Neolamprologus brichardi. Here, we tested the members of this network in the blockhead cichlid, Steatocranus casuarius, which displays conspicuously elongated dorsal and moderately elongated anal fins. Our study provided evidence for differences in the anatomy of fin elongation and suggested gene regulatory divergence between the two cichlid species. Only a subset of the 20 genes tested in S. casuarius showed the qPCR expression patterns predicted from the GRN identified in N. brichardi, and several of the gene-by-gene expression correlations differed between the two cichlid species. In comparison to N. brichardi, gene expression patterns in S. casuarius were in better (but not full) agreement with gene regulatory interactions inferred in zebrafish. Within S. casuarius, the dorsoventral asymmetry in ornament expression was accompanied by differences in gene expression patterns, including potential regulatory differentiation, between the anal and dorsal fin.Comparative analyses of gene regulation inform about the molecular basis of phenotypic trait evolution. Here, we address a fin shape phenotype that evolved multiple times independently across teleost fish, including several species within the family Cichlidae. In a previous study, we proposed a gene regulatory network (GRN) involved in the formation and regeneration of conspicuous filamentous elongations adorning the unpaired fins of the  Additionally, comparative studies of gene regulation across different levels of biological organization and diversification  address the divergence and conservation of regulatory networks and their roles in evolutionary processes4. For instance, parallel phenotypic variation across populations or species may be controlled by parallel regulatory changes, as demonstrated in the adaptive evolution of stickleback populations6 and for leaf shape complexity in Solanum7. In contrast, distinct changes in gene expression patterns were shown to underlie similar pigmentation patterns across Drosophila species8.Gene regulatory networks (GRNs) describe the regulatory relationships among genes involved in the development and maintenance of phenotypes. A large body of work concentrated on selected developmental models, for instance sea urchins9. Most studies focussed on the caudal fin of the zebrafish Danio rerio, which exhibits a particularly well developed regeneration capacity9. An additional reason to study the caudal fin of fishes in a comparative approach is to gain understanding about the evolution of body shape, for instance regarding the transition from the dorsoventrally asymmetrical  caudal fin of acipenseriformes, gars and bowfin to the outer symmetry displayed by the homocercal caudal fins of teleost fish10. Furthermore, within teleosts, variation in the outer shape and anatomy of caudal fins provided opportunities to identify genes and molecular mechanisms involved in fin shape formation13. The examination of dorsoventral (a)symmetry in fish fin morphology can be extended to the dorsal and anal fins, e.g. by comparing the molecular correlates of ornamental filaments14 or colour patterns15 between these fins. In a previous study, we investigated the molecular mechanisms involved with the formation of elongated fin filaments, which adorn the dorsal, caudal and anal fins of the cichlid fish Neolamprologus brichardi16. The extent of the outgrowth is similar across the unpaired fins in this species , which are required for angiogenesis or neurogenesis during fin formation24. Moreover, we identified six potential upstream regulators of the coexpression module 16, among which myc, irf8 and foxd3 were already indicated in fin/tissue regeneration studies28. Importantly, the expression patterns of foxd3 and its significant expression correlation with all members of the coexpression module led us to conclude that foxd3 is a main upstream regulator of the GRN in N. brichardi16. The expression patterns of some of the network genes in N. brichardi differed from expectations based on the functions of these genes in zebrafish. These include cx43, which, in zebrafish, is related with fin ray segment growth and the regulation of mmp9 and sema3d20. In N. brichardi, differences of cx43 expression levels between fin regions were not associated with differences in segment length, and correlations with mmp9 and sema3d were in the opposite directions than in zebrafish16.In fish, the molecular mechanisms controlling fin growth and regeneration have received particular attention not least due to their relevance for biomedical researchies Fig. . Using qSteatocranus casuarius. This species is particularly interesting because the unpaired fins of males differ with respect to the presence and extent of filament elongation  and the short (S) regions of the anal and the dorsal fins, using both intact and regenerating fins. All of these genes had shown significant L/S expression differences in N. brichardi, which suggested that their differential expression was associated with the growth pattern producing the fin elongations. Furthermore, to investigate the regulatory linkages inferred in N. brichardi, we tested for pairwise gene expression correlations in the anal and dorsal fins of S. casuarius. We predicted that the L/S expression differences and the gene expression correlations observed in N. brichardi would be detected in the distinctly elongated dorsal fin of S. casuarius, but not necessarily in its anal fin. We complemented the genetic analyses of fin elongation with an anatomical characterization of the phenotype, and measured fin ray segment length in elongated and short fin regions to determine whether elongation of rays was associated with lengthening of ray segments. Our findings suggest divergence in the regulation of the fin elongation phenotype between the two cichlid species.In the present study, we tested the members of the GRN in another cichlid fish, the blockhead cichlid ion Fig. . The dor−4, second-longest rays: t = 8.57, p = 1.32 * 10−13; Fig. nd-shortest rays of each fin: t = −4.15, p = 6.36 * 10−5, 3rd-shortest ray: t = −3.86, p = 1.85 * 10−4; Fig. We measured the lengths of fin ray segments in elongated and short regions of the anal and dorsal fins in order to determine whether fin elongation was associated with an elongation of ray segments. Fin ray segments differed in length both between and within fins. The segments of the long rays were longer in the dorsal fin than in the anal fin  and elf1a with lowest expression (highest Cq) 16 for differential expression between elongated (L) and short (S) fin regions in the distantly related cichlid, Steatocranus casuarius. The 20 target genes  of the network found in N. brichardi16. Additionally, we tested esco2, which is an upstream regulator of cx43 and sema3d in zebrafish34. Gene expression levels were compared between L and S regions within each fin type 9 at three regeneration stages. Stage 0 represents the original fin with expression patterns required for the maintenance of the phenotype, while stage 1 and 2 represent regenerating fin tissue. At stage 1 (20 days of regeneration), fin ray elongation was starting to become apparent, while at stage 2 (40 days of regeneration), the length difference between L and S regions was already obvious but not yet fully recovered , L/S expression did not differ at any of the individual developmental stages, which reduced our focal gene set to 12 genes  as well as for sema3d (lower L-expression). Additionally, foxc1 and myc showed higher L-expression in the anal fin . Note that a significant result in the LMM did not necessarily imply significant L/S expression differences at each of the individual developmental stages; vice versa, genes that showed a significant L/S expression difference at only one developmental stage were not necessarily picked up by the LMM analyses. Among the 12 focal genes, only angptl5 showed significant L/S expression differences (higher L-expression) in both fin types and at all developmental stages. For cx43, significantly higher L-expression was detected in all contrasts except at stage 2 in the anal fin. These results suggest that differential L/S expression of these genes may be involved in both maintenance and regeneration of the anal and dorsal fin elongations  or the dorsal fin , which may reflect fin-specific roles of these genes in the maintenance of the fin phenotypes. In contrast, differential L/S expression of angptl7 and gnao1a , and mmp9  occurred only during regeneration and may not be required for the maintenance of the elongated fin phenotypes in the original fin (stage 0)  than in the anal fin /(RQL + RQS) for each of the 20 network genes in each fin type separately, and then used paired Mann-Whitney U-tests to compare RED values between fin types  of angptl7, cd63, foxc1, foxd3 and myc. Additionally, cx43 had shown L > S expression in both fins in the LMM analysis, and the significant RED differences between fin types during late regeneration (stage 2) indicate that L/S differences persist longer in the dorsal than in the anal fin. Finally, RED values of wnt5b differed significantly between fin types during early regeneration (stage 1), although neither fin type had shown significant L/S differences in the LMM analyses. While we note that stage-specific differences between fins may also be influenced by possibly asynchronous regeneration of the two fin types, our observations suggest that the different phenotypes of the anal and dorsal fins (moderate versus distinct elongation) are associated with fin-specific expression patterns of some of the tested network genes.The above comparisons of L- versus S-expression in the dorsal and anal fins suggested fin-specific expression patterns for several of the investigated genes. To further explore this, we calculated relative L/S expression differences as RED =  involved in fin formation and/or regeneration during both maintenance and regeneration stages16. The proposed GRN included some functionally characterized members such as cx43, sema3d and mmp9, which are known to be involved in the formation and growth of fin ray segments in zebrafish20. Expression patterns in N. brichardi supported a scenario in which foxd3 acts as transcriptional activator of cx43 and mmp9  and repressor of sema3d16. Here, we investigated whether the same GRN might be involved in the formation of a similar phenotype, i.e. anal and dorsal fin elongation, in another cichlid fish, S. casuarius. Fin elongation in S. casuarius is far more pronounced in the dorsal fin than in the anal fin, which furthermore allowed us to examine whether gene expression patterns in the two fin types reflect the different elongation phenotypes of the anal and dorsal fin.In our previous study of fin elongation in the Princess cichlid, S. casuarius contrasted with predictions based on the patterns observed in N. brichardi. Regarding the links between foxd3 and cx43, mmp9 and sema3d, we found that while L/S expression differences of cx43 and mmp9 were congruent in N. brichardi (L > S), L-expression was higher for cx43 but lower for mmp9 in S. casuarius fins. Unlike in N. brichardi, no expression correlation was found between foxd3 and mmp9, nor between foxd3 and sema3d. Furthermore, while all of the 20 investigated genes had shown significant L/S expression differences in N. brichardi, only a subset of these genes showed significant L/S expression differences in S. casuarius, some of which were in the opposite direction than in N. brichardi . Pooling individual tissues into biological replicates is expected to reduce the variation among replicates, and indeed, coefficients of variation  were substantially lower in N. brichardi (mean CV ± sd = 0.13 ± 0.08) than in S. casuarius . However, we found no relationship between the coefficients of variation for individual genes and the failure or success to detect significant L/S expression differences and expression correlations in S. casuarius. Therefore, while non-significant contrasts and correlations do not necessarily imply the absence of expression differences and correlations in S. casuarius, there is no indication that the different experimental designs systematically affected the comparison between the two studies.Gene expression patterns in N. brichardi, expression patterns of some GRN genes did not conform to inferences on their regulatory interactions in zebrafish16, whereas expression patterns of some of these genes in S. casuarius are consistent with the zebrafish data  are a sister lineage to the Lake Tanganyika cichlids and the other Great Lakes radiations29. It now remains to be tested whether the gene functions and GRN composition observed in N. brichardi are unique to this species, shared by other Lamprologini (some of which sport elongated