The core gut microbiomes of different feeding habits were also previously discovered in animal‐associated and corresponding habitat samples. Certain energy metabolism pathways were enriched in herbivore/omnivore and zooplanktivore/zoobenthivore fishes, whereas lipid metabolism and glycan metabolism were enriched in zoobenthivore/piscivore fishes. Moreover, substantial taxonomic variability was found between the gut microbiomes of fish and animals, indicated by their low degree of shared microbiota. The data and observations reported herein pave the way for further investigations on the co‐evolution of fish gut microbiomes and their hosts, the physiological functions of gut microorganisms and the development of probiotics for improving the nutrition and health of aquaculture fish species.Microorganisms in the gastrointestinal tract of animals play vital roles in food digestion, homeostasis and immune response regulation. Globally, there are 33,700 fish species, representing almost half of all vertebrate diversity and a wide range of physiologies, ecologies and life histories. To investigate gut microbiomes with high coverage, we performed 16S rRNA gene amplicon sequencing with 115 samples of 20 common marine fish species. The fish gut microbiome is a remarkably simple community with low microbial diversity (a maximum of 300 amplicon sequence variants only) and has up to 70% of unknown species in some fish species. The gut microbial community structure was significantly shaped by the combined influence of host‐associated factors, including the fish taxon ( The fish digestive tract houses many diverse microorganisms, including bacteria, archaea and fungi, which jointly create a complex microbial ecosystem  facility at the School of Biological Sciences of the University of Hong Kong to determine the trophic level of the fish species. Dorsal muscle samples were taken from each fish specimen and dried using a freeze drier, and the dried tissues were then ground. Approximately 1.00 mg (±0.10 mg) of dried sample was weighed and subjected to SIA with SIRMS . δs et al. .2.3® SPIN Kit for Soil  following the protocol described by the manufacturer, as our previous study showed that the performance of this kit was better than that of other commercially available kits in terms of the recovery and purity of microbial DNA   in IBM SPSS Statistics for Windows, version 22  based on Bray–Curtis similarity matrix was conducted to test the importance of fish taxon, feeding habit and trophic level as factors to influence the fish gut microbiomes using PRIMER‐E PERMANOVA + software  according to the KEGG Orthology    Greengenes‐closed‐referenced biom files  harboured a considerable proportion of Tenericutes (average 35%), a phylum of gram‐negative, obligate cell‐associated bacteria that have lost their cell walls and many biochemical pathways, becoming highly dependent on their host cells, indicating a classical host–microbiome association.The bacterial richness in the gut represented by the cumulative ASVs revealed moderately diverse gut communities among the 20 sampled marine fish species Figure . The maxClostridium, Photobacterium, Ralstonia and Acinetobacter, were the dominant genera, as they had both a high abundance (average >2.5% of sequences) and high prevalence percentage (existing in >50% of samples), while Propionibacterium was a representative low‐abundance general genus. An interesting finding was that the genus Thermus comprised approximately 13.4% of the total gut bacteria in the marine fish Thryssa chefuensis, which is distinguishingly higher than that in other fishes.Among the fraction of ASVs annotated at the genus level (average 56%) Figure , six genRalstonia and the cyanobacterial lineage ML635J‐21, being commonly found in over 47% of all specimens  (Table p < .05). Since Scorpaeniformes mainly includes piscivorous fish at a relatively high trophic level, the main metabolic differences related to the lack of consumption of vegetation might explain this trend.According to the PERMANOVA results, fish species identity significantly explained the differences in the overall bacterial community composition in the fish gut based on Bray–Curtis distance matrixes (4) Table . When th4) Table . At the p < .05), canonical correlation analysis (CCA) was used to help evaluate whether the gut microbial communities were influenced by the difference in composition between orders or within orders. The three orders Aulopiformes, Clupeiformes and Scorpaeniformes were well separated, indicating that their gut microbiomes had distinguishing composition and structure patterns related to the taxonomic lineage of their hosts . To further identify the critical bacteria differentiating the fish orders, discriminative bacterial clades among the fish orders . This observation again conforms to the pattern that carnivore gut microbiomes are less diverse than herbivore gut microbiomes , suggesting that they have dissimilar microbiome composition and structure and the occurrence of a strong nutritional–environmental effect. Along CCA axis 1, a dietary gradient was observed: distance typically increased from herbivores/omnivores, zooplanktivores/zoobenthivores, zoobenthivores and piscivores to zoobenthivores/piscivores. This trend alone explained most of the taxonomic diversity among the gut microbial communities  and significantly discriminated the herbivores/omnivores from the other feeding habit groups.The gut microbial communities associated with the different feeding habits, especially the herbivore/omnivore, zooplanktivore/zoobenthivore and piscivore feeding habits, were significantly separated at the ASV level , 12 indicators for zooplanktivores/zoobenthivores , and 8 indicators for piscivores   zooplanktivore/zoobenthivore indicators were mainly composed of Hyphomicrobiaceae, Desulfobulbaceae, Microthrixaceae and Ellin6529; and (3) the indicators for piscivores primarily consisted of Acinetobacter johnsonii, Photobacterium damselae and Shewanella. The gut microbiomes of fish with these three feeding habits had characteristic fractions of the corresponding indicator bacteria that were significantly higher than those associated with the other feeding habit groups  Table . The thrs Figure . It was 3.4δ15δ15N is transferred from prey to predator, resulting in stepwise enrichment with an increase in trophic position. In practice, the measured trophic levels of individual fish species in this study were not entirely consistent with their feeding habit patterns as suggested by the literature. For example, the average trophic level of the herbivore Siganus canaliculatus was higher than that of the zoobenthivore/piscivore Inegocia japonica . The diversity of the fish gut microbiomes was found to slightly decrease as the trophic level increased . Twenty discriminative ASVs were selected as indicators, primarily comprising Erysipelotrichaceae for Level 1 consumers, Clostridium perfringens and Sva0725 (in phylum Acidobacteria) for Level 2 consumers, Desulfovibrionaceae for Level 3 consumers and Enterobacteriaceae for Level 4 consumers  Erysipelotrichaceae and Desulfovibrionaceae in the herbivore/omnivore fish, (2) Ralstonia and Gaiellales in the zooplanktivore/zoobenthivore fish, (3) Ralstonia and ML635J‐21 in the zoobenthivore fish, (4) Photobacterium in the zoobenthivore/piscivore fish and (5) Clostridium and Cetobacterium in the piscivore fish.Zooplanktivores/zoobenthivores, zoobenthivores and zoobenthivores/piscivores, respectively, contained two, two and five core ASVs , whereas herbivore/omnivore and piscivore fish did not show a clear core set of ASVs because of the high level of divergence between the most abundant and the most prevalent ASVs Figure . Most ofPoecilia reticulata) gut microbiome that approximately 60% of the core gut ASVs were also found in animal‐associated samples and that the percentage was 30–40% for environmental samples , corresponding to 294 KEGG L3 pathways, 40 KEGG L2 pathways and 6 KEGG L1 pathways Figure . In geneForty pathways related to digestion were identified, including those associated with carbohydrate, glycan, protein and amino acid, energy and lipid metabolism, of which 31 pathways exhibited significant differences in abundance among the five feeding habits Figure . Some paPhotobacterium, ML635J‐21 class, Ralstonia and Acinetobacter guillouiae. Photobacterium exhibits a distinct positive correlation with glycosaminoglycan degradation and other glycan degradation, while and Acinetobacter guillouiae was found to be negatively correlated with photosynthesis and photosynthesis proteins pathway. Ralstonia and ML635J‐21 class were strongly and positively correlated with 12 pathways . Photobacterium, Ralstonia and ML635J‐21 were three of the most dominant bacterial taxa across the surveyed fish gut communities in this study. In a study of Atlantic cod gut microbiomes, Photobacterium iliopiscarium and Photobacterium kishitanii were found to be particularly abundant   Figure . This an3.7Microbacteriaceae, Enterobacteriaceae and Peptostreptococcaceae. Microbacteriaceae was not commonly reported in the fish gut samples but was found in the intestine of xylophagous beetle larvae  were picked from the publicly available data sets of animal gut microbiomes and the fish gut microbiome in this study by closed‐reference clustering against the Greengenes 13.8 database, among which 999 OTUs were observed in fish gut microbiomes Figure . The gutIn addition, over half of the OTUs (595/999) in the fish samples were uniquely to the fish samples without being detected in other animal samples Figure . The num4Siganus fuscescens include Proteobacteria, Cyanobacteria and Firmicutes. Dehler et al. , a model organism for the study of genes and pathways related to development, metabolism and disease , Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes are the most abundant phyla   influence which bacterium to grow or decline. Thus, developing rankings or quantifying the relative contributions of affecting factors instead of limiting studies to qualification also benefits the exploration about the mechanisms of gut bacteria assembly. Surrounding environments such as water and sediment are considered to be major routes of gut microbiota acquisition and enrichment, as demonstrated in grass carp  may contribute to cellulase and amylase production. Likewise, zooplanktivore/zoobenthivore‐discriminative bacteria (e.g. Microthrixaceae) may be associated with chitinase, while piscivore‐discriminative bacteria (e.g. Shewanella) might be digestive enzyme secretors of lipase, protease and peptidase. In addition, enzyme‐producing bacteria isolated from fish intestines have previously been reported, including Bacillus, Acinetobacter, Aeromonas, Flavobacterium, Photobacterium, Pseudomonas, Vibrio, Microbacterium, Micrococcus, Staphylococcus