Acinetobacter baumannii (XDRAB), isolated in Thailand. These results revealed multiple antimicrobial-resistant genes, each involving two sequence type 16 (ST16) isolates, ST2, and a novel sequence type isolate, ST1479.Here, we report the complete genome sequences of four clinical isolates of extensively drug-resistant  Acinetobacter baumannii is one of the multidrug-resistant bacteria listed as a priority by the World Health Organization, as it exhibits resistance to most commercially available antibiotics and causes hospital-acquired infections (–Acinetobacter baumannii (CRAB) infections present limited therapeutic options and are associated with high morbidity and mortality as well as longer hospitalization (A. baumannii (XDRAB) infections have been reported worldwide, including in Thailand (n = 3) and bile (n = 1) samples by a tertiary hospital in northeastern Thailand between 2017 and 2018 (gyrB multiplex PCR (n = 2) and no clonal group (n = 2) based on a multiplex PCR assay . Quality control of ONT reads was undertaken using NanoPlot v1.28.1 (https://github.com/wdecoster/NanoPlot). For the Illumina platform, the sequencing library was generated using the NEBNext Ultra II DNA library prep kit for Illumina  following the manufacturer’s recommendations. We applied Fastp v0.19.5 (https://github.com/relipmoc/skewer). Quality checking of the Illumina reads was performed using FastQC v0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Hybrid assemblies with the ONT and Illumina data were performed using Unicycler v0.4.8 (https://github.com/rrwick/Unicycler/releases), and the genome sequences were checked for quality using QUAST v5.0.2 (http://quast.sourceforge.net/). Complete circular DNA structures of the bacterial chromosomes and plasmids were automatically produced using Unicycler software. The genome sequences were submitted to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.12 for annotation. Default parameters were used for all software unless otherwise specified.Bacterial genomic DNA samples extracted using ZymoBIOMICS DNA kits  were sequenced using the Oxford Nanopore Technologies (ONT) and Illumina platforms. Library preparation for ONT sequencing followed the rapid barcoding DNA sequencing protocol with the SQK-RBK004 kit without DNA size selection, to preserve the plasmid DNA, and the libraries were sequenced using a single R9.4.1/FLO-MIN106 flow cell on a MinION Mk1B sequencer. We base called and demultiplexed the raw data using Guppy v3.4.5 (ONT), specifying the high-accuracy model (-c dna_r9.4.1_450bps_hac.cfg). The ONT adapters were trimmed using Porechop v0.2.4 ( v0.19.5  (https:/r v0.4.8  (https:/T v5.0.2  (http://https://cge.cbs.dtu.dk/services/ResFinder/) and CARD (https://card.mcmaster.ca/): blaNDM-1, blaOXA-23, blaOXA-66, blaTEM1D, blaVEB-1, blaIMP-14, blaADC-25, sul1, sul2, cmlA1, mphE, msrE, ARR2, tetB, tet(39), aac(3)-IId, aadA1, ant(2″)-Ia, aph(3′)-Ia, aph(3″)-Ib, aph(3′)-VI, aph(6)-Id, and armA. The multilocus sequence type (MLST) findings using the PubMLST database indicated that two isolates were ST16 (or ST355), and the other isolates were ST2 (or ST195) and ST1479 (new sequence type), according to the Pasteur MLST scheme (with the Oxford MLST scheme in parentheses) (https://pubmlst.org/abaumannii/).All isolates contained plasmids, and we detected seven plasmids in one isolate . From thand CARD  (https:/ntheses)  (https:/PRJNA647677. The accession numbers and assembly statistics are provided in These sequences have been submitted to GenBank under BioProject accession number


Doppler echocardiographic (Echo) measurements of the mitral and pulmonary venous flow have been used to assess diastolic dysfunction (DD). Velocity-encoded cardiac magnetic resonance (Ve-CMR) can measure similar parameters and may potentially be used for the assessment of DD.To assess the correlation of Ve-CMR with Echo in assessing mitral inflow E/A ratio, left atrial (LA) size, and pulmonary vein (PV) flow.We prospectively enrolled 22 outpatients with a mitral inflow pattern by Echo that was normal (8), impaired relaxation (7), pseudo-normal (2), or restrictive (5). The mean left ventricular ejection fraction (LVEF) was 60.8 and two patients had left ventricular hypertrophy on echo. Ve-CMR was performed within 2 hours of the Echo. The following indices were measured: LA size, mitral inflow E-velocity (E), A-velocity (A), E/A ratio, PV waves .The mean age of the patients was 51 years. The average Echo mitral inflow absolute velocities were close to double the Ve-CMR {Echo E-velocity = 85.8 cm/sec vs. Ve-CMR E velocity = 41.4 cm/sec, Echo A-velocity 70 cm/sec vs. Ve-CMR A-velocity 32.5 cm/sec}. The average E/A ratio was 1.41 by echocardiogram and 1.48 by Ve-CMR. There was a significant correlation in LA size and Mitral inflow E/A ratio between Ve-CMR and Echo . There were no significant correlations in PV waves . Figure 1Ve-CMR has a very high correlation with Echo in the assessment of mitral inflow E/A ratio and LA size. Ve-CMR maybe useful for the assessment of diastolic dysfunction.