fins) or even across the large East African lacustrine cichlid radiations, or whether perhaps S. casuarius regained some of the zebrafish-like gene functions independently. Remarkably, in a comparative analysis of the evolution of regulatory elements across African cichlid species representing the Great Lakes radiations, the number of ‘accelerated conserved noncoding elements’ (CNEs evolving at a significantly accelerated rate) was highest in N. brichardi35. Before this background, it may be hypothesized that between-species differences observed in our study are due to the accelerated regulatory evolution in the lineage hosting N. brichardi.The differences between the two cichlid species in relation to the zebrafish model are interesting in a phylogenetic context. S. casuarius coincided with differences in gene expression patterns. The dorsal fin displays a more pronounced elongation and a larger difference in fin ray segment lengths between elongated and short fin regions than the anal fin  was observed only in the dorsal fin, where the difference in segment length between elongated and short fin rays was more pronounced than in the anal fin. Similarly, the transcription factor foxd3, which was associated with the exaggerated fin outgrowth (the sword) of male sword-tail fish11 and proposed as a main upstream regulator of the fin elongation GRN in N. brichardi16, showed significant L > S expression only in the dorsal fin of S. casuarius. In zebrafish, foxd3 is implicated in the dedifferentiation associated with tissue regeneration through its balancing effects on BMP and Wnt signals38. Together, these data support the hypothesis that expression of foxd3 promotes fin outgrowth in different fish species; however, the GRN comparison between S. casuarius and N. brichardi suggests that it may do so via different pathways. Likewise, divergent expression patterns of the transcription factors irf8 (see above) and foxc1  hint at variation in the regulatory pathways between species. Interestingly, however, positive correlations between sema3d and foxc1 were identified in both S. casuarius  and N. brichardi, indicating potential regulation of sema3d by foxc1 in both cichlid species. Whereas the majority of pairwise gene expression correlations were detected in both the anal and dorsal fin of S. casuarius, the small number of fin-specific correlations, including the one between sema3d and foxc1, may indicate regulatory differentiation between the two fin types, although non-significant correlations may also be due to small sample sizes.The different phenotypes of the dorsal and the anal fins of fin Fig. . Accordimyc was puzzling with regard to the function of this gene in other animals. myc encodes a nuclear phosphoprotein which can induce pluripotency in differentiated cell types in vertebrates39 and its expression is elevated in blastemal cells of regenerating Xenopus tails25. Accordingly, myc expression was higher in the elongated fin regions of N. brichardi than in the short regions16. Surprisingly, this was not the case in the distinctly elongated dorsal fin of S. casuarius, whereas the moderately elongated anal fin showed the expected L > S expression difference. This confounding result might be partially explained by complex auto-regulatory characteristics of myc, through which negative feedback loops limit its function43. In addition, it has been shown that prolonged expression of myc in murine cells can lead to premature induction of apoptosis rather than stimulate cell proliferation44. Similarly, another study has shown that activation of the canonical WNT pathway together with overexpression of myc induces a proliferative status in cancer cells, whereas prolonged overexpression of myc alone leads to apoptosis45. The modulation of the balance between myc-dependent cell proliferation/growth and apoptosis in different cell types and physiological conditions has been reviewed in mammalian cells47. Taken together, these observations propose a model in which the increased L-expression of myc during early fin formation can induce a proliferative status and subsequently slight elongation in the L region of the anal fin of S. casuarius. Later, its prolonged expression may have self-limiting effects through increased apoptosis which in turn constrains the elongation process in the anal fin.Finally, the fin-specific expression pattern of the transcription factor N. brichardi. Here, we found that some of those genes, particularly cx43, mmp9 and sema3d, are also differentially expressed in the elongated fins of another cichlid species, S. casuarius. However, despite the superficial similarity of fin shape in the two species, the anatomical basis of the elongations as well as the gene expression patterns of most (75%) candidate genes and transcription regulators differed between N. brichardi and S. casuarius. While this suggests that there is considerable regulatory divergence between the two cichlid species with regard to the formation of their convergent fin phenotypes, we emphasize that not all of the observed expression differences between the two cichlid species are necessarily linked to differential regulation of fin elongation. The members of the GRN were proposed based on qPCR expression profiling in N. brichardi, but were not validated for their involvement with fin elongation by functional analyses. Therefore, although the candidate genes tested in N. brichardi had been carefully selected based on their roles in fin growth and regeneration, some of them may display L/S expression difference in a context other than control of fin shape. This needs to be investigated by future work. The fact that we find anatomical differences with respect to how elongation is achieved in the two species, however, predicts that the underlying genetic mechanisms should also differ between them. Fin elongations evolved several times independently within Cichlidae, providing an excellent phylogenetic background for further comparative analyses. According to current evidence, regulatory divergence between species is mainly driven by cis evolution48, which suggests that the molecular basis of the observed differences may be identified in the upstream regions of the differentially regulated genes.In a previous study, we proposed 20 genes as putative members and regulators of a GRN associated with the formation and regeneration of ornamental fin elongations in the cichlid fish S. casuarius with total lengths of 10–12 cm were used to conduct the experiment. The fish were kept in one large tank (approx. 500 L) together with a group of 18 females. Each male was allocated a shelter consisting of a cluster of two to three PVC plastic tubes. Males stayed near their shelters most of the time and showed little movement and no aggression. Prior to taking the fin biopsies, fish were anesthetized in a solution of 0.04 g MS–222per 1 L water. To obtain tissue for gene expression analyses, the dorsal and anal fins were cut anterior to the first bifurcation of the fin rays . The branch was either selected randomly or by avoiding irregularities in the segmentation pattern, which sometimes occurred in one of the branches. Using the same criteria, we selected one branch of the second-shortest ray and one of the third-shortest ray for segment length measurements in the short fin region. The very shortest ray was avoided because it was only rudimentarily developed in some fins.To measure the length of fin ray segments in elongated and short regions, fin tissue samples were taken from five adult males and stained with alizarin red. We modified the acid-free double staining protocol described byAnaesthesia and fin biopsies were performed under permit number BMWFWF-66.007/0028-WF/V/3b/2017 issued by the Federal Ministry of Science, Research and Economy of Austria in accordance with the guidelines and regulations of BMWFW.RNA was isolated from a total of 72 tissue samples  using the ReliaPrep™ RNA Tissue Miniprep System Kit (Promega). Tissue samples were transferred to tubes containing 1.4 mm ceramic spheres and 250 µl of LBA buffer mixed with the recommended volume of 1-Thioglycerol. The tissues were homogenized using FastPrep-24 Instrument  and RNA isolation steps were performed according to manufacturer’s ReliaPrep™ protocol for RNA extraction from fibrous tissues. The ReliaPrep™ protocol includes genomic DNA removal (DNase treatment). The purification steps are conducted via a filtered column without the use of phenol-chloroform extractions or ethanol precipitations. The protocol is well-adjusted to isolate high quality RNA from small amounts of fibrous tissues, and allowed us to obtain a sufficient amount of RNA from each of the small tissue samples. After eluting the isolated RNA in 25 µl nuclease-free water, we measured the concentrations with a Nanophotometer . The quality of 20 randomly selected samples was checked in a R6K ScreenTape System on an Agilent 2200 TapeStation (Agilent Technologies) and exceeded 8 RIN (RNA integrity number) in all samples. The first strand cDNA was synthesized from 400 ng of RNA by following the manufacturer’s protocol supplied with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and cDNA was diluted 1:3 times in nuclease-free water as input for qPCR.S. casuarius fins, we selected one sample with a high RNA integrity number from each fin type and then pooled together with equal quantity for transcriptome sequencing. The library preparation was performed using a Standard TruSeq Stranded mRNA Sample Prep Kit (Illumina), following the manufacturer’s protocol, with an RNA input of 1200 ng. Subsequently, the quality of the library preparation was checked using a D1000 ScreenTape and reagents (Agilent) on a TapeStation 2200 (Agilent). Finally, the library was diluted and the sequencing was performed by the NGS Facility at Vienna Biocenter Core Facilities  to produce 125 bp paired-end reads. A quality control of the raw reads was performed using the bioinformatic tool FASTQC50. The removal of low quality reads and the quality trimming of the raw reads was carried out using the software Trimmomatic51. Finally, the transcriptome of S. casuarius was assembled de novo, based on the quality trimmed paired-end reads, using the Trinity software package53. The generated sequence read archives and assembly are available at NCBI under SRA Accession No. SUB4649276.To obtain a comprehensive list of transcripts from 56. As target genes, we selected 20 members of a gene regulatory network involved in fin and tissue morphogenesis and/or regeneration , one species in Ectodini tribe , one species in Lamprologini tribe (Neolamprologus brichardi) and four species from diverse Haplochromini tribe 58. Next we used the aligned sequences to identify conserved regions across the species  and at the exon/exon boundaries . The primers with short amplicon sizes (<250 bp) were designed using Primer Express 3.0  and their dimerization and secondary structure formation was checked through OligoAnalyzer 3.1 (Integrated DNA Technology) (Supplementary Data To identify stable reference gene gene(s), we selected 8 candidate reference genes with abundant expression in a range of tissues, which have already been investigated as reference genes in fins or other tissues containing skeletal structures or/and epidermis in fish  was performed at the end of the amplification step. Finally, we calculated primer efficiencies  using the LinRegPCR v11.0 programme60 (Supplementary Data We followed the manufacturer’s protocol for Maxima SYBR Green/ROX qPCR Master Mix 2X)  to set up qPCR reactions, which were performed in 96 well-PCR plates on an ABI 7500 real-time PCR System (Applied Biosystems). Each biological replicate was conducted in two technical replicates for each gene and we followed a sample maximization methodX  based on Cq values of the fin regions was calculated by BestKeeper to determine the expression variation for each reference gene. In addition to ranking, BestKeeper also determines the stability of reference genes through a correlation calculation or BestKeeper index (r). GeNorm calculates mean pairwise variation between each gene and other candidates  in a stepwise manner and NormFinder identifies the most stable genes  based on analysis of inter- and intra-group variation in expression levels variations31. In this study, the geometric mean of the Cq values63 of two most stable reference genes in both fins types, Cq reference, was used to normalize Cq values of target genes in each sample (ΔCq target = Cq target − Cq reference). In order to calculate ΔΔCq values, we randomly selected one biological replicate of the elongated fin region of the anal and the dorsal fin , and subtracted the ΔCq from the calibrator ΔCq value . Relative expression quantities  were calculated as 2−ΔΔCq 64.Candidate reference genes were ranked according to expression stability by three different algorithms, BestKeeper65. Next, we compared L/S expression at each developmental stage of the anal and dorsal fin using paired t-tests . Similarities in expression patterns between the target genes across tissues and developmental stages were examined by calculating pairwise Pearson correlation coefficients.RQ values were log-transformed for statistical analyses. For each target gene, differences in expression levels between elongated (L) and short (S) regions of the anal and the dorsal fin were tested in linear mixed models with log(RQ) as dependent variable, length (L or S) as fixed factor and developmental stage nested within biological replicate as grouping factor /(RQL + RQS); i.e. corrected for differences in the absolute expression levels between fins. This yielded, for each gene, six RED values for the anal fin and six RED values for the dorsal fin , which were then compared using paired Mann-Whitney U-tests .In order to compare the L/S expression To compare fin ray segment length across rays and fin types, we used linear mixed models with segment length as dependent variable, ray or fin type as fixed factor , and the sampled fish as random factor.All experimental protocols related to the fishes used in this study were approved by the Federal Ministry of Science, Research and Economy of Austria, permit numbers<b>Supplementary Figures</b><b>Supplementary Table 1</b><b>Dataset 1</b><b>Dataset 2</b>