and some unidentified anaerobic bacteria, as potential contributors to amylase, cellulase, protease, lipase, phytase, tannase, xylanase and chitinase production  or culture‐independent methods . In our study, the bacteria associated with different feeding habits might serve as potential producers of corresponding enzymes in hosts. More specifically, the herbivore/omnivore‐discriminative ASV Group I bacteria (e.g. Clostridium, Photobacterium, Ralstonia and Acinetobacter were dominant genera. The fish gut microbial community is a microorganism reservoir that is worthy of study like other microbial communities, as a large proportion of ASV reads remain unidentified at the species level. Fish taxon, feeding habit and trophic level are significant factors shaping gut microbiomes, although their contributions to the variation in community composition and structure are relatively small. The gut microbial community may not be a direct reflection of host‐related factors and environmental factors but a comprehensive result of the interaction of these factors. An inverse relationship between the diversity of gut microbiomes and trophic level of fishes was shown. The predicted functional pathways of the gut microbiomes revealed that some energy metabolism pathways were enriched in herbivores/omnivores and zooplanktivores/zoobenthivores, lipid metabolism and glycan metabolism pathways tended to be enriched in zoobenthivores/piscivores, and protein and amino acid metabolism pathways were primarily abundant in zoobenthivores, zoobenthivores/piscivores and piscivores. The low proportion of shared OTUs between fish and other animals suggests a large distinction existing in the different animal gut microbiomes. The characterized indicator microbiota of different fish species could be further applied as host‐tracking markers in biodiversity surveys using environmental DNA methods in future studies. The identified discriminative microbiota of the different feeding habits are potential digestive enzyme producers, and future studies are required to unravel and validate the functions of the microbiota for the development of probiotics for the fish farming industry. Our work contributes a unique set of data for further understanding the role of gut microbiota in host metabolism and paves the way for manipulative studies on the application of gut microbiota as probiotics in maricultural practices.In summary, we conducted a study on the gut microbiome diversity among 20 marine fish species to explore their general relatedness with host taxon, feeding habit and trophic level, trace the common habitats of core gut microbiomes, compare them with other animal gut microbiomes and link gut microbiomes with digestion‐related functions. Proteobacteria and Firmicutes accounted for approximately 70% of the gut microorganisms in these fishes, and K.M.Y.L., T.Z. and Q.H. initiated and designed this study. Q.H., R.C.T.S., Y.D., Y.P.M. and C.X.W. conducted the sampling, laboratory work and data analyses. Q.H. drafted the main manuscript text. K.M.Y.L. and T.Z. supervised the work and contributed to the data analysis and interpretation as well as the manuscript preparation. All authors reviewed the manuscript.Figs S1‐S10Click here for additional data file.Tables S1–S7Click here for additional data file.Appendix S1Click here for additional data file.


Pfcrt codons 72, 74, 75, and 76), sulfadoxine , and pyrimethamine . No parasites harbored Pfcrt point mutations. Prevalence of the Pfdhfr S108N single mutation was 47%, and we found the triple mutant Pfdhfr haplotype  in a single isolate. We observed no Pfdhps variants except in 1 isolate (A437G mutation). These data confirm the lack of highly resistant chloroquine and SP alleles in Haiti and support the continued use of chloroquine and SP.Haiti is striving for zero local malaria transmission by the year 2025. Chloroquine remains the first-line treatment, and sulfadoxine/pyrimethamine (SP) has been used for mass drug-administration pilot programs. In March 2016, nationwide molecular surveillance was initiated to assess molecular resistance signatures for chloroquine and SP. For 778 samples collected through December 2017, we used Sanger sequencing to investigate putative resistance markers to chloroquine ( SP works to inhibit the protozoal folate pathway, can be administered inexpensively in a single dose, is generally well-tolerated by the recipient population  in a P. falciparum isolate’s Pfdhps and Pfdhfr genes have been shown to be a robust predictor of parasite resistance to SP .As Haiti moves toward malaria elimination, verification of drug efficacy will be crucial for ensuring http://premiermedcorp.com), CareStart Malaria HRP2 (Pf) , and SD Bioline Malaria Ag Pf . Patients who were clinically unstable and requiring urgent medical care were ineligible for participation. We selected a health facility in each of Haiti’s 10 administrative departments, plus an 11th site in Grand Anse  Center for Global Health and approved as a nonresearch surveillance activity. Blood specimens were collected only when participants (parents or guardians for children) consented to participate.Because the sentinel health facility in Artibonite (Centre de Santé Clinique Jolivert) had not provided any malaria-positive specimens during 2016–2017, we chose samples from a separate malaria prevalence survey conducted in southern Artibonite in April 2017 from 2 health facilities ≈1 km apart to represent genotypes from this important malaria transmission area. This survey in Artibonite was approved by Haiti’s Ministry of Public Health and Population Bioethics Committee and the institutional review boards of Tulane University and the London School of Hygiene and Tropical Medicine. Adult participants provided written consent, consent for children (<18 years) was provided by a parent or guardian, and children >6 years of age gave written assent to participate. Persons 16 or 17 years of age who were married, a head of household, or a parent were considered mature minors and consented directly. Thumbprint consent or assent (countersigned by a witness) was used for illiterate participants. Persons <6 months of age or who required immediate medical attention were excluded.https://www.gelifesciences.com). To ensure consistency in blood sample collection, all health facility workers had previously been trained on appropriate dried blood spot sample preparation. Each filter paper was air-dried overnight and individually stored in a sealed plastic bag containing a desiccant packet. Filter papers were stored at health facilities at room temperature away from sunlight until transfer. Three times per year, sentinel site samples were transferred to Haiti’s National Public Health Laboratory . For the separate Artibonite survey, samples were transported to LNSP on a weekly basis. For consenting participants in sentinel surveillance and cross-sectional survey study sites, health facility workers drew a sample of ≈200 μL by fingerprick onto Whatman 903 Protein Saver cards  according to the manufacturer’s protocol, with the exception that double the amount of filter paper blood and proteinase K were used. After elution, DNA samples were kept at 4°C for short-term storage and −40°C for long-term storage.At LNSP, filter paper cards were cut in half, with 1 section remaining at LNSP and 1 shipped to CDC  for molecular analysis and sequencing. On the Whatman 903 card, the center circle was not filled with blood to allow cutting of the card down the middle without introducing DNA contamination risk. Scissors were wiped with 70% ethanol between card cuttings. DNA extraction was performed by using the DNA Mini Kit  real-time assay as described previously  on the basis of the beginning of the log phase increase in fluorescence intensity, and we considered a Ct value <40.0 to be DNA-positive signal. We converted Ct values to estimated parasite densities on the basis of a separate DNA dilution series of 3D7 and Dd2 P. falciparum blood cultures that had been dried on Whatman 903 filter paper and on the basis of DNA extracted according to the same protocol we have described. We estimated parasite density by extrapolating from Ct values on the basis of the average regression curve of the 2 P. falciparum culture strains  and the Sigma dNTP kit , with addition of 1μL of each primer (at 15 mmol/L) and 2 μL DNA per reaction, yielding a final reaction volume of 20 μL. We subjected amplified products to a sequencing PCR with the Applied Biosystems Big Dye kit  with 1 μL of 15 mmol/L primer (https://macvector.com).We performed Sanger sequencing for eviously  Table 1.L primer  Table 1.For samples collected during March 2016–December 2017, 757 were analyzed for molecular drug resistance markers associated with chloroquine and SP resistance. Of 11 sentinel sites participating in the program, 9 provided samples . A totalP. falciparum infections to be at low estimated parasite densities; 299 (38.4%) were estimated at a parasite density of <1,000 p/μL blood and 544 (69.9%) <5,000 p/μL blood  of all samples analyzed provided results for codons 50, 51, 59, 108, and 164  and ranged from 6% to 67% of isolates sequenced from each health facility  samples providing interpretable results. Not considering the 540 codon, we successfully 465 (59.8%) samples analyzed sequenced for Pfdhps. The only mutations in Pfdhps were for the five considered codons, except for a single polymorphism of A437G found in 1 sample from the Les Anglais facility in Sud department.Of 778 samples analyzed, we successfully sequenced 741 (95.2%) for reporting of , and 76  Table 2. and 164  Table 2.Pfcrt, Pfdhfr, and Pfdhps genes was largely dependent upon the estimated parasite density via P. falciparum DNA content in the sample. At parasite densities >1,000 p/μL blood, all codons were reported for Pfcrt in >98% of samples  >75% of the time, and in samples >25,000 p/μL blood sequencing was successful 89% of the time (if not including Pfdhps K540E at 79%). However, at densities <1,000 p/μL blood, the 5 Pfdhfr codons were only sequenced in 50% of samples and the 5 Pfdhps codons in 17% of samples.The rate of success for sequencing the  samples . Even atOur study aimed to describe Haiti’s nationwide sentinel site surveillance program for monitoring antimalarial drug resistance for samples collected during 2016–2017  2 for primary diagnosis, any P. falciparum isolates lacking functional HRP2 and HRP3 production might have been overlooked through this surveillance strategy , for this initial report to establish a molecular baseline for polymorphisms in the P. falciparum drug-resistant haplotypes in the country outside of the catchment areas of these health facilities  to the population in areas with a