EGFR mutation-positive NSCLC at a major cancer center. This study showed the efficacy of 1G or 2G EGFR-TKIs as the 1L treatment, and subsequent therapy including 3G EGFR-TKIs in the real-world setting.The present study showed the comprehensive analysis of disease characteristics and treatment patterns in uncommon EGFR) mutations in non-small-cell lung cancer (NSCLC) are uncommon EGFR mutations. Although the efficacy of second (2G) or third generation (3G) EGFR tyrosine kinase inhibitors (EGFR-TKIs) in the patients with uncommon EGFR mutation has been proven, further studies are warranted to define the optimal treatment approach for uncommon EGFR mutation-positive NSCLC. This study retrospectively investigated the treatment patterns and outcomes of patients with uncommon EGFR mutation-positive NSCLC from January 2011 to December 2019 at the Samsung Medical Center, Seoul, Korea. During the study, 2121 patients with EGFR mutation-positive NSCLC received first-generation  or 2G EGFR-TKI (afatinib) as the first-line (1L) systemic therapy. Of this, 135 (6.4%) patients harbored uncommon EGFR mutations. Of 135, 54  patients had overlapping mutations with major EGFR mutations. The objective response rate (ORR) for the 1L EGFR-TKI was 63.3%. The median progression-free survivals (PFSs) were 8.6 months (95% CI: 3.8–13.5), 11.7 months (95% CI: 6.6–16.7), 7.7 months (95% CI: 4.9–17.4), and 5.0 months (95% CI: 3.7–6.1) for major uncommon EGFR mutation , compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The median overall survivals (OSs) were 25.6 months (16.9–34.2), 28.8 (95% CI: 24.4–33.4), 13.5 months (95% CI: 7.4–27.8), and 9.4 months (95% CI: 3.4–10.5) for major uncommon EGFR mutation (G719X), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The response rate, median PFS, and OS were 63.3%, 16.3 months (95% CI: 15.6–16.9), and 37.5 months (95% CI: 35.4–39.6) for common EGFR mutation-positive NSCLC. After failing 1L EGFR-TKI, repeated tissue or liquid biopsy were carried out on 44.9% (35/78) of patients with T790M detected in 10/35 (28.6%) patients. With subsequent 3G EGFR-TKI after failing the first-line EGFR-TKI, the ORR and PFS for 3G EGFR-TKI were 80% and 8.9 months (95% CI: 8.0–9.8). These patients showed a median OS of 34.6 months (95% CI: 29.8–39.4). The ORR, PFS and OS were poorer in patients with uncommon  than those with common EGFR mutations. T790M was detected in 28.6% of the uncommon EGFR mutation-positive patients for whom prior 1G/2G EGFR-TKIs failed and underwent repeat biopsy at the time of progression.Approximately 10% of the epidermal growth factor receptor ( Uncommon EGFR mutations account for 7–23% of EGFR mutation-positive NSCLCs. Uncommon EGFR mutations can be categorized as follows: (i) de novo T790M; (ii) exon 20 insertions; (iii) “major” uncommon mutations Gly719Xaa (G719X), Leu861Gln (L861Q), and Ser768Ile (S768I); (iv) compound mutations; (v) other uncommon mutations [Exon 19 deletions (Del19) and epidermal growth factor receptor EGFR mutation-positive NSCLC has been revolutionized with the development of next-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs). Currently, five EGFR-TKIs are available as the first-line (1L) therapy for advanced EGFR mutation-positive NSCLC—first-generation (1G) reversible EGFR-TKIs, gefitinib, and erlotinib; second-generation (2G) irreversible ErbB family blockers, afatinib, and dacomitinib; third-generation (3G) irreversible EGFR-TKIs, osimertinib. Of the EGFR-TKI prospective randomized trials undertaken to date, only Iressa Pan-Asia Study (IPASS) [EGFR mutations. However, these studies included a small number of cases with uncommon EGFR mutations. Thus, it is unclear whether it is the best practice to use 2G or 3G EGFR-TKIs as the treatment of choice.The treatment of neration G irreverneration G irrever (IPASS) ,5 includEGFR-mutation positive NSCLC, recently, the highly anticipated findings of the phase III FLAURA trial showed 38.6 months of overall survival (OS) with frontline osimertinib, a 3G EGFR-TKI, versus 31.8 months with erlotinib or gefitinib [EGFR