The aim of this study was to detect the severity of ulnar variance (UV) compared with contralateral hand on postoperative wrist function in patients with distal radius fracture. 116 cases with unilateral distal radius fracture were retrospectively analyzed and divided into high or low UV severity groups . The following parameters were used to evaluate the effect: palmar tilt, radial inclination, VAS score, DASH score and wrist function. The severity of UV existed widely, accounting for 93.1% (108 cases). The severity of UV correlated with palmar tilt, radial inclination, grip strength, VAS score, DASH score and the wrist function (P < 0.05). Log-rank analysis showed that the severity of UV, palmar tilt, radial inclination were important factors influencing the joint function postoperatively (P < 0.0 5). Multivariate analysis confirmed that the severity of UV was an independent and significant factor on wrist function (P = 0.010). And the palmar tilt was also an important factor influencing wrist function (P = 0.047). The severity of ulnar variance compared with contralateral hand is an independent and significant factor on wrist function, which should be considered as an important step during preoperative plan. Most of them destroyed the anatomy of distal radioulnar joint and antibrachio-carpal joint, resulting in severe chronic pain, wrist stiffness, grip strength weakening and dysfunction2. In latest years, most newly surgical methods  are used3, a certain adverse prognosis rate is still not uncommon4.The distal radius fractures are the most common upper extremity fractures, accounting for about 16%5. Jianli B used 11 fresh cadaver specimens to investigate the effect of radial shortening on wrist joint mechanics, and confirmed that the ulnar variance affected the wrist function. The transmitted load distribution between the radius and ulna was measured. The negative ulnar variance brought 94% load transmission in radial side and 6% in the ulnar side, while positive ulnar variance with 69% load transmission in radial side and 31% of the ulnar side6. Tomaino MM found that ulnar impaction syndrome occurred not only in ulnar positive wrist, but also in wrists with either ulnar negative or neutral variance7. All these reminded us that individual change of UV might be an important prognostic factor.Previous studies had shown that the change in ulnar variance (UV) up to 2.5 mm usually resulted in a significant change from the normal biomechanics of wrist, which brought higher risk of severe wrist painTo our knowledge, there is little study about the severity of UV affecting the clinical prognosis in patients with distal radius fracture. We hypothesized that the severity of UV was an important prognostic factor affecting wrist function. The aim of this study was to detect the severity of UV compared with contralateral hand on postoperative wrist function.This clinical trial was designed and approved by the Ethics Review Committee of Changzhou Traditional Chinese Medical Hospital, affiliated to Nanjing University of Traditional Chinese Medicine. All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained.8, 13 of type I, 63 of type II, 30 of type III and 10 of type IV. The inclusion criteria were over 18 years old; underwent open reduction and internal fixation of unilateral distal radius fracture; no disorders affecting the healthy side upper limb function; no obvious mental disorders and could accomplish a complete clinical follow-up. Patients were excluded if they had disorders affecting contralateral limb function disorder or older than 18 years; if they had a history of trauma to contralateral hand in the past years. According to the variance of UV , all enrolled patients were divided into high UV severity group (45 cases) and low UV severity group (71 cases).From Jul. 2010 to Jul. 2015, 116 cases, ASA I-III, aged 19–68  years, unilateral distal radius fractures were retrospectively analyzed. All these patients  enrolled were recommended to undergo open reduction and internal fixation by palmar approach. Of them, 64 patients got surgical treatment within 48 hours after trauma, and 52 cases in 3–8 (5.7 + 2.1) days after trauma. According to Femandes typeThe operations were performed by one senior experienced orthopaedic surgeon major in upper limb fracture. Mobilization started from the second day postoperatively with the same rehabilitation scheme. On the following days, patients attended rehabilitation twice a day.All patients got followed-up at 1, 3, 6, 12 months postoperatively. Follow-up duration was defined as the date of operation to the date of latest follow-up. Pre-operative assessment involved bilateral wrist standard radiographs. Post-operative assessments involved radiographic evaluations  and clinical evaluations , joint function, visual analogue scales (VAS) score and the Disabilities of the Arm, Shoulder, and Hand (DASH) test).9.Follow-up radiographs were assessed for fracture congruity, restoration of radial inclination and palmar tilt. Fracture union was established radiographically by the presence of bone continuity or bridging callus on both the posteroanterior and lateral views associated with no signs of implant loosening10. The data analysis was independently evaluated by two different radiologists using PACS system. The final results were the mean value.Ulnar variance was measured in the standard posteroanterior x-ray of the wrist. A line, perpendicular to the long axis of the bone, was drawn through the most ulnar point of distal articular surface of the radius. The distance between this line and the ulnar dome in millimeters was defined as ulnar varianceSeverity of UV = post-operative UV − contralateral UVThe positive indicated the UV increased compared with the contralateral side, while the negative with decreased variance.Wrist joint mobility11.Wrist joint mobility was measured using a goniometer12Visual analogue scales(VAS)All patients were asked to mark their level of pain on a 10 cm visual analogue scale. Score 0 represented no pain, while 10 represented the worst pain.Grip strength was measured using a calibrated hydraulic hand dynamometer  and compared with the contralateral wrist.Disabilities of the Arm, Shoulder, and Hand (DASH) test13.The DASH test was a self quantified questionnaire to evaluate the upper extremity symptoms and the ability to engage in daily activities, including 30 projects such as physiological activity, severity of symptoms, damages on the social activities and so on. The DASH score less than 15 was regarded as fine2 test. Associations between prognostic variables and excellent rate were analyzed by log rank test. Multivariate analysis employed Cox’s proportional hazards regression model with a forward conditional stepwise procedure to determine the acting independent factor. The positive statistical significance was set at P < 0.05.The SPSS statistical package  was employed for statistical analyses. The normality of distribution for continuous numeric variables was assessed by Kolmogorov-Smirnov test. According to normally distributed or not, the variables were presented as means with SD, and otherwise as medians with inter-quartile ranges . Student’s t-test for normally distributed continuous variables, while others using Pearson’s χAll 116 patients got bone united uneventfully, and no secondary displacement, superficial or deep infection was found. The average time to clinical and radiographic union was 8.1 ± 2.3 weeks . The patients resumed their previous work at an average of 12 ± 3.6 weeks  post-operatively. The total average DASH score was 9.7 ± 5.6, representing high level of satisfaction.The severity of UV existed widely in the patients with distal radius fracture, accounting for 93.1% (108 cases). Of them, 100% in high UV variance group and 88.73% in low UV variance group.No significant correlation was found in age, gender, injury side, fracture type, surgical preparation time and bone graft or not . All these reminded us that individual change of UV might be an important prognostic factor.However, the UV is not unchangeable in all human beings. Nathan PA 21. These results just identified the main predictive factors on secondary displacement in distal radius fractures, but the effect of radial inclination on the functional prognosis was not confirmed. Stahl S and Goeminne S had concluded that negative UV negatively correlated with wrist function in Kienbock diseases23. Kodama N et al. had concluded the same results with our study. They assessed the radiographic findings of patients with no wrist dysfunction and revealed that palmar tilt and UV were the main prognostic factors for clinical outcomes. These parameters usually brought worsen outcomes when exceeding a tolerable range24.In present study, the severity of UV was calculated compared with contralateral hand, which commendably avoided individual variation. Multivariate analysis confirmed that the variance of UV was an independent and significant factor on wrist function (P = 0.010). At the same time, the study also found that the palmar tilt was an important factor influencing wrist function (P = 0.047). Tahririan MA pointed out that in patients with closed reduction, radial shortening of more than 6.5 mm, loss of radial inclination of more than 6.5 degrees and age above 52 were the most important predictive factors for in stabilityet al.25 compared the radiographic and functional outcomes of 318 patients with distal radius fractures. And they found higher values for radial inclination, radial length and volar tilt correlated with better functional outcome as measured by disabilities of the arm shoulder and hand (DASH) and patient rated wrist evaluation (PRWE) scores. Lower values for ulnar variance correlated with better functional outcome. However, the relationship between ulnar variance and other radiographic parameters wasn’t confirmed. P Dario et al.26 evaluated 51 patients treated with volar locked plate for articular unstable distal radius fractures and found that ulnar variance and volar tilt are the most important radiographic parameters influencing the final functional outcome. Kodama et al.24 reported similar results: volar tilt and ulnar variance were significantly correlated with clinical outcomes, although radius height was not evaluated. In our study, the severity of UV and palmar tilt were also the important parameters affecting function.A number of reports have provided the importance of radiographic parameters on wrist function, but no data in the literature concerning accurate association between different parameters. Stephen A. Brennan This study has some inherent limitations. It should be pointed out that the sample size enrolled is relatively small, although illustrating the prognostic value of severity of UV compared with contralateral hand on postoperative wrist function. The second critique maybe the patients came from just one single centre, multicentre and more cases studies are needed.Therefore, the severity of ulnar variance compared with contralateral hand plays an important role in the prognosis of wrist joint function in patients with distal radius fracture. The surgeons should consider it as an important step during preoperative plan.