high level of transmission, with particular focus on the southern peninsula  is required for high-level resistance (Pfdhfr gene and might lose efficacy if multiple codon mutations would arise in the gene (P. falciparum parasite population in Haiti is currently only showing incomplete penetrance of the S108N mutation, if SP were used for MDA in Haiti, then this ongoing drug-resistance surveillance program will be crucial to detect any emergence of Pfdhfr double or triple mutants, especially in conjunction with the A437G and K540E mutations in the Pfdhps gene (Pfdhfr triple mutant in Cazale and 1 Pfdhps single mutant in Les Anglais is notable, but both represent well under 1% of the parasite isolates in the our study, which is consistent with previous reports (One of the most consistent findings in this 2016–2017 study was the pervasive prevalence of the Pfdhfr and Pfdhps genes, although Pfcrt Sanger sequencing performed well at any level of DNA content. Using this strategy of DNA quantification will assist in excluding samples for Pfdhfr and Pfdhps Sanger sequencing that would have a low likelihood of interpretable results. Although the success rate for Sanger sequencing of Pfdhfr and Pfdhps was low compared with the success rate for Pfcrt, especially in low-density parasitemia samples, resistant chloroquine or highly resistant SP resistant genotypes clearly are very rare across Haiti. Furthermore, our results support the rationale for using SP for MDA in certain areas of Haiti with the aim of interrupting P. falciparum transmission as part of malaria elimination efforts. If different antimalarial drug strategies are proposed for Haiti in the future, this nationwide sentinel surveillance system could also be used for monitoring of other putative genetic markers of resistance. In addition, employing next-generation sequencing methods will also enable the comprehensive characterization of this parasite population for well-established resistance markers in other target genes besides only Pfcrt, Pfdhfr, and Pfdhps. Continued monitoring for drug-resistance markers, especially during MDA, will be critical for detecting any potential emergence of highly resistant genotypes associated with resistance to SP and chloroquine.Categorizing the samples by quantified parasite DNA levels was very predictive of success in Sanger sequencing for the Plasmodium falciparum drug-resistance alleles to chloroquine, sulfadoxine, and pyrimethamine, Haiti, 2016–2017.Additional information about nationwide monitoring for


Microfluidic wet spinning has gained increasing interest in recent years as an alternative to conventional wet spinning by offering higher control in fiber morphology and a gateway for the development of multi-material fibers. Conventionally, microfluidic chips used to create such fibers are fabricated by soft lithography, a method that requires both time and investment in necessary cleanroom facilities. Recently, additive manufacturing techniques were investigated for rapid and cost-efficient prototyping. However, these microfluidic devices are not yet matching the resolutions and tolerances offered by soft lithography. Herein, we report a facile and rapid method using selected arrays of hypodermic needles as templates within a silicone elastomer matrix. The produced microfluidic spinnerets display co-axially aligned circular channels. By simulation and flow experiments, we prove that these devices can maintain laminar flow conditions and achieve precise 3D hydrodynamic focusing. The devices were tested with a commercial polyurethane formulation to demonstrate that fibers with desired morphologies can be produced by varying the degree of hydrodynamic focusing. Thanks to the adaptability of this concept to different microfluidic spinneret designs—as well as to its transparency, ease of fabrication, and cost-efficient procedure—this device sets the ground for transferring microfluidic wet spinning towards industrial textile settings. Different from conventional wet spinning where the precipitation of polymer fibers occurs in coagulation baths, microfluidic wet spinning confines polymer and coagulant solutions within co-axial laminar flows. This technology permits a higher control of the diffusion interface amid the two laminar flows, thus allowing the solidification of polymer fibers in a more regulated manner . SeveralConventional wet spinning of fibers entails a broad range of polymers, namely acrylics, rayon, polyurethane-urea copolymers, aromatic polyamides, and polyacrylonitrile, which have been routinely utilized in the textile industry . ConversDespite the existence of many fabrication methods for microfluidic wet spinning devices, none of them can fulfill all the above-mentioned requirements. For instance, soft lithography has been widely used in a laboratory setting for producing microfluidic chips ,9,10, buRecent advances in additive manufacturing have allowed rapid prototyping of devices with high versatility and precision . While cIn this study, we propose a novel fabrication platform for microfluidic wet spinning via the development of a simple and scalable templating method for the production of a spinneret. Templating methods have been used to fabricate devices consisting of tapered glass capillaries molded within transparent silicone-based elastomers ,31,32,33To demonstrate the versatility and potential for industrial applications of our platform, we developed a lab-based microfluidic wet spinning stage connected with a tensile drawing apparatus. We picked a thermoplastic polyurethane formulation (BASF’s Ellastolan series) for the spinning trials since they are industrial formulations used in a range of applications from nonwoven fabrics to injection molding. The resultant fiber diameter, surface morphology, and porosity differ by varying the extent of hydrodynamic focusing.Furthermore, we extended the versatility of this device with the spinning of poly(acrylonitrile) (PAN) and poly (PVA) fibers, ,7. We haThe design and fabrication of the spinneret are shown in ® BBraun, Carl Roth, Arlesheim, Switzerland) and 25G  hypodermic needles were blunted and cut to length by a rotary tool  attached with a cutting disc . These needles were then cleaned from burs if necessary, with a cutting disc, cleaned with water and acetone  and left to dry. As depicted in During the molding phase, two-component Dow Corning Sylgard 184  was used thoroughly with a 1:10 cross-linker/prepolymer ratio. The resin was transferred to 15 mL Falcon tubes  and centrifuged for 30 s to remove air bubbles trapped inside. A glass Petri dish with the appropriate size was used as a negative mold and filled with the resin until reaching half of the desired spinneret thickness. The resin was partly cured for 5 min at 150 °C in an oven . After the first PDMS layer was partly cured, a 25G needle was slightly inserted into a 21G needle and laid in the middle of the dish. Two 21 G needles were positioned perpendicularly to the axially aligned 25G and 21G needles to a cross shape. Afterward, the rest of Sylgard 184 resins was poured in the Petri dish. The air bubbles inside the needles were removed by placing the glass dish into a desiccator connected to a vacuum pump. Full curing of the first and the second resin layers was attained by placing it in the oven and heating up to 150 °C for 10–15 min. After cooling down to room temperature (RT), the PDMS with needles embedded inside was removed from the glass dish by using methanol  as a lubricant and a spatula for detaching it from the glass (assembly phase). With methanol as a lubricant, PDMS was cut by a utility knife to expose the needles, which were subsequently removed with tweezers, yielding the microfluidic spinneret.3, and 7 × 10−10 m2/s, respectively.A two-dimensional, axisymmetric model is implemented in COMSOL Multiphysics (version 5.4a) to investigate the laminar flow behavior and the resultant hydrodynamic focusing in the microfluidic reactor. Finite element modeling is used to solve the governing equations. An axisymmetric model is justified by the symmetry of the geometry. A schematic of the computational model together with the applied boundary conditions are provided in the 3 (C% = 100%) and 0 mol/m3 for core and sheath inlets, respectively. Atmospheric pressure condition is imposed at the outlet boundary. Appropriate grids were built based on a grid sensitivity analysis. Grid refinement has been performed toward the core needle, to enhance numerical accuracy and stability, and toward the measuring section to have a better resolution in the results. Finally, laminar flow and transport of diluted species were solved together as a stationary problem. Different combinations of core and sheath flow rates were evaluated. The tolerances for convergence were selected based on sensitivity analysis in such a way that increasing the tolerance further did not change the results (core flow diameter) anymore. The simulation model  were used as the model fluids. A few drops of saturated aqueous methylene blue  solution was utilized to enhance the boundary region amid the core and sheath flow. The two fluids were loaded into syringes  that were attached to three microfluidic syringe pumps . The three syringes linked to the core flow channel and the two sheath flow channels were connected to 1 mm inner diameter silicone tubes by utilizing 18G  blunt needles. The microfluidic spinneret was prepared by firstly tapering 21G and 25G needles with a rotary tool and then inserting them into the respective channels. All inlets were connected to the pumps via silicone tubes. The study was performed during continuous flow and microscopy images were taken for analysis and the diameter of the core flow was measured using imaging software (Leica Application Suite LAS EZ v3.4.0) at three different locations along the main channel. For this study, the total flow rate of 300 µL/min was chosen with a sheath/core flow ratio (r) of 0.4, 0.5, 1, 2, 5, 10, 20, 50, 100. Four additional high-speed tests were performed at 1000 µL/min with sheath/core flow ratio (r) of 10, 50, 100, 200.Laminar flow in the reactor, as well as hydrodynamic focusing, has been studied by flow experiments and visualized with an overhead microscope  . For thiw/v of TPU  in DMF . To prepare this solution, TPU was dissolved in DMF by heating and stirring until reaching a clear solution, which was then cooled down to RT to yield a slightly cloudy viscous solution. Meanwhile, 6% w/v PEO  in distilled water was prepared by stirring at 70 °C until complete dissolution. The solution was then diluted 1:1 with DMF to obtain a cloudy solution of 3% w/v PEO, which was subsequently used for sheath flow. Syringes and tubes in this section were connected similarly to the flow experiments. The spinning dope consisted of 15% ® BBraun, Carl Roth, Arlesheim, Switzerland) connected to the sheath flow channels of the reactor by a 21G needle while spinning dope was filled into a 5 mL disposable syringe  connected to the core flow channel with a tapered 25G needle. Sheath fluid was filled into two 20 mL disposable syringes  in water and 5% w/v PAN  in DMF were used for core flows, while PEG (Mw 3350 Da) in ethanol  at 20% w/v or in EtOH/DMF (2:1) at 13% w/v were used for sheath flow to induce the coagulation of PVA and PAN, respectively. The microfluidic device used in these experiments only differed in the main channel diameter of 1.2 mm, which was derived from the outer diameter of 18G needles . 