mutation-positive NSCLC who received sequential afatinib/osimertinib. The median OS was 41.3 months : 36.8–46.3) in the total population and 45.7 months (90% CI: 45.3–51.5) in patients with Del 19 [As to which treatment strategy is the best for the patients with major EGFR-TKIs and best sequential treatment, the bulk of these trials were limited to patients whose tumors harbored common EGFR mutations.While robust data from clinical trials have demonstrated the efficacy, tolerability, and benefits of EGFR-TKIs, followed by sequential treatment including 3G EGFR-TKI and cytotoxic chemotherapy in patients with uncommon EGFR mutation-positive NSCLC. In this study, we describe the real-world data of practice pattern and treatment outcomes in patients with uncommon EGFR mutation-positive NSCLC, including objective response rate (ORR), progression-free survival (PFS), and OS.To the best of our knowledge, there have been no prospective studies on the use of 1G or 2G EGFR mutation-positive NSCLC received gefitinib, erlotinib, or afatinib as 1L chemotherapy at the Samsung Medical Center. A total of 135 (6.4%) patients harbored uncommon EGFR mutations such as G719A/G719C/G719X, L861Q, S781I, H351I, E709K, and de novo T790M. The types of uncommon EGFR mutations are described in EGFR insertion 20 who received EGFR-TKIs as a 1L treatment. We classified patients with uncommon EGFR mutation into the major uncommon EGFR mutation , L861Q alone (n = 18), compound mutation with major EGFR mutation , other compound mutations (n = 13), other uncommon mutations (n = 2) and T790M (n = 15). There were 47.4% (64/135) male, and ex- or current smokers were 44.4% (60/135). About 25.9% of patients had symptomatic or asymptomatic brain metastasis at initial diagnosis.From January 2011 to December 2019, 2121 patients with EGFR-TKIs was 63.3% (76/120). The median PFS was 11.1 months (95% CI: 7.2–15.0) for uncommon EGFR mutation-positive NSCLC , compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), compound mutation , and other uncommon mutations , respectively. The median OS were 25.6 months (16.9–34.2), 28.8 (95% CI: 24.4–33.4), 13.5 months (95% CI: 7.4–27.8), and 9.4 months (95% CI: 3.4–10.5) for major uncommon EGFR mutation (G719X), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), compound mutation, and other uncommon mutations, respectively. From October 2014 to December 2019, in the uncommon EGFR mutation-positive NSCLC group, the median PFS was 15.1 months (95% CI: 12.5–17.7) for afatinib and 7.7 months (95% CI: 1.3–14.1) for gefitinib or erlotinib (p = 0.165). The median OS was 25.6 months (95% CI: 18.2–33.0) for uncommon EGFR mutation-positive NSCLC  for afatinib and 15.5 months (95% CI: 9.0–22.0) for gefitinib or erlotinib (p = 0.03). There was no significant difference in sequential treatment after failing 1L treatment among the three EGFR-TKIs.The ORR  for the 1L 1G or 2G ve NSCLC . The medrlotinib A  patients (EGFR-TKIs (8/10 patients had overlapping EGFR mutations with Del 19 or EGFR exon 21 p.L858R), seven patients who did not have T790M received other EGFR-TKIs , 41 patients received cytotoxic chemotherapy, and 20 patients did not receive any sequential treatment.Among 120 patients (except de novo T790M), 78 experienced disease progression until January 2020. Among them, patients . T790M wEGFR-TKIs and cytotoxic chemotherapy, respectively (p = 0.05). In 58 patients who experienced disease progression and received sequential treatment, the median OS2 was 15.1 months (95% CI: 9.0–21.2) and 11.0 months (95% CI: 5.1–16.9) for 3G EGFR-TKIs and cytotoxic chemotherapy, respectively (p = 0.214). The median OS was 34.6 months (95% CI: 29.8–39.4), 24.4 months (95% CI: 17.4–31.4), and 5.3 months (95% CI: 2.9–7.7) for 3G EGFR-TKIs, cytotoxic chemotherapy, and no sequential treatment, respectively (p < 0.001).In 58 patients who experienced disease progression and received sequential treatment, the median PFS2 was 8.9 months (95% CI: 8.0–9.8) and 4.2 months (95% CI: 2.3–6.2) for third-generation ectively A (p = 0.ectively B (p = 0.EGFR-TKIs after failing