Bos indicus) heifers.Impaired fertility in cattle limits the efficiency of livestock production systems. Unraveling the genetic architecture of fertility traits would facilitate their improvement by selection. In this study, we characterized SNP chip haplotypes at QTL blocks then used whole-genome sequencing to fine map genomic regions associated with reproduction in a population of Nellore , representing the daughters from 78 sires. After performing marker quality control, 64800 SNPs were retained. Haplotypes carried by each sire at six previously identified QTL on BTAs 5, 14 and 18 for heifer pregnancy and BTAs 8, 11 and 22 for antral follicle count were constructed using MTERF2, RTMB, ENSBTAG00000037306 (miRNA), ENSBTAG00000040351, PRKDC, and RGS20. The potential causal mutations found in the present study were associated with biological processes such as oocyte maturation, embryo development, placenta development and response to reproductive hormones.A total of six (BTA 5), six (BTA 14) and five (BTA 18) sires were heterozygous for heifer pregnancy QTL whereas six (BTA 8), fourteen (BTA 11), and five (BTA 22) sires were heterozygous for number of antral follicles’ QTL. Due to inadequate representation of many haplotype alleles in the sequenced animals, fine mapping analysis could only be reliably performed for the QTL on BTA 5 and 14, which had 641 and 3733 concordant candidate sequence variants, respectively. The KEGG “Circadian rhythm” and “Neurotrophin signaling pathway” were significantly associated with the genes in the QTL on BTA 5 whereas 32 MeSH terms were associated with the QTL on BTA 14. Among the concordant sequence variants, 0.2% and 0.3% were classified as missense variants for BTAs 5 and 14, respectively, highlighting the genes MTERF2, RTBC, miRNA ENSBTAG00000037306, ENSBTAG00000040351, PRKDC, and RGS20, which are known to have influence on reproductive biological processes, were detected.The identification of heterozygous sires by positionally phasing SNP chip data and contrasting haplotype effects for previously detected QTL can be used for fine mapping to identify potential causal mutations and candidate genes. Genomic variants on genes  Bos indicus) cattle, where heifers take longer to reach puberty compared to Taurine (Bos taurus) animals [Fertility is a major determinant of female reproductive efficiency, particularly in Zebu  spread across the genome, which can be rapidly and inexpensively genotyped . Such SNHaplotype blocks (haploblock) result from the joint inheritance of nearby loci without the occurrence of recombination events within blocks. At population level, there are many possible combinations of alleles at nearby loci and therefore many potential alternative haplotypes. Haplotype alleles may exhibit major effects on animal performance, often being associated with traits of economic interest , 9. KnowA deeper understanding of the effects of haplotype alleles on target phenotypes can be achieved by determining the causal quantitative trait nucleotides (QTN), i.e. polymorphisms that explain the effect of a QTL. With the advent of whole-genome sequencing technology, tens of thousands of base pairs that are positional candidates for the QTL can be readily compared between individuals known to be segregating the QTL . CurrentIn this study, we phase genotypes to reconstruct haplotypes in regions of previously reported QTL associated with heifer pregnancy (HP) or number of antral follicles (NF) in a Nellore cattle population. We contrast the two haplotype alleles carried by each sire at each QTL to determine those sires that are segregating the QTL effects and therefore segregating the causal mutation. Positional whole-genome sequence data were then used to identify those sequence variants that were concordant with the QTL segregation status.The dataset used for this study was previously described by Oliveira Júnior et al. , and conBos indicus HD array (74677 SNPs), which is a subset of the 777962 SNPs from the Illumina® BovineHD BeadChip, chosen for being particularly informative in Bos indicus cattle. The heifers were the offspring from 78 sires, among which 42 of these were previously genotyped using the BovineHD BeadChip. The sire genotypes were reduced to the same filtered set of markers that passed quality control for the heifers (64800 SNPs).The 1337 heifers were genotyped on a GGP The genotypes of sire-offspring pairs as recorded in the pedigree were tested for opposite homozygosity using FImpute 2.2  to detecContemporary groups (CG), that represented the group of animals that were born and managed together, were formed as the subclass combinations of birth year within herd, resulting in 12 herd-year groups. Records from any CG without phenotypic variability were eliminated from the analyses. Furthermore, animals with age exceeding 3.5 standard deviations from the overall mean age in the CG were excluded from the dataset. More details about the dataset and edits can be found in Oliveira Júnior et al. .Some 36 of the 78 sires with genotyped offspring were not themselves genotyped. Among those, the 19 ungenotyped sires that had more than five genotyped progeny , 19 wereThe accuracy of imputation was tested by similarly imputing the genotypes (64800 SNPs) of the 42 genotyped sires that had more than five progeny. The accuracy of their imputation was quantified as the correlation between observed and imputed genotypes .findhap [Haploblocks considered in this paper were limited to those reported in Oliveira Júnior et al. . These rfindhap , consideHaplotype mismatches were identified when supposed half sib progeny did not receive either of the paternal haplotype alleles carried by their putative sire at the given haploblock region. Observations for heifers where any haplotype allele did not match its sire, or any that were observed in less than three offspring of any particular sire, were omitted from subsequent analyses.The effects of all the paternal haplotypes were estimated for each haploblock by fitting models with the dosage of all the haplotype alleles in that haploblock as fixed effects, using Gensel software . In the yi is the trait record for heifer i; b is the vector that includes class effects of the contemporary groups and a regression coefficient for heifer age (months) at artificial insemination; F is the incidence matrix relating b to y; h is the vector of the fixed effects of each haplotype allele fitted, the incidence matrix H comprises a column for each haplotype allele representing the dosage  of the allele for each individual; n is the number of SNP chip loci outside the QTL region (i.e. not in the haploblock); xijk is the genotype covariate of animal i at SNP chip locus j, with genotype allele dosage indicator k ; sj is the allele substitution effect of SNP chip marker j, assuming δj = 1, and δj = 0; δj is an indicator variable for locus j; and ei is the random residual effect for animal i, assuming δj was:where th sample from the following 88000 samples were used for inference. The posterior distribution of the contrast between the effects of the two haplotype alleles for each sire was constructed from the Markov chain samples of each haplotype allele to identify sires that were segregating alternative QTL alleles at the haploblock, using a posteriori probability  < 0.10 as the threshold level.Heifer pregnancy was analyzed using a threshold model, which related the observed categorical success or failure scores to an underlying continuous normal scale, whereas NF was modeled as a continuous variable. Samples from the first 2000 iterations of the Markov chain were discarded (burn-in) and every 100The haplotype alleles were identified in further sections using the notation Trait_BTA_Allele, .Whole-genome sequencing (WGS) data of 26 Nellore sires was available, comprising of six bulls that were among the 42 genotyped sires included in the previous analyses, and another 20 bulls that had more distant degrees of relationships with the heifers, mainly as grandsires. The WGS data was obtained using paired-end sequencing from an Illumina HiSeq 2500 platform, following recommended manufacturer protocols . A total of 26115176 variants were identified to be segregating in these 26 animals, including 91.6% that were SNPs and another 8.4% that were genomic insertions or deletions. The overall average depth of sequencing coverage was 14.7X.Bos taurus reference genome assembly (UMD 3.1) using BWA-MEM software (v0.7.15) [http://picard.sourceforge.net/). Mapped regions were analyzed in the variant calling process using GATK v3.6 Haplotype caller [A visual quality control (QC) of raw data was done using the FastQC package . Sequencv0.7.15) . The PCRe caller . The ideThe presence or absence of any positional sequence variants in every sire haplotype allele at a QTL were imputed using linear regression. The dosage of the sequenced variant  at each locus in the QTL region was regressed on the dosage of the haplotype alleles at that QTL. This regression was repeated for every sequence variant that was mapped in any of the six QTL regions. For this purpose, haploblocks that spanned several Mb were partitioned into narrower fragments (~ 1 Mb) otherwise it was rare for sequenced sires to share the same haplotype alleles. These analyses were only undertaken for haplotype fragment (~ 1 Mb) that had been observed on at least three occasions. The following model was fitted separately at each sequence locus within its corresponding QTL region:y is a vector of length equal to the total number of sequenced sires and contained the dosage of the alternate allele  at a sequenced locus considered as a phenotype; Q is a matrix containing haplotype allele dosages  for each sequenced sire for all N haplotype fragments at the QTL; d is the vector of regression coefficients for the sequenced allele in each of the N haplotype alleles, which is expected to be 0 or 1 providing there were no errors in the sequencing calls, no phasing errors in the QTL haplotypes, and all copies of each haplotype allele carried the same sequence SNP; and e is the vector of residual imputation error. There were six different Q matrices representing the six different QTL regions. There were many vectors of phenotypes representing sequence variants for each QTL region. Regression coefficients that were exactly 0 or 1 and with standard errors < 0.0001 were used to impute the sequence SNPs to haplotype alleles whereas sequence loci with regression coefficients of intermediate values or with larger standard errors were assumed to be unreliably imputed and were therefore not considered in any further analyses.where As in Weller et al. , it was Ensembl v.88 Variant Effect Predictor (VEP) [The candidate causal