21G and 25G hypodermic needles were used as sheath and core nozzles respectively in a spinneret with the same geometry as previously described. During the experiment, the total flow rate was set as 500 µL/min for both fibers. PVA fibers were spun into an acetone  bath and collected manually with a tweezer. PAN fibers were spun at r = 20 and 100, and collected by the mini-wet-spinner.6% The SEM images of the produced fibers were taken with a JEOL Hitachi S-4800  SEM at 2 keV with 5 nm of Au-Pd sputtering prior to loading in SEM sample chamber. Cross-sections were obtained by cutting the fibers with scissors in a liquid nitrogen bath and imagined by tilting the SEM stage.The microfluidic spinnerets we are demonstrating in this study were manufactured by using hypodermic needles as templates of the channels. These needles can be sourced from commercial suppliers and are offered with dimensional tolerances as low as ±12.7 µm (34G), making them excellent templating materials for smooth and circular microfluidic channels. The needles can be cut into required lengths with a rotary tool to obtain the desired spinneret channel templates and tapered to avoid sharp edges that would lead to flow instabilities. Co-axial alignment of these needles is achieved by selecting commercial needles with different Gauges (G) and coupling co-axially the fitting ones. Specifically, two pairs of needles were used for templating the co-axial channels, i.e., 25G/21G pair and 21G/18G pair A. Sylgard 184 was used as a thermoset resin for producing PDMS chips, since it can be molded for fast production of transparent objects, with a curing time of 10 min at 150 °C. The needles were employed as negative molds, encapsulated between two layers of PDMS for the construction of the microfluidic spinneret . This avDuring the initial trials, several spinnerets with different designs have been fabricated, including those with thicker channels or multicore spinnerets see . Howeversh/Qc, where Qsh and Qc represent the volume flow rate of the sheath and core flows, respectively) on co-axial flows of polymer core and coagulant sheath solution or so-called hydrodynamic focusing. These insights open up opportunities to optimize geometrical parameters of the setup , to define the set of boundary conditions where the microfluidic device can operate to produce fiber in a continuous fashion. Moreover, overall flow stability as well as uniformity around the nozzle, which is critical for 3D hydrodynamic focusing and stable fiber production, were evaluated.Fluid flow characteristics were analyzed by using laminar fluid dynamics simulations with the finite element method (FEM). This computational model aims to evaluate the impact of different sheath/core flow rate ratios  in the solution for core flow, while colorless PEG (Mw 400 Da) was used for sheath flow. PEG was chosen as a model fluid for the flow experiments as its viscosity resembles the one utilized in the subsequent experiments for fiber production. Compared to using water as the model fluid, this enabled us to evaluate the performance of the microfluidic spinneret under the conditions that are more relevant to fiber production. To compare experimental results with simulations, the same total flow rate of 300 μL/min was chosen. Hydrodynamic flow-focusing was characterized by varied sheath/core flow ratio (r) ranging from 0.5 to 200   solution in DMF/H2O (1:1) was used as sheath flow. This mixture of solvent and non-solvent in sheath flow ensures slow coagulation of TPU polymers in the core flow [As a proof of concept, thermoplastic polyurethane fibers were produced by microfluidic wet spinning. 15% ore flow , therefoore flow , leadingore flow . Additioore flow  or in enore flow  when theTo facilitate the fiber spinning and collection, the microfluidic spinneret was coupled to a customized automatic fiber drawing system  and poly(acrylonitrile) were also spun. We have also shown that the fabrication of individual few-micron-thick fibers can be considered realistic with this device. In addition, the present microfluidic device can be upgraded with two sets of sheath flows and a multicore channel with three differential channel diameters along the main channel. The upgraded version would enable the microfluidic production of complex fibers, including hollow fibers, core–shell fibers and liquid-core fibers. Such a cost, time and labor efficient fabrication of versatile microfluidic devices with these features will ease the translation of microfluidic wet spinning within industrial textile research facilities. Indeed, the fabrication technique developed here can make microfluidic research more accessible for not only the academic field, but also to the industrial sector.


The presence of obstructive sleep apnea (OSA), a novel cardiovascular risk factor, contributes to the development of peripheral arterial diseases (PAD). There is a lack of data showing how often these diseases coexist.The aim of the study was to determine the prevalence of OSA in the population of patients with PAD.Patients previously qualified for the first revascularization due to PAD were included in the study. All patients underwent an overnight sleep study to detect OSA. Diagnosis of OSA was made when the apnea–hypopnea index (AHI) was ≥5 per hour.2, p = 0.01) and lower hip circumference . There were no differences in the distribution of other investigated cardiovascular risk factors and diseases between these groups. There were no significant differences in OSA distribution or its severity between patients with lower extremity artery disease and carotid artery disease.From 141 patients , OSA was diagnosed in 68 patients (48%). OSA occurred in mild form (5 ≤ AHI < 15/h) in 39 cases (28%), in moderate form (15 ≤ AHI < 30/h) in 21 cases (15%), and in severe form (AHI ≥ 30/h) in 8 cases (6%). Patients without OSA had significantly lower body mass index (BMI; 26.9 ± 5.5 vs. 27.7 ± 5.3 kg/mThe prevalence of OSA in patients with PAD is very high, affecting nearly half of the studied population. Cardiovascular disease (CVD) is a major cause of mortality and disability in European society. The predominant etiology of CVD is atherosclerosis in various anatomical locations.  CoronaryObstructive sleep apnea (OSA) is one of the most common respiratory disorders.  OSA is cEvidence from numerous studies on animal models and clinical data suggest a direct and indirect effect of OSA on the development of atherosclerosis, and OSA is proposed as a novel cardiovascular risk factor. , 6 OSA aPAD patients are classified as a population at very high cardiovascular risk.  In a lar2, and any condition impairing the ability to participate in the trial. All patients included in the study were previously qualified for revascularization by vascular surgeons in accordance with current recommendations and the latest medical knowledge. The presence of PAD was previously confirmed by clinical and imaging studies. All patients on admission underwent a standard examination with an emphasis on the history of CVD and CV risk factors. In addition to standard clinical and biochemical tests, all subjects underwent an overnight sleep study to evaluate possible OSA.This study is an analysis of data from the PARADISE trial —an observational cohort study for which the protocol has been previously published.  Between All enrolled patients, regardless of symptoms suggesting OSA, underwent overnight polygraphy before revascularization intervention. A type 3 portable monitoring device was used in an unattended hospital setting—Embletta® MPR PG (Multi Parameter Recorder—Polygraphy); Flaga, Reykjavik, Iceland. Embletta® meets the American Academy of Sleep Medicine recommendations for evaluation of suspected OSA.  EmblettaSleep study recordings were analyzed and scored manually by a study physician trained in sleep medicine. According to the guidelines, apnea was defined as a reduction in airflow >90% compared to baseline lasting at least 10 s.  HypopneaArterial hypertension: repeated blood pressure measurements at least 140/90 mmHg or taking antihypertensive treatment.Heart failure (HF): the presence of typical HF signs and symptoms or left ventricular ejection fraction lower than 40%.Diabetes: fasting plasma glucose ≥7.0 mmol/L or 2-h plasma glucose during a 75-g oral glucose tolerance test ≥11.1 mmol/L or random plasma glucose ≥11.1 mmol/L with hyperglycemia symptoms or taking antidiabetic treatment.2 measured for at least 3 months.Chronic kidney disease: glomerular filtration rate <60 mL/min/1.73 mPositive family history: CAD or other atherosclerotic diseases occurring in men before 55 years of life or in women before 65 years of life.Previous myocardial infarction, stroke, asthma, chronic obstructive pulmonary disease: on the basis of medical records.2; overweight, ≥25 and <30 kg/m2; and obesity, ≥30 kg/m2.Body weight categories were determined on the basis of BMI: normal, ≥18.5 and <25 kg/mOn the basis of clinical history, disease and drug questionnaires, physical examination, body measurements, current blood test, and imaging studies, we determined CV risk factors and comorbidities. All disease entities were defined according to the latest medical knowledge. Selected definitions are presented below:The study protocol including questionnaires, clinical examinations, body measurements, blood tests, polygraphy studies, as well as patient information and informed consent were approved by the Bioethics Commission by the Medical University of Warsaw (KB/196/2015). Each patient was assigned a unique identification number for the protection of personal information. Researchers, statisticians, and controlling bodies were blinded to research data. No third parties had access to the research data.t test was used for data with normal distribution and Mann–Whitney U test was used for data deviating from a normal distribution. For frequency comparisons, depending on the sample size, χ2 test or non-parametric Fisher’s exact test was used. For comparison of mean values between more than two independent groups, depending on the distribution, analysis