the 1L EGFR-TKI treatment. The median PFS was 4.9 months (95% CI: 3.8–6.0), and the median OS was 24.0 months (95% CI: 0.7–4.8). Patients who received 3G EGFR-TKIs showed a median OS of 38.0 months (95% CI: 10.5–65.4).Among the 15 patients with de novo T790M mutation, 11 patients had an EGFR exon 21 p.L858R mutation and four patients had a Deletion 19 as a coexisting mutation. As a 1L treatment, seven patients received gefitinib, one patient received erlotinib, and seven patients received afatinib. Among them, eight patients received 3G EGFR mutation group, there were relatively more male patients and current/ex-smokers than in the common EGFR mutation group (EGFR mutation group was 66.6% (75% between October 2014 and December 2019), and in the uncommon EGFR mutation group it was relatively low at 44.9%. Although there was a difference between the two groups in the re-biopsy rate, the T790M detection rate in the patients who underwent re-biopsy was 63.5% in the common EGFR mutation group but was 28.6% in the uncommon EGFR mutation group. The response rate was 63.3% and 86.6% for the uncommon and common EGFR mutation groups, respectively. The median PFS was 16.3 months (95% CI: 15.6–16.9) and 11.1 months (95% CI: 7.2–15.0) for common EGFR mutation-positive NSCLC and uncommon EGFR mutation-positive NSCLC, respectively (p < 0.001).In the uncommon on group . During ectively  . In the patients who received 3G EGFR-TKIs after failing 1L EGFR-TKIs, the median OS was 44.4 months (95% CI: 38.9–49.9) and 34.6 months (95% CI: 29.8–39.4) for common EGFR mutation-positive NSCLC and uncommon EGFR mutation-positive NSCLC, respectively.In the overall population, the median OS was 37.5 months (95% CI: 35.4–39.6) and 25.6 months (95% CI: 18.2–33.0) for common ectively  , compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), afatinib showed superior OS results. In our study, less than one-third of the patients with uncommon EGFR mutation-positive NSCLC had a T790M mutation in the re-biopsy after failing 1L EGFR-TKIs. Most T790M mutation positive cases had overlapping mutations with major (Del 19 or EGFR exon 21 p.L858R) and uncommon EGFR mutations. The rate of T790M mutations after failing 1L EGFR TKI treatment in our study was only 28.6%, relatively lower than that of common EGFR mutation [EGFR mutation-positive NSCLCs than in common EGFR mutation-positive NSCLCs (27.1 % vs. 45.2%) [EGFR mutations after failing EGFR-TKI is lower than in patients with common EGFR mutations.The median ORR, PFS and OS of patients with uncommon ve NSCLC . The PFS G719X, L61Q, compmutation ,11. In a. 45.2%) . It needEGFR mutations are heterogeneous and have various sensitivities to EGFR-TKIs. In addition, the mechanism of acquired resistance to 1L EGFR-TKIs has not yet been well defined. Several retrospective studies and case reports of 1G EGFR-TKIs showed inconsistent responses in patients with uncommon EGFR mutation-positive NSCLC. A post-hoc analysis of prospectively collected data from the participants of the LUX-Lung 2, LUX-Lung 3, and LUX-Lung 6 trials showed the clinical activity of afatinib in patients with advanced uncommon EGFR mutation-positive NSCLC, especially G719X, L861Q, and S768I. However, it reported a low activity against T790M and exon 20 insertion mutations. Afatinib showed an ORR of 71% and a median PFS of 10.7 months (95% CI: 5.6–14.7), except for those with T790M or exon 20 insertion mutations, for whom ORR was 9–14%, and PFS was less than 3 months. The median OS was 19.4 months (95% CI: 16.4–26.9) [n = 315), it showed activity against major uncommon mutations , compound mutations , other uncommon mutations , and some exon 20 insertions  in EGFR-TKI naïve patients [Uncommon .4–26.9) . In a reEGFR-TKI naïve patients with uncommon EGFR mutation-positive NSCLC (KCSG-LU15-09), 22 of the 36 patients received osimertinib as the 1L treatment [EGFR-TKIs. Osimertinib conferred an ORR of 50%. The median PFS was 8.2 months (95% CI: 5.9–10.5), and the median OS was not reached. Furthermore, the response rate and median duration of response (11.2 