mutations were annotated using or (VEP)  and assior (VEP)  using thor (VEP) , 30. Theor (VEP) , a sequeP- value < 0.05) for reporting g significant genes in a certain functional term was estimated by:Functional terms, defined as groups of genes that share a biological process or molecular function, can be used to categorize genes using over-representation analysis (ORA). We evaluated ORA considering two databases: Medical Subject Headings (MeSH)  and KyotS is the total number of target genes, N is the total number of genes that were analyzed, and k is the total number of genes in the term considered [meshr [ppiPre package [MeSHSim [where nsidered , 35. Thed [meshr  while th package . Semanti[MeSHSim .Pedigree inconsistencies were detected from the presence of opposite homozygous SNP chip genotypes in about 9% of the sire-offspring pairs. All genotyped sires had genotyped offspring that were consistent with the pedigree-records, suggesting there had not been any sample mismatch errors in the sires’ genotypes. The average (±standard deviation) SNP chip imputation accuracy for the 42 genotyped sires was 0.96 (±0.03). Accordingly, a total of 19 non-genotyped sires with at least five genotyped progeny were imputed for the 64800 SNPs.alpha < 0.10) that were associated with HP and 20 alleles that were associated with NF  was present in three sires . For all these sires, HP_5_1 had a favorable effect on HP when compared to those sires’ alternate haplotype alleles. Similarly, alleles HP_5_5, HP_5_7 and HP_5_9 in sires 4, 5 and 6 had favorable effects on HP in relation to their alternate alleles  had a smaller effect in two different sires (sires 8 and 9), suggesting it contained an unfavorable allele for this trait.Six sires (sires 7 to 12) had significant differences between their haplotype alleles for HP at the BTA 14 QTL  had a favorable result when contrasted with HP_18_24 in sire 12, it was inferior to HP_18_22 in sire 11. The HP_18_24, in turn, had inferior values in both sires 12 and 14, where it was contrasted with HP_18_27, suggesting that it was an unfavorable allele for HP.In addition to segregating the QTL on BTA 14 for HP, sires 11 and 12 were segregating the QTL on BTA 18  had positive effects on NF in sires 16 and 8, whereas allele 34 (NF_8_34) had negative effects in sires 17 and 18.For NF, the contrast analyses for the BTA 8 QTL resulted in the identification of six sires  with haplotype alleles significantly different from their corresponding alternate alleles  had a positive effect on five animals , suggesting the haplotype carried a favorable sequence for NF. In contrast, allele 45 (NF_11_45) showed inferior results in five different animals , suggesting it harbored an unfavorable allele for NF. Moreover, alleles 41 (NF_11_41) and 42 (NF_11_42) had inferior effects on the alternative alleles in three  and two (sires 20 and 24) animals, respectively. Some sires were also heterozygous for QTL associated with HP. Sires 1, 2, 4 and 5 were segregating the QTL on BTA 5, and sires 8 and 15 were segregating the QTL on BTA 14 and 18, respectively.Of the 20 haplotype alleles that positively affected NF, half were on BTA 11, carried by 14 different sires  positively affecting NF  as possible causal mutations affecting HP. From these, 527 variants were in sire 1 and 468 in sire 4, with 354 common to both. Among these 354 common variants to both sires, 202 fell within genes. For the alleles on BTA 14, there were 3733 variants (0.09% of the sequence SNP in the window) that were detected, being 3432 variants from sire 7, another 3184 from sire 8 and 3709 from sire 10. The majority of the identified variants where shared between them. Some of those variants (1128) were annotated as being within genes  of the genomic variants were in intergenic regions which can control genes nearby  Figs. 11 and 2. MTERF2  gene.The missense variant found for the QTL on BTA 5 is the SNP rs137779125 located at base pair 70,597,511, and is present on the Illumina® BovineHD BeadChip, with MAF of 0.19 in a sample of genotyped sires. The marker is in a coding region of the PRKC (3), PPDPFL (1), RB1CC1 (1), RGS20 (1) and RP1 (4). Of these, the markers rs137722134 (23998860 bp – MAF: 0.22) and rs109065397 (21104637 bp) are present on the BovineHD BeadChip and BovineSNP50 BeadChip, respectively.The 11 missense variants identified on the QTL of BTA 14 were located within coding region of the genes ENSBTAG00000040351 (1), alpha < 0.10). Israel et al. [Weller et al.  proposedl et al.  similarlThe reconstructed haploblocks spanned the previously reported significant genomic windows of each chromosome and trait presented in Oliveira Junior et al. . The numAPAF1 (apoptotic protease activating factor 1) gene located in the haplotype HH1 on BTA 5 (~ 63 Mb). In addition to HH1, Cole et al. [The detection of haplotype alleles associated with the impairment of female fertility can have an important economic impact. Adams et al.  reportede et al.  reportede et al. , HH4 [45e et al. ). MoreovMTERF2, RTBC, miRNA ENSBTAG00000037306, ENSBTAG00000040351, PRKDC, and RGS20, which are known to influence reproductive biological processes.In the present study, the strategy of regressing the sequence variants present on the SNP chip haplotype alleles in QTL regions of heterozygous sires has been used. The haplotype alleles identified to be segregating for NF were not well represented in any of the sequenced sires, and consequently, no fine map sequence variants were able to be reported for that trait. For HP, the fine mapping the QTL regions identified 641 sequence variants on BTA 5, and 733 sequence variants on BTA 14, that were associated with the trait. Among those, one was classified as missense variants for BTA 5, and eleven had the same classification for BTA 14. These variants were detected in the genes The identification of new genetic variants allows us to better understand the biological complexity of traits of interest. Knowledge of causal mutations should increase accuracy of genomic predictions as well as the genetic progress of the population . AccordiImputation procedures are normally used to increase SNP density for fine mapping studies , 49. HowCRY1 (cryptochrome circadian regulator 1) is one of the circadian genes which are known to encode transcription-suppressing factors that control the circadian clock in mammals [CRY1 gene (Table CRY1 transcripts (such as other circadian genes) are important for the development of oocytes and preimplantation embryos in cattle. Reiter et al. [The over-representation analyses were used to identify classes of genes or proteins that are over-represented in a large set of genes or proteins and that may have an association with the phenotypes. Among the detected MeSH terms associated with HP, “Prolactin” D011388) is well known to be related to mammary tissue development, immune function, heat tolerance, and reproduction. Polymorphisms of prolactin gene have been associated as mediators of physiological responses of heat stress on cattle, playing a role in the reproductive performance of dairy cows managed in tropical environmental conditions , 52. Ley mammals . Of the 88 is welr et al.  also disr et al.  concludeMTERF2 gene. Although this gene is not well characterized, other genes of this family (MTERF3 and MTERF4) have been related to embryonic death and a phenotype that is lethal to mouse embryos [The VEP classification identified a missense variant (G/A) at 70597511 base pair of BTA 5. This mutation is a known SNP (rs137779125), included in Bovine HD Illumina BeadChip, and within a codon region of the  embryos , 61.RTBC gene, which is associated with biological processes such as utero embryonic development and placenta development [Two novel putative causal variants were detected on BTA 5. The first one was in the upstream region  of the elopment . The secelopment .PRKDC, and RGS20. The SIFT approach uses sequence homology to predict whether a substitution affects protein function and consequently the phenotypes [There were 11 missense-variants identified in protein coding regions in the haploblock of BTA 14 Tables  and 4. Senotypes .The gene ENSBTAG00000040351 encodes Vomeronasal type-1 receptor protein, which presents the molecular function of G-protein coupled receptor and transducer. G-protein coupled receptors mediates most of the physiological responses to hormones, neurotransmitters and environmental stimulants being directly related to reproductive hormones mainly in females . There iPRKDC) gene encodes DNA-dependent protein kinase (DNA-PK), which is a nuclear protein serine/threonine kinase that is a molecular sensor of DNA damage. A previous study demonstrated that PRKDC expression increases due to embryonic genome activation and acts enzymatically activating the existing proteins in blastocysts [PRKDC is needed during early bovine embryo development.Protein kinase, DNA-activated, catalytic polypeptide (stocysts . Based oRGS) contains regulatory and structural components of G protein-coupled receptor complexes, which mediate several cellular processes. RGS20 was previously identified as a hub gene in a transcriptional profile study on pre-implantation of bovine embryos developed in vivo. RGS20 have been described as an important regulator of gene expression and stage transition in early embryo development [The family of regulators for G protein signaling .Additional file 2: Figure S2. Posterior distribution of the differences in estimated values of heifer pregnancy between two haplotype alleles of the chromosome 14 in those six Nellore sires where the contrast was significant (posterior Alpha < 0.10).Additional file 3: Figure S3. Posterior distribution of the differences in estimated values of heifer pregnancy between two haplotype alleles on chromosome 18 in five Nellore sires where the contrast was significant (posterior Alpha < 0.10).Additional file 4: Figure S4. Posterior distribution of the differences in estimated values for number of antral follicles between two haplotype alleles one chromosome 8 in six Nellore sires where the contrast was significant (posterior Alpha < 0.10).Additional file 5: Figure S5. Posterior distribution of the differences in estimated values for number of antral follicles between two haplotype alleles on chromosome 11 in fourteen Nellore sires where the contrast was significant (posterior Alpha < 0.10).Additional file 6: Figure S6. Posterior distribution of the differences in estimated values for number of antral follicles between two haplotype alleles on chromosome 22 in five Nellore sires where the contrast was significant (posterior Alpha < 0.10).Additional file 7: Table S1. Significant  MeSH terms related to genes located in the QTL of chromosome 14.