of variance (ANOVA) or the Kruskal–Wallis test was used. All statistical calculations were made using SPSS v.21.0 software .Patients were characterized using standard statistical tools; for continuous parameters, mean values and standard deviations or medians were used. Parameters with discreet character were presented in frequency tables. Normality of distribution was determined on the basis of D’Agostino–Pearson and Kolmogorov–Smirnov tests. To compare mean values between groups, Student’s Of 141 consecutive patients enrolled, the majority were men (60%) at mean age of 69.6 ± 9.5 years. A majority of patients  were qualified for revascularization due to LEAD. The rest of the study population  was referred for carotid artery revascularization. OSA was diagnosed in 68 patients (48%), mild in 39 cases (28%), moderate in 21 (15%), and severe in 8 (6%) . We also observed a trend toward a more common presence of heart failure in the LEAD group. Compared to LEAD patients, patients with carotid artery disease had statistically lower LDL-C levels  and CRP levels . In anthropometric measurements, the only significant difference was in arm circumference. In tested subgroups, no statistically significant differences were found in the distribution of other CV diseases or CV risk factors, including OSA prevalence or OSA severity , significantly lower hip circumference , more common normal body weight , higher total cholesterol , and were less often treated with statins .Analysis of study population subgroups separated on the basis of presence of OSA is presented in Table n = 82), of which 28% cases were mild, 28% moderate, and 29% severe. [n = 59), of which 35% were mild, 27% moderate, and 38% severe. [This study constitutes the largest cohort of patients with PAD tested for the presence of OSA. This is the first study including both LEAD and carotid artery disease populations. The main finding of the trial was a high prevalence of OSA in patients with severe PAD in the Polish population. This prevalence substantially exceeds that previously reported in the general population. For example, in the Wisconsin Sleep Cohort Study, the prevalence of sleep-disordered breathing defined as AHI ≥5/h was estimated at 9% of women and 24% of men between 30 and 60 years old.  In our s severe.  Schahab  severe.  These di severe. , 16 ReasAs expected, the prevalence of other risk factors for CV diseases is very high in patients with PAD exceeding that reported in the general population. Two of the most common risk factors are hypertension and tobacco use, which affect about three fourths of patients with PAD undergoing primary arterial revascularization. Also, a high prevalence of diabetes and a family history of cardiovascular diseases contribute negatively to the risk profile of these patients. It is important to note that despite high prevalence of CV risk factors in this population with atherosclerosis, recommended statin treatment was prescribed for only one third. Improvement in the application of pharmacotherapy prior to surgery may potentially contribute to postponing treatment, improving quality of life as well as prognosis in this group of patients.2, prevalence of OSA has been reported between 40% and 90%. [Obesity is the most frequently documented risk factor for the development of OSA. In population with BMI >40 kg/mand 90%.  This obsand 90%.  Similar and 90%.  The prevand 90%.  In our cOur study has important limitations. The cross-sectional design of the trial makes it impossible to establish a causal relationship. Our liberal exclusion policy, on the one hand, gives an opportunity to assess prevalence in a real life population with PAD, while on the other hand, the heterogeneous population with many existing comorbidities may overestimate the prevalence of OSA in populations with isolated PAD without serious comorbidities.We conclude that unrecognized OSA is highly prevalent in patients with both severe lower extremity artery disease and severe carotid artery disease. However, in most cases, the OSA is mild to moderate in severity. Because diagnosis and treatment of OSA may potentially affect outcomes in this population at very high cardiovascular risk, it is important to maintain a high index of suspicion for OSA and to assess these patients with sleep studies.


Bluetongue virus (BTV) epidemics are responsible for worldwide economic losses of up to US$ 3 billion. Understanding the global evolutionary epidemiology of BTV is critical in designing intervention programs. Here we employed phylodynamic models to quantify the evolutionary characteristics, spatiotemporal origins, and multi-host transmission dynamics of BTV across the globe. We inferred that goats are the ancestral hosts for BTV but are less likely to be important for cross-species transmission, sheep and cattle continue to be important for the transmission and maintenance of infection between other species. Our models pointed to China and India, countries with the highest population of goats, as the likely ancestral country for BTV emergence and dispersal worldwide over 1000 years ago. However, the increased diversification and dispersal of BTV coincided with the initiation of transcontinental livestock trade after the 1850s. Our analysis uncovered important epidemiological aspects of BTV that may guide future molecular surveillance of BTV. Outbreaks of arboviruses are not only a major public health threat but are often responsible for significant economic losses2. Effective molecular surveillance is crucial to characterize patterns of spread and facilitate pathogen control. For example, phylodynamic (ref) and phylogeographic models have provided fundamental insights into arbovirus spread across the globe3, yet for many arboviruses these models have yet been utilized.Understanding the patterns of pathogen spread at a global scale is notoriously difficult. This is particularly the case for arthropod-borne multi-host viruses (hereafter arboviruses) that infect livestock. Often the reservoir species that are important for maintaining arbovirus prevalence in landscapes are unknown2. BTV causes severe infection in ruminants, especially sheep5. Domestic cattle have been implicated as an important reservoir for the disease6, yet wild ruminants has been also identified as a potential reservoir for BTV as well4, but the relative roles of each host species in BTV circulation remain unresolved. Midges (Culicoides spp.) are major vectors for the transmission of BTV between ruminants7, however instances of other modes of transmission, such as oral route, of BTV are possible9. In general, the geographical distribution of BTV serotypes worldwide is mostly determined by the location of distinct ecosystems of different midge species, including tropical and temperate regions11. BTV belongs to the family Reoviridae (genus Orbivirus) and has a double-stranded RNA genome consisting of 10 linear segments12. BTV is a rapidly evolving pathogen with a marked mutation rate ranging from 0.52 × 10–4 to 6.94 × 10–4 substitution rate per site per year for segments 2, 3, 6, and 1013. Segment 10 encodes non-structural protein NS3/NS3A and comprises approximately 800 nucleotides14. While segment 10 is the smallest among other gene segments, it was shown to be suitable for Bayesian phylogeographic analyses13. Currently, BTV is classified into 27 antigenically distinct serotypes15. Serotypes 1–24 are likely to be predominantly transmitted by midges16, while there are uncertainties around the role of midges in the transmission of serotypes 25 and 2618. BTV control and eradication is heavily dependent on vaccination and movement restriction. However, the use of BTV live attenuated vaccines is a consequential anthropological factor for the dissemination of the disease on an intercontinental scale19.Bluetongue virus (BTV) is one arbovirus that infects livestock worldwide and is responsible for global losses of agriculture of up to US$ 3 billion20. However, the vast geographical expansion of vector range and animal movement through trade led to the subsequent spread of BTV throughout Africa21. In 1924, BTV outbreaks were observed among sheep in Cyprus21. Subsequently, after 1950 the disease has been identified in Asia, Europe, Australia, and North America and thus, has been recognized as an emerging transcontinental disease21. The disease became endemic in many regions worldwide, where it either causes outbreaks through the year in tropical countries or distinctly seasonal outbreaks in temperate countries16. Beside animal movements, climate change has been recognized as the major factor for the transformation of BTV global distribution and epidemiology23. Subsequently, the geographical distribution of BTV outbreaks is also shifting especially in Europe, where exotic and novel strains have been introduced from multiple continents26. During the 2000s, BTV moved from southern to northern Europe causing severe outbreaks and unprecedented economic losses, mostly by serotypes 1, 4 and 823. Over 15 serotypes and 108 novel strains have been reported in India and China, making both countries also of potential importance for BTV emergence and spread29. However, very few studies have attempted to reconstruct the evolutionary history of BTV on either local or regional scales32. Given the continuous emergence of novel BTV strains and the subsequent impact of their outbreaks on livestock populations worldwide, especially Europe, exploring the global evolutionary epidemiology of BTV is a critical element for designing risk-based intervention programs, such as identifying and targeting areas infected with emerging strains.BTV was first described in South Africa in 1876 when European sheep intensive farming was introduced in the region34. The objective of our study was to infer geographical origins, intercontinental spread, significant dispersal routes, and reservoir dynamics of BTV using phylodynamic analyses on all publicly available segments 10 (S10) and 6 (S6) gene sequences. We reveal new evolutionary insights into BTV spread and transmission cycles between hosts across the globe. Such findings may be used to inform and enhance current molecular surveillance efforts of BTV worldwide and subsequently reduce outbreaks caused by both emerging and endemic strains.Here we applied Bayesian phylodynamic methods to reveal the global spatiotemporal and cross-species characteristics of BTV using a commonly sequenced segment in an integrated statistical framework. Phylodynamic methods have become well-established and have been shown to be the most robust tool for tracing historical origins and transmission of rapidly evolving pathogens like BTV–4/site/year (95% highest posterior density (HPD): 3.38 × 10–4–5.09 × 10–4), while for S6 was 5.02 × 10–4/site/year (95% highest posterior density (HPD): 4.42 × 10–4–6.71 × 10–4).Our demographic reconstruction of BTV through time revealed that the approximate divergence time of the virus was inferred around 1000 years ago Figs. A,C usingThe results of the global phylogeographic analyses suggest that BTV emerged from East Asia (root state posterior probability (RSPP) = 0.48 and 0.23 for S10 and S6, respectively; Fig. The Bayesian stochastic search variable selection (BSSVS) approach suggested significant non-directional dispersal routes for BTV between Asia, Africa, Europe, Australia, Middle, South, and North America Fig. A. Most sThe results of both gene segments agree on Europe having the highest forward and reverse transitions in terms of viral jumps between continents Fig. A,B. Yet,Using both gene segments, we found strong evidence that goats were the likely ancestral host of the virus . Indeed, during the past three decades, a whole new-generation of BTV vaccines were introduced and were shown to be more effective in terms of providing a protective immune response and cross-protective against more than serotype39. That said, the continuous emergence of new strains worldwide indicates that vaccine development barely keeps up with the rapid BTV reassortment40.We estimated that BTV has circulated in ungulate populations for at least 1000 years based on the S10 gene and potentially more than 2000 years based on the S6 analysis  inferred between Indonesia and Australia  library66. The final data comprised a total of 700 and 559 sequences for S10 and S6 gene segments, respectively, collected from 52 countries worldwide. We used year of collection to estimate divergence times, and therefore, sequences with no time information were excluded from the analyses . Vaccine and laboratory strains were also excluded from the sequence dataset .We retrieved all complete and near complete S10 and S6 gene sequences of BTV isolated worldwide between 1937 and 2016 from GeneBank. We also retrieved associated metadata, which included, isolate name, collection date, country of origin, isolate type, and host species. Additional sequence metadata were retrieved from the related published literature and the online phylogenetic sequence analysis and improved diagnostic assay system for viruses of the family 67 and MUSCLE version 3.8.368 to align and confirm the reading frame by examining the amino-acid translation of 558 S10 and 487 sequences. The length of the final alignment of the S10 sequences ranged from 690 to 822 bp, and 1572–1638 bp for S6. We mapped the geographical distribution of the sequences and used the GTR + Γ substitution model implemented in RAxML version 8.069 to assess their phylogenetic tree topology  approach in order to identify the best fitting demographic model for our dataset. We used the stepping-stone sampling and path-sampling methods79 to estimate the marginal-likelihood of each candidate model. We then used the BF comparisons from the resulting marginal-likelihood estimates to select among the relaxed-clock models.We used the relaxed molecular-clock models implemented in BEAST version 1.10.480. For the S6 gene, we had to combine three MCMC simulations with a total of 900 million cycles using LogCombiner to achieve model convergence. We used TreeAnotator version 1.10.4 to discard the first 10% of the samples from each MCMC chain and summarized the resulting posterior probability density as a maximum clade credible (MCC) tree. We used tracer to generate plots for the estimates of the effective population over time from the Bayesian Skygrid coalescent tree with the uncorrelated lognormal branch-rate models.We used Bayesian Markov chain Monte Carlo (MCMC) simulations for 300 million cycles and sampled every 3000th state to infer the posterior evolutionary parameters of BTV’s phylogeny under each candidate model. We used duplicate MCMC runs for each model to assess the stability of their marginal-likelihoods estimates. Convergence of the posterior parameters was assessed through effective sample size  using Tracer version 1.7.181. To assess the impact of the spatial trait resolution on the inferred phylogeographic history, we specified a total of 8 and 17 discrete traits to model spatio-temporal diffusion of BTV between continents and countries, respectively , China (aggregated with Taiwan and Japan), Middle America , Netherland (aggregated with Germany), Nigeria (aggregated with Cameroon), North Africa , Spain , South America , Turkey (aggregated with Cyprus), and France (aggregated with the UK), while non-aggregated countries included India, Israel, Italy, Australia, South Africa, and the USA. We aggregated South Africa with Nigeria and Cameroon for the S6 region as only one sequence from each country was available , goats (Capra aegagrus hircus), cattle (Bos taurus), and deer  as discrete traits. The best fitting coalescent tree model and branch-rate prior combinations to the sequences data, selected as described above, was used for the subsequent ancestral-trait reconstruction analyses. We used the Bayesian stochastic search variable selection (BSSVS) method, implemented in BEAST, to identify significant migration routes and their directionality between countries and hosts. Also, we assessed the fit of the sequence data to two candidate discrete-trait models, namely the symmetric model (reversible transitions) and the asymmetric model (irreversible transitions) using the BF comparisons, as described above. Using the Markov-jump (MJ) method83, we inferred the intensity of backward and forward transitions within discrete trait matrices, as a proxy for the mean number of viral jumps between countries or hosts.To model the transmission between host species, we used sheep (84 to summarize the root state posterior probabilities (RSPP) of the discrete traits at the internal nodes of the MCC trees inferred from the selected phylodynamic models. SPREAD3 version 0.9.6 was used to assess the statistical significance (BF > 3) of viral dispersal routes between countries and hosts. The spatio-temporal evolutionary history of the MCC tree and significant dispersal routes between countries were mapped using JavaScript Object Notation (JSON) maps and SPREAD3 version 0.9685. Finally, we assessed the statistical significance of the association index (Ai) and parsimony scores (Ps) for the selected discrete traits using the Bayesian Tip-Significance Testing (BaTS) software version 2.086 to evaluate their contribution to the structure of the inferred posterior MCC trees.We used FigTree version 1.4.4Supplementary Information.


Glis glis, did not show apoptosis or necrosis. Moreover, their nuclei were typical of quiescent cells, showing similar amounts and distributions of heterochromatin, pre-mRNA transcription and processing factors, as well as paired box protein 7 (Pax7) and the myogenic differentiation transcription factor D (MyoD), as in euthermia. However, the finding of accumulated perichromatin granules  in SC nuclei of hibernating dormice suggested slowing down of the nucleus-to-cytoplasm transport. We conclude that during hibernation, SC nuclei maintain similar transcription and splicing activity as in euthermia, indicating an unmodified status during immobilization and hypometabolism. Skeletal muscle preservation during hibernation is presumably not due to SC activation, but rather to the maintenance of some functional activity in myofibers that is able to counteract muscle wasting.Satellite cells (SCs) participate in skeletal muscle plasticity/regeneration. Activation of SCs implies that nuclear changes underpin a new functional status. In hibernating mammals, periods of reduced metabolic activity alternate with arousals and resumption of bodily functions, thereby leading to repeated cell deactivation and reactivation. In hibernation, muscle fibers are preserved despite long periods of immobilization. The structural and functional characteristics of SC nuclei during hibernation have not been investigated yet. Using ultrastructural and immunocytochemical analysis, we found that the SCs of the hibernating edible dormouse,  Satellite cells (SCs) represent a population of postnatal mononucleated stem cells  that areHibernation is an adaptation to adverse winter conditions adopted by several mammals , which iGlis glis, Gliridae), with myonuclei showing morphological evidence of transcriptional activity [Over the last several years, the morphology of the cell nucleus during the hibernation/arousal cycle has been studied in several tissue types of different species in our laboratory ,25,26,27activity . In the This is a retrospective study conducted on specimens obtained from six male edible dormice captured in 1998/1999 for the purpose of multiple investigations upon permission from the Regione del Veneto . Adult (approximately 1–2-year-old) wild-living animals were trapped and maintained in an outdoor animal house supplied with food and bedding material. Under such conditions, they spontaneously began to hibernate in November and awoke in March. Three animals were killed during the euthermic period (June–July), and three during deep hibernation . Euthermic animals were decapitated under deep anesthesia; hibernating animals were taken from the cage and immediately decapitated.Samples of the right quadriceps muscle were processed for transmission electron microscopy for either morphology or immunocytochemistry.v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M Sørensen phosphate buffer pH 7.4 at 4 °C for 2 h, post-fixed with 1% (v/v) OsO4 at 4 °C for 1 h, dehydrated through graded acetone and embedded in Epon 812. Ultrathin sections (70–80 nm in thickness) were placed on copper grids coated with a Formvar layer and stained with Reynold’s lead citrate prior to observation.For morphological analysis, muscle samples were fixed by immersion in 2.5% (v/v) paraformaldehyde in 0.1 M Sørensen phosphate buffer at 4 °C for 2 h, washed, treated with 0.5 M NH4Cl in PBS 0.1 M pH 7.4 to block free aldehydes, dehydrated with ethanol and embedded in LR White resin polymerized under UV light. Ultrathin sections (70–80 nm in thickness) were placed on nickel grids coated with a Formvar-carbon layer and treated with the following probes: mouse monoclonal antibodies directed against the active phosphorylated form of RNA polymerase II  and the DNA/RNA hybrid molecules [w/v) bovine serum albumin  and 0.05% (v/v) Tween 20. After rinsing, sections were floated on NGS, and then reacted for 30 min at room temperature with the specific secondary 12 or 6 nm gold-conjugated antibody  diluted 1:10 in PBS. Finally, the sections were rinsed and air-dried. The control grids were treated as above, but the primary antibody was omitted from the incubation mixture, and then processed as described. After the