months) were lower than those observed with osimertinib in patients with common mutations . In our study, the median PFS of 1G or 2G generation EGFR-TKI was 11.1 months (95% CI: 7.2–15.0), and the median OS was 25.6 months (95% CI: 18.2–33.0) in patients with uncommon EGFR mutant-positive NSCLC.In the phase II study of osimertinib for reatment . A totalEGFR mutations because of low sensitivity compared to the NGS [EGFR mutation detection accuracy [P53) in common EGFR mutation-positive NSCLC. However, in our study, few patients had an NGS result; therefore, further studies are needed to determine the prognostic effect of co-mutations in uncommon EGFR mutation-positive NSCLC.This study has several limitations due to its retrospective nature and single center experience. We had a small number of patients with brain metastasis at initial diagnosis. This study could not evaluate for CNS-specific outcomes or subgroup analysis. Among other things, only 44.9% of the patients were evaluated by repeated tissue or liquid biopsy at the time of progression because there was no accessible site for biopsy . Furthermore, the polymerase chain reaction-based or direct sequencing methods might have had limitations in detecting compound  the NGS ,16. Succaccuracy . It is aEGFR mutation-positive NSCLC, who started 1L gefitinib, erlotinib, or afatinib treatment for recurrent or metastatic NSCLC at the Samsung Medical Center between January 2011 and December 2019. This non-interventional observational study through big data analysis retrospectively collected de-identified patient data from a clinical data warehouse (CDW) using a unique algorithm with Standard Query Language (SQL) called the ROOT project. Patient demographic characteristics, such as age, sex, smoking history, performance status, and EGFR mutation type, were reviewed. We classified the uncommon EGFR mutation-positive NSCLC into the group with major uncommon EGFR mutations , compound mutations with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutations, other uncommon mutations  and T790M. Demographic information was obtained when the 1L EGFR-TKI treatment was initiated. EGFR mutations were identified using a peptide nucleic acid (PNA)-clamp kit and real-time polymerase chain reaction, COBAS (cobas®EGFR Mutation Test v2 [TM).This study included patients with  Test v2 , or nextThis study was reviewed and approved by the Institutional Review Board (IRB) at the Samsung Medical Center (IRB No. 2018-05-130). The trial was conducted following the Declaration of Helsinki (as revised in 2013).EGFR-TKIs until progression or death resulting from any cause. PFS2 was defined as the first date of sequential treatment after failing 1L EGFR-TKIs until progression or death resulting from any cause. OS was defined as the first date of EGFR-TKIs until death resulting from any cause. OS2 was defined as the first date of sequential treatment after failing 1L EGFR-TKIs until death resulting from any cause. All p-values were two-sided, and p-value < 0.05 was considered statistically significant. The Korea Food and Drug Administration approved afatinib in October 2014. Among the three 1G or 2G EGFR-TKIs, PFS and OS were analyzed in patients who received EGFR-TKIs between October 2014 and December 2019.The all-data cut-off date for the analyses was February 2020. PFS and OS were calculated using a Kaplan–Meier estimator and compared using the log-rank test. PFS and OS were presented as median values, with two-sided 95% CIs. PFS was defined as the first date of EGFR-mutant NSCLC showed lower ORR, PFS, and OS than the patients with common EGFR-mutant NSCLC. In particular, among the uncommon EGFR mutation, compared to the group with compounding mutation with major EGFR mutation and the group with major uncommon EGFR mutation, the group with other compounding mutation or other uncommon EGFR mutation showed inferior outcomes. Compared to common EGFR mutation-positive NSCLC, a lower percentage of patients underwent repeated biopsy and showed a lower detection rate of T790M. Less than one-third of the patients received 3G EGFR-TKI after failing the 1L EGFR-TKIs. However, like the common EGFR group, patients with T790M mutation after failing the 1L EGFR-TKI who received 3G TKI showed a favorable OS compared to other sequential treatments in the uncommon EGFR group. With the availability of more EGFR TKIs and a better understanding of tumor biology, further prospective studies are warranted to define the optimal treatment approach for uncommon EGFR mutant-positive NSCLC.The patients with uncommon