Salmonella is one of the major foodborne pathogens worldwide. The antimicrobial resistance (AMR) of this foodborne pathogen has raised a great concern in recent years. Studies on the frequency and characterization of Salmonella serotypes can help to improve our knowledge on the epidemiology of this pathogen. The purpose of this study was to compare the serotypes, AMR and genetic profiles of Salmonella isolates recovered from raw beef throughout the beef production chain and from human feces associated with clinical cases of salmonellosis. The serotype, AMR and pulsed-field gel electrophoresis profile of 243 Salmonella enterica isolates recovered from beef carcasses (n = 78), ground beef (n = 135), and human feces from clinical cases of salmonellosis (n = 30) were compared. Forty-three different Salmonella serotypes were identified and regardless of the source, the top five corresponded to Typhimurium, Give, Group B , Infantis and Anatum. Twelve serotypes from beef carcasses were also found in ground beef, showing their presence throughout the beef production chain. Salmonella Typhimurium, Infantis, Anatum and Montevideo were the only serotypes identified in all sample types. Resistance to tetracyclines was the most frequent (41.2%) followed by resistance to aminoglycosides (37%), folate pathway inhibitors (21%), quinolones (20.2%), phenicols (17.1%), penicillins (15.6%) and cephems (7%). Multidrug resistance was observed in 28.8% of the isolates, and those from human feces showed resistance to a larger number of antimicrobials. Great concern arises from the resistance and reduced susceptibility observed to quinolones and cephalosporins because these drugs are the first line of treatment for invasive Salmonella infections. Twenty-seven distinct pulse-types were detected among 238 isolates. Clustering analysis for the most frequent serotypes identified groups of isolates with similar AMR profiles. Multidrug resistance spreading throughout the food production chain should be continually monitored and its importance emphasized. Salmonella is one of the major foodborne pathogens worldwide. The World Health Organization (WHO) has measured the global burden of foodborne diseases and estimated that non-typhoidal Salmonella enterica accounted for more than 78 million cases of foodborne illnesses worldwide with approximately 59,000 deaths in 2010  of foodborne medicine . The surSalmonella strains isolated from foods are important because they can help to improve our knowledge on the epidemiology of this pathogen. The characterization of Salmonella isolates from foods, animals and humans often describe their serotypes, AMR and genetic profiles, providing valuable epidemiological information to improve food safety policies. In Mexico, these reports are scarce and limited to a few regions of the country  associated with clinical cases of diarrhea occurring in health-care institutions; their serotypes, AMR and PFGE profiles were analyzed for this investigation. The H isolates were characterized at the Food Safety Laboratory , and kept on tryptic soy agar  slants at 4 °C for testing. Each isolate was individually grown in tryptic soy broth  at 35 °C overnight, then they were streaked on brilliant green sulfa agar (BD Diagnostic Systems); plates were incubated at 35 °C for 24 hours. Typical colonies were biochemically tested and confirmed as Salmonella by a multiplex polymerase chain reaction (PCR) assay to detect the presence of invA (544 bp) and fimA (686 bp) genes, using previously described primers and methods  profiles of 243 ch group ; from th methods . The isoE. coli ATCC 25922 was used as quality control. Salmonella isolates showing resistance to three or more antimicrobial classes were classified as multidrug resistant (MDR) , gentamicin , tetracycline , trimethoprim-sulfamethoxazole , chloramphenicol , ceftriaxone , ciprofloxacin , kanamycin , nalidixic acid , streptomycin , and cephalothin . The test was performed according to the disk diffusion method on Mueller-Hinton agar  using cont (MDR) . To deteSalmonella enterica isolates according to the standard protocol developed by the Centers for Disease Control and Prevention  was digested with restriction endonuclease XbaI, and it was used as a size marker. Pulse times were ramped from 2.2 to 63.8 s over 21 h with an angle of 120° at 6.0 V cm−1. Genomic-DNA profiles or ‘pulsetypes’ were manually analyzed. Distinct pulsotypes were assigned to isolates when any differences in electrophoretic mobility were found. Following separation, EtBr staining, and image capture, PFGE banding patterns were visually inspected to detect polymorphic segments. DNA fragments exhibiting sizes above 54.7 S were taken into consideration to define informative characters, based on a side-by-side comparison with the reference Salmonella Braenderup H9812 strain, as recommended by the CDC guidelines. DNA fragments showing size variation were coded into a presence/absence matrix to obtain a dataset of informative characters. A total of 27 polymorphic DNA fragments were detected by PFGE and were later used for cluster analysis; 13 out of the 27 fragments were commonly found in S. Braenderup and the other 14 fragments were found only among the analyzed isolates, for a total similarity matrix of 27 informative characters and 238 taxa in the final dataset. The PFGE profiles of five isolates were not obtained.PFGE was performed for the 243 evention . Brieflyhttp://genomes.urv.cat/UPGMA)  algorithm as implemented in a web-provided platform (t/UPGMA) . The Peat/UPGMA) . DetectiSalmonella enterica strains isolated from C (n = 78), G (n = 135) and H (n = 30) samples based on their serotype, AMR and PFGE profile. Forty-three different serotypes were identified among 238 Salmonella isolates , Give, Infantis and Anatum. The larger serotype diversity was observed among isolates from ground beef (G samples) with 33 different serotypes identified from 135 isolates; S. Group B, Anatum and Infantis were the most common in this type of samples. From beef carcasses (C samples), 17 serotypes out of 78 isolates were identified and the most frequent were S. Give, Typhimurium and Group B. Among 30 isolates recovered from human feces (H samples), 15 serotypes were identified and the most common were S. Typhimurium, Enteritidis and Muenchen.The present investigation consisted of a descriptive study on the diversity and relatedness of 243 isolates  and fiveSalmonella Typhimurium, Infantis, Anatum and Montevideo were identified on H, C and G; on the contrary, S. Enteritidis and Muenchen were frequent on H, but not on C or G samples.Twelve of the serotypes found on C were also present on G samples, showing their presence throughout the beef production chain. Some other serotypes were probably introduced into ground beef at the butcher’s shops because fabrication of ground beef, ground pork, and sometimes ground chicken, using the same grinder during the same working day was previously identified as a common practice in these establishments . Ten outSalmonella isolates was compared. Resistance to at least one antimicrobial class was present in 52% (n = 127) of the isolates. In general, resistance to tetracyclines (TET) was the most frequent as it was present in 41.2% of the isolates (p > 0.05).In addition to serotyping, the AMR profile of the 243 isolates , followep < 0.05). For aminoglycosides, the frequency of intermediate-susceptible isolates was significantly higher on those obtained from C samples (p < 0.05).Reduced susceptibility to quinolones was frequent on isolates from all types of samples . Furtherp > 0.05). Three isolates (1.2%) showed resistance to the seven classes of antimicrobials tested. Salmonella Typhimurium was the most common MDR serotype followed by Group B, Anatum and Derby  for serotypes Typhimurium (75%) and Group B (70.9%).MDR was present in 70 (28.8%) of 243 isolates  and no dnd Derby . Two Sal classes . The proS. Typhimurium), and seven isolates from C and G samples .Thirteen isolates had MDR phenotypes that included resistance to CIP, and 17 isolates showed MDR phenotypes with resistance to at least one cephalosporin . Ten isoS. Typhimurium, Infantis, Anatum and Montevideo) and UPGMA clustering analysis was conducted on the final dataset based on the pulse-types. Out of the total 238 PFGE pulse-types identified, 28 corresponded to serotype Typhimurium, 19 to Infantis, 15 to Anatum and five to Montevideo, and they were observed to cluster in good agreement to their AMR profiles. Two isolates of S. Anatum were untypeable by PFGE.In order to compare genetic relatedness among isolates belonging to serotypes identified in the three types of samples , four of the most common serotypes were selected , G (eight isolates) and H (six isolates) samples were analyzed by PFGE and UPGMA and two major monophyletic clusters were identified , G (12 isolates) and H (one isolate) samples , G (one isolate) and H (one isolate) samples were compared  in human and cattle feces, and raw beef samples collected at four States of Mexico, which represent 17% of the national territory; Salmonella Typhimurium and Enteritidis were the most commonly found in human feces, while Anatum, Meleagridis, Agona and Typhimurium were most frequent in cattle feces and raw beef samples. In other countries like the US, Salmonella Agona, Infantis, Uganda, Anatum and Dublin were the top five serotypes isolated from beef carcasses in 2011, while S. Montevideo, Dublin, Cerro, Newport and Muenchen were the top five from ground beef in 2014  showed decreased susceptibility to this drug . Salmonella infections caused by MDR phenotypes are more likely to result in treatment failure and adverse health outcomes  and a significantly larger proportion of MDR isolates was observed among outcomes .The WHO Global Action Plan on AMR that was endorsed at the Sixty-Eight World Health Assembly made a call to improve awareness and understanding of AMR, and to strengthen knowledge through surveillance and research as strategic objectives . ImportaSalmonella isolates through UPGMA together with AMR profiling allows detecting spread of resistance along the food production chain. The finding of similar serotypes on the three types of samples support the observation that some Salmonella serotypes are widespread throughout the beef production chain but a high genetic and phenotypic AMR variability was observed intra-serotypes. This could suggest phenotypical changes in the pathogen possibly related to the usage of antimicrobial substances as therapeutic agents and growth promoters in cattle . A good agreement between AMR and pulse-types were found, which could suggest that both analyses provide evidence of phenotypic features underlying genetic variation valuable for adaptation to the selective pressure caused by the use of antimicrobials. As noticed in S. Typhimurium, and partially in S. Infantis and S. Anatum  according to results from PFGE experiments and genetic analysis of the isolates. Columns correspond to number of informative characters. Lines correspond to each isolate analyzed.Click here for additional data file.


Several new treatment options have become available for pancreatic ductal adenocarcinoma (PDAC), but the support for their use for resectable, borderline resectable and locally advanced PDAC is unclear.A survey was distributed to the members of the European-African Hepato-Pancreato Biliary Association (E-AHPBA) and the pancreas group of the European Organization for Research and Treatment of Cancer (EORTC) regarding 1) definitions of local resectability, 2) indications for neoadjuvant therapy and 3) case-vignettes regarding the resectability and treatment of PDAC.In total, 114 participants from 37 countries were registered. About 35% of respondents, each, were of the opinion that borderline resectability is defined by any venous tumor contact and venous involvement < 180° or > 180°, respectively. The majority (75.4%) of participants believed that borderline resectable PDAC has a high risk for R1 resection and that neoadjuvant therapy might increase the R0-resection rate (79.8%) and improve oncological patient selection (84.2%). Chemotherapy was regarded useful to convert locally advanced to resectable PDAC by 55.7% of respondents. In the cases with resectable, borderline resectable, and locally advanced PDAC, 10 (8.8%), 78 (68.4%), 55 (48.2%) of participants would start with chemotherapy, respectively.Although definitions for borderline resectability differ among European surgeons, there seems to be a rather strong support for preoperative chemotherapy in PDAC aiming at minimizing R1 resections while increasing resection rates.The online version of this article (10.1186/s12885-019-5889-5) contains supplementary material, which is available to authorized users. The treatment of pancreatic ductal adenocarcinoma (PDAC) has seen large changes during recent years. While surgery was only an option for resectable and non-metastatic PDAC in the past , modern Several expert panels and associations have established criteria for resectability of PDAC . These dIn parallel, neoadjuvant therapy is gaining popularity, but again indications may vary. Current literature suggests that a significant proportion of locally unresectable PDAC  may undergo radical resection with favourable outcome after adequate treatment response . RecentlThe aim of this survey was to analyze the current understanding of experts in the field regarding treatment aims and attitudes in the (neoadjuvant) management of pancreatic cancer.A web-based survey was distributed through the administrative offices of the European-African Hepato-Pancreato Biliary Association (E-AHPBA) and the pancreas group of the European Organization for Research and Treatment of Cancer (EORTC) to the respective members using Surveymonkey™. In addition to questions regarding the individual experience in the treatment of pancreatic cancer, the survey was designed to evaluate the attitude of European experts regarding the definition of local resectability and the indications for neoadjuvant therapy of PDAC of the pancreatic head.Since participation to this European survey was anonymous and participation was voluntary and only offered to experts in the field, an approval of an ethical review board was not considered necessary.The SurveyMonkey™ platform was used. The survey contained 21 questions, of which 9 questions served to assess the individual experience of each participant in the management of PDAC, the understanding of definitions of resectability, and effects of neoadjuvant therapy on resectable, borderline resectable and unresectable PDAC , The Netherlands (n = 10), Great Britain (n = 9), Germany (n = 9) and Italy (n = 9). The vast majority of participants was hepato-pancreato-biliary (HPB) surgeons, and most of the participants reported an experience of more than 10 years in the management of patients with PDAC  that borderline resectability is associated with a high risk of R1 resections. In addition, a significant proportion of surgeons felt that borderline resectability inherits a higher surgical morbidity and requires a particular technical expertise Table .Table 2VWhile 39.5% of participants believed that any tumor contact > 180° defines borderline resectability, 32.5% each considered a venous contact up to 180° or any venous involvement as criteria for borderline resectability. Moreover, 42% of participants considered an arterial involvement up to 180°, and 34.2% any tumor contact to the superior mesenteric (sma) or hepatic artery as borderline resectable disease  considered locally unresectable tumors without metastases as locally advanced disease. Six participants did not fully agree with the offered definitions  was of the opinion that palliative therapy is associated with a prolongation of survival. Interestingly, the 80% of oncologists (12/15) associated a palliative treatment with a prolongation of survival, while on a 25.3% of surgeons (25/99) had the same association. Moreover, (86.7%) of oncologists (13/15) and 63.6% of surgeons (63/99) were of the opinion that the aim of a palliative therapy is to relief symptoms. The vast majority of participants, however, was of the opinion that the aim of adjuvant therapy is to reduce the recurrence risk after complete tumor resection , while palliative treatments should relief symptoms .Being asked, which treatment aims are associated with palliative and adjuvant therapies in pancreatic cancer, 23 (20.2%) participants considered any postoperative treatment as adjuvant, all of which were surgeons - except one participant. Only eight participants believed that palliative treatment has a potential for patient cure, and about a third of participants .Considering neoadjuvant therapy, 91 participants (79.8%) replied that neoadjuvant therapy has the aim to increase the R0-resection rate in borderline resectable cancer. Also, 45 (39.5%) respondents considered the treatment of micrometastases and 33 (28.9%) a decreased risk of metastases in resectable cancer as treatment aims of neoadjuvant therapy. Moreover, 31 (27.2%) participants believed that the aim of neoadjuvant therapy is to increase the size of the resection margin in resectable as well as borderline resectable cancer  considered neoadjuvant treatment to increase the R0-resection rate of borderline resectable PDAC than to convert unresectable to resectable PDAC (55.3%). We also found a tendency that oncologists more often believe that the treatment of micrometastases is the primary aim of neoadjuvant therapy. Since multiple answers were allowed to this question, we found a large overlap between the replies.n = 14) of respondents were of the opinion that the surgical morbidity is lower after neoadjuvant therapy. Nearly half of the participants felt that neoadjuvant therapy was better tolerated than adjuvant therapy. However, these differences did not reach statistical significance.The majority of participants  considered the oncological patient selection as the strongest advantage of neoadjuvant therapy over adjuvant therapy, meaning that surgery would be avoided in patients with progressive disease. A quarter of respondents believed that the blood supply to the tumor is better during neoadjuvant than adjuvant therapy. Only 12.3%  considered the resectable tumor as resectable, while three were not sure and five participants were of the opinion that the tumor was borderline resectable.n = 79; 69.3%), 11 respondents considered the tumor as upfront resectable, and 10 as unresectable. Only two were not sure.Similarly, most participants judged the borderline resectable tumor as true borderline resectable  with upfront surgery followed by adjuvant chemotherapy (65%). However, 11% recommended surgery-only (without adjuvant therapy), and 9% would start with a neoadjuvant therapy  Fig. . In this(case 2), about half of the respondents (48%) would start with a neoadjuvant treatment, of which 15 (13.2%) would treat the patient with chemotherapy, and 27 (23.7%) with chemoradiotherapy. Only 20 participants (17.5%) would primarily explore the patient with the aim of a primary resection, and 19 of them would also give adjuvant therapy after an apparently curative resection , about half of participants  would start with a neoadjuvant therapy with consequent surgery in case of adequate tumor response, while 41 (36%) would apply palliative chemotherapy. As neoadjuvant treatment, chemotherapy was preferred  over chemo-radiotherapy , and five participants would use other treatments  as unresectable  disease. In contrast, 26 respondants (22.8%) considered this clinical scenario as primarily resectable, and 12 as borderline resectable disease. Only two participants were not sure  liver metastasis ure Fig. .This first pan-European survey performed by E-AHPBA and EORTC on neoadjuvant treatment in pancreatic cancer found that the definition of borderline resectability (and unresectability) varies among European experts. These different definitions often do not affect the clinical management of patients with PDAC. This study confirms a paradigm shift in the understanding and treatment of patients with locally advanced and oligo-metastatic cancer, but also a broad heterogeneity in the management of such patients. For resectable PDAC, primary resection followed by adjuvant chemotherapy was the preferred concept, and a neoadjuvant approach was only recommended by a few participants. About half of the respondents would initiate neoadjuvant therapy in borderline resectable and in LAPC. Finally, neoadjuvant therapy was only recommended by a minority of participants for metastatic PDAC.Following most classifications, a resectable tumor has a maximal tumor contact to the pv/smv of less than 180° without any contact to the celiac or superior mesenteric artery. Borderline resectability includes tumors which have more than 180° contact to the pv/smv and up to 180° contact to the celiac/superior mesenteric arteries. Tumor infiltration of the celiac/superior mesenteric arteries beyond 180° defines unresectable disease . AlthougThe heterogeneity in treatment concepts for unresectable or oligo-metastatic cancer is even more pronounced and mainly reflects the controversial literature: a few recent publications suggest favorable long-term outcome of patients with unresectable or even oligo-metastatic disease after response to FOLFIRINOX , 12, 13.neoadjuvant treatment should be to decrease the recurrence risk after a curative resection, which may be attributed to fewer lymph node metastases as well as the treatment of micrometastases and circulating tumor cells as indicated by most participants. Further beneficial effects may be larger resection margins, a lower R1 resection rate and smaller tumors. In locally unresectable tumors or whenever the risk of an R1 (wide) resection is high, the primary treatment aim of a preoperative therapy is to shrink the tumor and convert a  unresectable to a resectable disease. In this scenario, the treatment should be considered a down-sizing or conversion therapy [In contrast to well established definitions of adjuvant and palliative treatment aims, a clear definition of treatment aims for a neoadjuvant treatment has not been established, yet. Accordingly, about half of the recipients considered the conversion from an unresectable to a resectable disease as aim of neoadjuvant therapy, while ¾ of them considered the increase in the R0-resection rate as aim of neoadjuvant therapy. Considering the aim of an adjuvant treatment (reduction of tumor recurrence) and the homology of the terms “adjuvant” and “neoadjuvant”, the primary aim of a  therapy . If seco therapy –16. Also therapy –19.neoadjuvant therapy could be given with the aim of an improvement of recurrence-free and overall survival in this scenario. The same idea is the basis of neoadjuvant therapy in resectable tumors [conversion therapy with an estimated lower secondary resection rate (depending on the tumor response rate). The same would be true for a borderline resectable tumor with tumor contact to the superior mesenteric artery – a scenario with a high risk of R1- or even un-resectability. Moreover, in the case of a resectable primary tumor with resectable liver metastases, the aim of a neoadjuvant therapy would be to decrease recurrence rate (e.g. new metastases) after the resection of both the primary tumor and metastases (e.g. case 4).Such differentiation in the terminology of treatment concepts would particularly increase the comparability of results in the borderline resectable group: a borderline resectable tumor (e.g. infiltration of the smv) according to current definitions could be treated by a neoadjuvant therapy with an expected high resectability rate – since it is most probably primarily resectable and would be resected upfront in many experienced centers. Since a deep infiltration may be associated with impaired outcome , a real e tumors . A primaThese two separate definitions of “neoadjuvant” treatment would not necessarily change the management of the patient, since the same treatment is often applied in both situations. However, they might help to better define inclusion criteria for clinical studies and the reading of the literature. If a high response rate is required for a locally unresectable tumor, the conversion therapy should be a maximally potent local treatment (including chemo-radiation), while a systemic treatment could be more suitable to prevent metastatic recurrence in some borderline resectable tumors .In conclusion, this first E-AHPBA/EORTC pan-European survey documents a shift in attitudes regarding the management of advanced PDAC among HPB surgeons and oncologists with fairly large support for neoadjuvant treatment. Moreover, this survey depicts different understandings of borderline resectability and neoadjuvant therapy which should trigger a discussion on an adaption of definitions in the setting of PDAC.Additional file 1:Questions of the survery . (PDF 323 kb)Additional file 2:Case vignettes (provided including CT scans). (PDF 362 kb)


Proteobacteria, Actinobacteria, Bacteroidetes, and Cyanobacteria were dominant in both lakes, with different relative abundances between lakes, and Parcubacteria (OD1) was dominant in air samples for all sampling times, except two. We also identified certain shared taxa between lake water and the air above it. The results obtained on these communities in the present study provide putative candidates to study how airborne communities shape lake water bacterial compositions and vice versa.Very few studies have attempted to profile the microbial communities in the air above freshwater bodies, such as lakes, even though freshwater sources are an important part of aquatic ecosystems and airborne bacteria are the most dispersible microorganisms on earth. In the present study, we investigated microbial communities in the waters of two high mountain sub-alpine montane lakes—located 21 km apart and with disparate trophic characteristics—and the air above them. Although bacteria in the lakes had locational differences, their community compositions remained constant over time. However, airborne bacterial communities were diverse and displayed spatial and temporal variance.  Microorganisms are ubiquitous and govern biogeochemical cycles on a global scale . They haet al.  based on epidemiological studies  and Yuan-Yang Lake (YYL) are the only freshwater sub-alpine lakes in north-central Taiwan. They share similar geographical characteristics—latitude, altitude , and limited anthropogenic activity—and are both in protected areas. In addition, rainfall is the only source of water for these lakes. They have different nutrient levels because TFL is oligotrophic  and YYL In the present study, we used a 16S rDNA Illumina sequencing approach to characterize the bacterial communities in and directly above the two lakes at four time points between January and February, 2015. Our goal was to investigate bacterial compositions and attempt to decipher the influence (if any) of airborne bacteria on lake water communities and vice versa.Yuan-Yang Lake  is located 1,670 m above sea level, while Tsuei-Feng Lake  is 1,840 m above sea level. The two lakes are approximately 21 km apart, and both are sub-alpine montane lakes in Taiwan’s Hsinchu and Yilan counties, respectively.−1. After sampling, the cellulose acetate membrane was carefully transferred to a 15-mL polystyrene tube and placed at −20°C until DNA extraction in the laboratory. There was no rainfall at any of the sampling times (±3 h), even though humidity was high on certain days  connected to a portable battery  and cellulose acetate membranes . Air samples were collected for 1 h during each sampling time on the shores of the lakes at a height of 1 m. The flow rate was measured using the Dwyer RMA-21-SSV Flow meter  and was 2 L minain days .n=3) were collected at each sampling point. Samples were kept at −20°C before DNA extraction in the laboratory.Surface water (depth of 1 m) samples were collected from a boat at the deepest points of the lakes at the same time as air samples. Two liters of lake water was initially filtered using a piece of gauze (to remove large debris such as leaves), followed by an 11-μm filter to remove sand, dust particles, and planktonic organisms, and 200 mL of water was then filtered with a 0.22-μm  filter. Time replicates  following the manufacturer’s protocol. The quality and quantity of extracted nucleic acids were measured using a Scandrop spectrophotometer .16S rRNA gene amplification was performed using the bacterial universal primers 968F (5′-AACGCGAAGAACCTTAC-3′) .Ex Taq HS , 5 μL of 10× Ex Taq buffer, 4 μL of 2.5 mM deoxynucleotide triphosphate mixture, 1 μL of each primer (10 μM), and 1–5 μL (10–20 ng) of template DNA in a volume of 50 μL. PCR was performed under the following conditions: 94°C for 5 min, 30 cycles of 94°C for 30 s, 52°C for 20 s, and 72°C for 45 s, with a final extension of 72°C for 10 min.The reaction mixture contained 1 μL of 5 U TaKaRa The PCR amplicons of the bacterial 16S rRNA gene’s V6–V8 region were verified by DNA agarose gel electrophoresis with a 1.5% agarose gel and 1× TAE buffer. The expected DNA band (~320 bp) was cut from the gel, DNA was recovered with the QIAEX II Agarose Gel Extraction Kit , and quality was verified with the Scandrop spectrophotometer (Thermo Scientific).DNA-tagging PCR was used to tag each PCR product of the bacterial 16S rRNA gene’s V6–V8 region  was performed on pooled 40-ng lots of uniquely marked samples . A TruSeq DNA-PCR Free library was prepared for 2×300 bp paired-end reads by Yourgene Biosciences. Raw reads were sorted and primers removed before further analyses.Reads obtained from Illumina sequencing were quality filtered using Mothur v1.38.1  on a perOperational Taxonomic Units (OTUs) were generated from filtered and non-chimeric reads using the UPARSE pipeline  with cloAir and surface water samples were rarefied separately (based on minimum read counts) due to large variations in their sequence counts. Air samples had 140,235 sequences ranging between 3,572 and 10,128. Surface water samples had 605,007 sequences that ranged between 8,375 and 51,720. Air samples were rarefied with 3,572 sequences and surface water samples with 8,375; rarefaction was performed in Mothur. Diversity measures  were calculated with 1000 iterations on rarefied data with Mothur.The relative abundance (RA) of OTUs was calculated on unrarefied data using an in-house R script. A matrix of the bacterial RA of OTUs at the genus level was used to calculate the Bray-Curtis distance between each sample (air and surface water combined). Clustering with the group average method was performed at the phylum and class levels to reveal differences in taxa between air and surface water samples from two sampling sites via Primer6  . A non-mShared OTUs (unrarefied data) among the four habitats were identified in all possible combinations and a Venn diagram was plotted using Venny 2.1. Shared OTUs were classified into four groups: Group A, present only in the air; Group B, present only in the lake; Group C, present in lake and air samples; Group D, present in all four habitats. Taxonomic distributions for all groups were analyzed and pie charts were prepared of distribution at the phylum level.Since Group D OTUs were found in all four habitats, a Blastn  analysisBLAST against a non-redundant nucleotide database  was performed and hits were filtered based on a percent identity ≥90% and e-value <1e–05, with a maximum of five hits being obtained using the max_target_seqs option of standalone BLAST. FASTA sequences were extracted from the NCBI nucleotide database based on the accession IDs of the hits. Multiple sequence alignment was performed using MUSCLE  in MEGA7et al.  Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) Model and the Global Data Assimilation System (GDAS1) meteorological database , 49. TraP<0.05), Simpson and Evenness diversities showed no significant differences with the factor of two lakes and the factor of four sampling times.In total, 690 OTUs were identified in surface water and air samples when analyzed together, with 662 OTUs in surface water and 68 OTUs in air samples when analyzed separately. When rarefied, there were 4 to 8 air sample OTUs and 76 to 182 surface water sample OTUs . When diProteobacteria, Actinobacteria, Bacteroidetes, and Cyanobacteria were dominant. However, RAs in these phyla were different between the two lakes. In terms of Proteobacteria, Betaproteobacteria was the dominant class in YS, whereas Betaproteobacteria had a lower abundance than Alphaproteobacteria in TS  was dominant in two samples from TA (TA1 and TA2) and all samples from YA  and had 18 OTUs that overlapped in four habitats, Parcubacteria (OTU6) was the most abundant.Shared OTUs in various habitats were categorized into four groups . AlphaprAlthough bacterial OTUs in Group D were identified in the lakes and air at the two locations, they were divided into two groups based on their RAs in lakes or air . OverlapCandidatus Sonnebornia vantaiensis  was a clone sequence of taiensis , Table 1Backward trajectory results indicated that air masses at each sampling time had different trajectories during the 72 h prior to reaching the two lakes. Air masses in the first and second sampling times at TFL (TA1 and TA2) and YYL (YA1 and YA2) mainly came from South-East Asia through a mix of oceanic and land travel . HoweverCyanobacteria and members of the hgcI clade (Actinobacteria), whereas YYL was abundant in Betaproteobacteria, including the genera of Albidiferax, Polynucleobacter, and Limnohabitans. In addition, Gammaproteobacteria, Sphingobacteria, and a few other groups were detected.The present study focused on bacterial community compositions in the water of and air above two sub-alpine lakes (TFL and YYL). Bacterial communities significantly differed between the water of the two lakes, possibly because the lakes had different trophic states  at different sampling times. Terrestrial routes came from different regions of China, passing over the East China Sea ; oceanic routes came from the Pacific Ocean ; and hybrid routes combined the terrestrial and oceanic areas and traveled longer distances . These differences in routes may also affect airborne bacterial compositions above the two lakes. Airborne bacterial communities were similar during the first and second sampling, but varied significantly in the third and fourth sampling, suggesting a dynamic pattern influenced by air masses coming from different sources.Parcubacteria and had >99% similarity with Candidatus Sonnebornia yantaiensis from a freshwater pond in China (In terms of the bacterial composition in air, OTU6 was the most dominant taxon among in China . Since 1Lake shores are potential sources for airborne bacteria near lakes and above a lake’s water. Furthermore, terrestrial environments play a vital role in contributing bacteria to air. The identification of shared bacterial OTUs between air and lake water may contribute to the identification of bacteria species with the ability to inhabit both environments. In the present study, the diverse habitats of shared bacterial species provided an example of the transport of bacteria with air as a medium. One caveat in identifying shared and unique OTUs based on unrarefied data is variations due to the sequencing depth. If we sequence at a higher depth, the count of OTUs shared between habitats may increase in number. Bacterial dispersal and consequent deposition have been reported as a major cause of shifts in bacterial communities during dust storms in Asia  and EuroIn conclusion, we observed variations in bacterial communities in air over time above both lakes and hypothesize that different trophic states account for these differences in the communities between the 2 lakes. We also identified certain bacterial species that have diverse original habitats and may have originated from soil on the shore or water from the lakes to air, as suggested by the MHI analyses. These are putative candidates, and long- and short-term interval observations may provide a deeper understanding of their roles in shaping the bacterial communities of lake water.