Older adults self-administer prescribed medication regimens to treat chronic diseases which can lead to mismanagement, medication related harm and hospitalizations. We examined the extent to which source of purchased medications influenced the occurrence of self-reported medication mistakes and hospitalizations in community-dwelling participants who managed medications independently (N= 3899). The majority (65%) picked-up medications, 18% had medications delivered, and 17% used both (picked-up and delivery). Compared to those picking up their medications, those using delivery only were less likely to have a hospital stay (OR=0.691 [95% CI 0.507-0.943]) and no difference in odds of medication mistakes (OR=1.051 [95% CI 0.764-1.445]), while those using both methods were more likely to report hospital stays (OR=1.429 [95% CI 1.106-1.846]) and medication mistakes (OR = 1.576[95% CI 1.078-2.304]). Older adults who picked-up medications from a local pharmacy and had medications delivered were more likely to report medication mistakes and hospitalizations.


Scientific Reports 10.1038/s41598-019-39851-6, published online 27 February 2019Correction to: This Article contains errors in the legend of Figure 7.http://imaging.org.au/AMBMC/Model.”“a) Association matrix for calculating modules. The modules in (b) non-fasted and (c) fasted groups. Background image from should read:http://imaging.org.au/AMBMC/Model.”“Association matrix for calculating modules in (a) non-fasted and (b) fasted groups. The modules in (c) non-fasted and (d) fasted groups. Background image from


Mycobacterium abscessus strain Taiwan-45 that lytically infects M. abscessus strain BWH-C; phiT45-1 also infects M. abscessus ATCC 1997 but not Mycobacterium smegmatis. Phage phiT45-1 has a 43,407-bp genome and carries a polymorphic toxin-immunity cassette associated with type VII secretion systems.Mycobacteriophage phiT45-1 is a newly isolated bacteriophage spontaneously released from  Mycobacterium abscessus strain Taiwan-45 that lytically infects M. abscessus strain BWH-C; phiT45-1 also infects M. abscessus ATCC 19977 but not Mycobacterium smegmatis. Phage phiT45-1 has a 43,407-bp genome and carries a polymorphic toxin-immunity cassette associated with type VII secretion systems.Mycobacteriophage phiT45-1 is a newly isolated bacteriophage spontaneously released from  Mycobacterium abscessus is often antibiotic resistant and refractory to treatment. M. abscessus infections are frequent among cystic fibrosis patients and those with bronchiectasis and can disseminate in immunosuppressed patients  strains  are mycobacterial species that do not cause tuberculosis or leprosy . Among tpatients , 3. The  strains . The risernative .M. abscessus to contain prophages (M. abscessus Taiwan-45 onto a lawn of M. abscessus strain BWH-C (both provided by Chidiebere Akusobi and Eric Rubin) on solid medium at 37°C using standard methods (http://cobamide2.bio.pitt.edu)  . Putativitt.edu)  and HHpritt.edu) , 15. No itt.edu) . All tooM. smegmatis  (48 and 49) and several predicted HNH endonucleases  and immunity repressor (35) is consistent with phiT45-1 being temperate. Interestingly, phiT45-1 codes for a polymorphic toxin (PT) cassette, including an immunity protein (30), a polymorphic toxin (31) with RipA-like and WXG-100 domains (32)  to phages isolated on megmatis , althougviridae) . Early l domains , 21, andins (32) , situateMW570842 and BioProject accession no. PRJNA488469. The sequencing reads are available in the SRA under accession no. SRX10050651.Phage phiT45-1 is available at GenBank under accession no.