immunocytochemical procedure, the sections were treated with Uranyl Acetate Replacement Stain  for 30 min and lead citrate for 45 s in order to weakly stain the heterochromatin and visualize the structural constituents in the interchromatin space. All grids were observed in a Philips Morgagni TEM operating at 80 kV and equipped with a Megaview III camera for digital image acquisition.For immunocytochemistry, muscle samples were fixed by immersion in 4%  of the immunolabelled samples as well as on the tissue of control samples. The same procedure was used to assess the density of perichromatin granules  in sections treated in the same run. Briefly, the surface areas of the nucleoplasmic region and the heterochromatin were measured in 10 randomly selected electron micrographs  of SC nuclei from each animal using a computerized image analysis system . The interchromatin space area was calculated, the gold particles present over the interchromatin space were counted and their density was expressed as number/μmpre-mRNA ) over thTo estimate the amount of heterochromatin, the percentage of the heterochromatin area within the total nucleoplasm area was calculated.p ≤ 0.05).For each analyzed variable, the Kolmogorov–Smirnov two-sample test was performed in order to verify the hypothesis of identical distribution among animals in each group, and then the mean ± standard error of the mean (SEM) was calculated. Comparisons of variables in the two groups (euthermic and hibernating) were performed with one-way ANOVA snRNP, PAX7 and MyoD was similar in SC nuclei from hibernating and euthermic dormice, being almost exclusively associated with PFs at the edge of the heterochromatin clumps . QuantitThe absence of morphologically recognizable apoptotic or necrotic nuclei in SCs suggests that hibernation does not negatively affect the viability of the SC pool; this is consistent with findings in hindlimb skeletal muscles of late torpid thirteen-lined ground squirrels showing that the number of SCs does not decrease during deep hibernation in comparison with euthermia .The SCs bordering the myofibers of hibernating dormice were morphologically similar to those found in euthermic animals; in particular, the structural features of their nuclei were typical of quiescent cells with low nuclear activity, i.e., showing abundant clumps of heterochromatin and a few PFs ,35. QualSimilarly, the in situ analysis of pre-mRNA transcription and processing factors did not reveal differences in both their intranuclear distribution and amount between euthermic and hibernating dormice.Activated RNA polymerase II and DNA/RNA hybrid molecules as well as snRNPs were specifically located in PFs. Similar results were obtained in myonuclei of the same edible dormice  as well Pax7 (a transcription factor marker of both quiescent and active SC) as well as MyoD , werThe presence of similar amounts of immunolabelling for both Pax7 and MyoD in SC nuclei of euthermic and hibernating edible dormice indicates the absence of changes in their activation state along the hibernation cycle, in accordance with findings by Brooks et al.  in grounInterestingly, no nuclear bodies were observed during hibernation inside the SC nuclei of edible dormice, whereas myonuclei of the same animals showed some amorphous bodies . DiffereIn conclusion, the SC nuclei of hibernating edible dormice maintain similar transcription and splicing activity as in euthermia, although the nucleus-to-cytoplasm transport undergoes a slowing down. Therefore, the SC activation state is unmodified during hibernation, supporting the idea that skeletal muscle preservation during this seasonal phase, characterized by prolonged inactivity and starvation, is not due to SCs, but rather to the maintenance of some functional activity in myofibers that is able to counteract muscle wasting ,28.


Lower limb joint angular displacement  explained the highest portion of point variability (89.3%), and three clusters were recommended . Delta flexion was significantly different between clusters and players were characterized as “stiff flexors”, “hyper flexors”, or “hip flexors”. There were no significant differences in jump height between clusters (p > 0.05). Multiple regression analyses indicated that most of the jumping height variance was explained by the same four variables,  regardless of the cluster (p < 0.05). However, each cluster had its own unique set of secondary predictor variables.Previous research has demonstrated large amounts of inter-subject variability in downward (unweighting & braking) phase strategies in the countermovement jump (CMJ). The purpose of this study was to characterize downward phase strategies and associated temporal, kinematic and kinetic CMJ variables. One hundred and seventy-eight NBA  players  performed three maximal CMJs. Force plate and 3D motion capture data were integrated to obtain kinematic and kinetic outputs. Afterwards, athletes were split into clusters based on downward phase characteristics ( The countermovement jump (CMJ) is an essential motor skill for a variety of sports including volleyball, soccer and basketball. For basketball in particular, CMJ performance may provide a technical/tactical edge in both offensive and defensive actions  . Though A defining feature of the CMJ is the downward phase performed prior to push off. According to McMahon et al., 2018) , the dow , the don = 11) [It has been assumed that elite athletes are able to self-select their movement strategies in order to maximize jump height ,16,17. In = 11) ,17, hampn = 11) ,19.Therefore, the purpose of this investigation was two-fold: (1) to determine possible differences in downward phase kinematics of the CMJ in 178 NBA players through a cluster analysis; and (2) to determine the temporal, kinetic and kinematic variables that are associated with different downward phase movement strategies.The present study used a cross-sectional design to examine the downward phase movement pattern of NBA players in the CMJ. Every athlete agreed to release their data and was on an active roster in the NBA at the time of data collection. Standard biomechanical procedures for analyzing the CMJ were implemented. Afterwards, athletes were clustered based on similarities in movement strategy during the downward phase of the CMJ. Downward phase movement strategies were evaluated through the following parameters: delta flexion , maximum flexion, average flexion velocity and maximum flexion velocity at the ankle, knee and hip joints, respectively. These variables were selected as they account for the rate and range traveled at each joint, which impact the efficiency of the SSC ,12,20,21One hundred and seventy-eight professional NBA players (mean ± standard deviation (SD): age 23.6 ± 3.7 years, height 200.3 ± 8.0 cm; mass 99.4 ± 11.7 kg; CMJ height 68.7 ± 7.4 cm) volunteered to participate in the study. To be eligible for inclusion, all subjects had to be on an active NBA roster and free from any musculoskeletal injuries at the time of data collection. All subjects signed an informed consent form going over the details of the procedures and inherent risks.Before testing, athletes underwent a standardized warm-up which consisted of a series of 16 dynamic movements performed on a twenty-yard indoor track. Movements included lunge and hinge variations and progressed to low intensity plyometric exercises. After the warmup, subject’s weight,  overhead reach,  standing height, and playing position were recorded by the principle investigator. Subjects were given practice trials followed by three maximal attempts, interspersed by ninety seconds. Subjects were instructed to jump as high as possible and knock away as many of the half inch veins as they could on the Vertec. Countermovement velocity and depth were self-selected. The jump with the greatest vertical displacement was used for further analysis.A nine-camera Simi  Motion analysis system in conjunction with dual Bertec  force platforms were integrated to collect jump data. A twenty lower extremity reflective marker set was used. Markers were placed on the first metatarsal joint, medial and lateral malleolus, heel, shank, medial and lateral condyles of the femur, anterosuperior iliac spines, greater trochanters, fourth lumbar vertebrae, and the sternum. The motion capture system synchronized motion (120 Hz) and force (1000 Hz) data. Identified marker mergences were split manually and gaps were reconstructed with a cubic spline interpolation for a maximum gap size of 20 frames . Raw dat−1) was defined as the integral of the force time curve divided by the subject’s body mass. Total movement time was calculated by subtracting the takeoff instant  from the initiation of the countermovement, which was determined as a drop in vertical force of 2 SD below average body weight. Peak relative unweighting force refers to the first local minimum (Fz) following the initiation of the countermovement [The following variables were used for further analysis: body weight, jump height, net relative impulse, relative un-weighting force, sum (left and right) braking force, relative sum (left and right) concentric force, total movement time, maximum joint flexion average, delta joint flexion, joint total range of motion, maximal joint flexion velocity, joint flexion acceleration, joint extension, joint extension velocity, joint extension acceleration, and time to maximum joint flexions and extensions. All kinetic variables were obtained from force plate vertical axis data and followed the methodological recommendations described by McMahon et al. (2018) . In briemovement  divided movement . Maximummovement . Peak jok-means cluster analysis was run with four sets of downward phase kinematic variables: delta flexion, maximum flexion angles, average flexion velocity and maximum flexion velocity at the ankle, knee and hip respectively. It should be noted that a considerable challenge facing cluster analysis is choosing the optimal variables, and number of variables, to create the clusters. We attempted to account for this by analyzing the aforementioned sets of downward phase kinematic variables independently and selecting the set of variables which created the most stable clusters. While downward phase movement strategies can be altered by both velocity and range, we decided to only account for one set of variables at a time as adding in additional variables may reduce the stability of the cluster. The ball hall index was used to determine the optimal number of clusters. Afterwards, a one-way analysis of variance (ANOVA) was run to test for between-cluster differences for each dependent variable. A one-way ANOVA was also run on descriptive characteristics ) to examine if there were any between-cluster differences in common CMJ variables. Additionally, a one-way ANOVA was run comparing the average position between clusters . Whenever a significant F-value was obtained, a post hoc test with a Tukey adjustment was performed for multiple comparison purposes. Finally, a stepwise regression was performed on the entire data set and for each individual subgroup, having jumping height as the dependent variable. The Bayesian Information Criterion (BIC) index was used to determine which variables were included in the final model. For each regression model, variables are listed with their partial and total adjusted R2 values. Inverse relationships are depicted by an *. To account for co-linearity between variables, a variance inflation factor (VIF) was run. For a variable to be included in the final output the VIF had to be below 10.0. Reliability was assessed using intraclass correlation coefficients (ICCs). Based on the recommendations of previous literature, obtaining an ICC of >0.70 was set as a minimum acceptable reliability [p < 0.05, and data were presented as mean and standard deviation, delta difference, percent difference, and Cohens d effect size utilizing the pooled SD.All temporal, kinetic and kinematic data were normalized  prior to analysis, a common practice in clustering and regression modeling . A k-meaiability . Data weThe ICCs for each kinematic and kinetic variable included in the regression models can be found below. Kinematic ICC values for each variable are listed from the hip, knee and ankle respectively: maximum joint flexion average , delta joint flexion , joint total range of motion , maximum joint flexion velocity , maximum joint flexion acceleration , maximum joint extension , maximum joint extension velocity , maximum joint extension acceleration . The ICCs for the kinetic variables are as follows; sum braking force (0.91), relative unweighting force (0.97), sum concentric relative force (0.91), total movement time (0.97).Delta flexion explained the highest portion of the point variability (89.43%) compared to the other three downward phase variables. Three subgroups explained most of the variance in the data according to the Ball Hall Index. The degree of delta flexion at the ankle, knee and hip joints within each cluster can be found in p = 0.041) and Cluster 2 . Furthermore, Cluster 2 demonstrated significantly greater delta ankle dorsi flexion compared to Cluster 3 . For delta knee flexion, Cluster 1 had significantly lower range of motion compared to Cluster 2  and Cluster 3 . Additionally, Cluster 2 traveled through more range compared to Cluster 3 . For delta hip flexion, Cluster 1 traveled through the least amount of range compared to Cluster 3  and Cluster 2 . However, Cluster 3 and Cluster 2 were not significantly different from each other, .Cluster 1 demonstrated significantly less delta ankle dorsi flexion compared to Cluster 3  −10.1, Effect Size (ES) 0.43, p > 0.5). For maximum knee extension velocity, Cluster 2 demonstrated significantly greater outputs compared to Cluster 1 . Furthermore, there were no statistical differences between Cluster 3 and Cluster 1 , or Cluster 3 and Cluster 2 . For concentric relative force Cluster 1 demonstrated significantly greater values compared to Cluster 2  and Cluster 3 . However, there were no statistical differences between Cluster 3 and Cluster 2 . For total movement time Cluster 1 had the shortest time compared to Cluster 2  and Cluster 3 . However, there were no statistical differences between Cluster 3 and Cluster 2 .The complete results for the one-way ANOVA on descriptive characteristics between clusters can be found in p = 0.76) as well as Cluster 1 and Cluster 3 . However, the average position number in Cluster 3: (1.87 ± 0.71) was significantly greater compared to Cluster2: (1.51 ± 0.65) . Further, we can infer that cluster three was composed of more forwards and centers compared to cluster two  “stiff flexors” travel through the least angular displacement at each joint. Cluster 2 (n = 49) “hyper flexors” travel through an above average angular displacement at each joint. Cluster 3 (n = 52) “hip flexors” travel through an average angular displacement at the ankle and knee joints but above average at the hip joint  during the downward phase can create three stable clusters. Cluster 1   who examFrom a physics standpoint, CMJ height is determined by the net vertical impulse  produced during the upward concentric phase ,27. As j2R value between stiff flexors, hyper flexors and hip flexors, respectively. Nevertheless, it is interesting to point out that each cluster has its own set of secondary, “cluster specific”, variables which also contribute to the adjusted 2R values. For example, stiff flexors have a negative relationship with maximal plantar flexion, maximal knee extension, and amount of time between peak ankle and knee flexion. Collectively, these additional variables account for 14.29% of the adjusted 2R value within stiff flexors. This suggests that stiff flexors cannot optimize jumping height when they increase the angular displacement at the knee and ankle joints. On the contrary, hyper flexors have a positive relationship with delta knee flexion, and amount of time between peak hip and knee flexion. These additional variables account for 22.73% of the adjusted 2R value within hyper flexors and suggest that, to optimize jumping height in this cluster, players demonstrate high angular displacement at the knee and take more time between peak hip and knee flexion. Lastly, hip flexors demonstrate a positive relationship with hip total angular displacement, and a negative relationship with maximum knee extension, ankle angular displacement and time of movement in which peak dorsi flexion occurs. These secondary variables account for 30.77% of the 2R value within hip flexors, suggesting that hip flexors go through more range at the hip, less extension at the knee and reach peak dorsi flexion earlier on in the movement while limiting total range at the ankle. Though these additional variables do not carry as much impact as the four variables which are consistent between clusters, they provide further support for clustering athletes based on similarities in movement strategies, as this type of analysis provides additional insights into cluster specific predictor variables that would have otherwise been washed out in a single group analysis.Regarding the mechanical variables associated with different downward phase strategies, our findings demonstrate that the same four variables  account for 85.71%, 77.27% and 69.23% of the adjusted Furthermore, as the net vertical impulse is what ultimately determines jump height, researchers have spent decades investigating the bodily strategies, temporal, kinetic and kinematic variables that are associated with high impulses ,19,28. IOur study has several inherent limitations. Firstly, jump height was determined based on the vertex reaching height rather than based on vertical ground reaction force data. The readership should be aware that reaching to a vertex may introduce some bias into the measurement ; however, contrary to untrained cohorts, our sample was composed of high level athletes that perform jump reaching tests routinely  . FurtherOur main findings demonstrate that elite basketball players tend to fall into one of three downward phase movement strategies during the CMJ: stiff flexors, hyper flexors, and hip flexors. Despite having significantly different downward phase strategies, athletes from each cluster are capable of producing similar jump heights. That said, the downward phase strategy does significantly impact how these jump heights are achieved. While the main jump height predictor variables for each cluster are similar, subtle differences between clusters do exist. Practitioners can utilize the specific information presented in this manuscript to develop a better understanding of how their athlete’s downward phase strategy affects common CMJ variables. Furthermore, the data presented can serve as a reference point for CMJ outputs at the highest level of basketball competition in the world. The descriptive nature of this study makes precise training recommendation difficult; however, practitioners may consider adding auxiliary exercises targeting some of the cluster specific predictor variables. One example may be adding half squats to stiff flexors training programs as increasing ability to produce force over minimal amounts of knee flexion may increase CMJ performance for this cluster. Another example may be adding additional posterior chain work for hip flexors as increasing the ability to produce force while traveling through a greater range of motion at the hip joint may increase CMJ performance for this cluster. In certain settings it may also be pertinent for practitioners to target kinetic/kinematic qualities that athletes in a given cluster may lack relative to other clusters. For example, as hyper flexors generally possess lower relative concentric force outputs, practitioners may incorporate general strength exercises into their programs targeting force development. Lastly, it is also important to address the demands of the game and the fact that if two athletes are competing for the same ball athletes who can A) better position themselves prior to performing the jump and B) perform the jump the fastest are likely to achieve a greater percentage of successful outcomes. Future research should look into whether these clusters have any correlation with game derived statistics, and may also attempt to determine if the differences in downward phase strategies reported herein are related to limited active range of motion in a specific joint, weakness of a given muscle group producing torque around a lower limb joint or demonstrate any relationship with prospective/ retrospective injuries.


This study aimed to complement previous studies on the risk of herpes zoster in the asthmatic adult population.The Korean Health Insurance Review and Assessment Service—National Sample Cohort (HIRA-NSC) from 2002 through 2013 was used. A total of 64,152 participants with herpes zoster were matched for age, sex, income, region of residence, hypertension, diabetes, and dyslipidemia with 239,780 participants who were included as a control group. In both the herpes zoster and control groups, previous history of asthma were investigated. The crude and adjusted odds ratios (ORs) and 95% confidence intervals (CI) of asthma for herpes zoster were analyzed using unconditional logistic regression analysis. Subgroup analyses were conducted according to age and sex.Approximately 16.2%  and 12.8%  of participants in the herpes zoster and control groups, respectively, had a previous history of asthma (P < 0.001). The herpes zoster group demonstrated a 1.32-times higher odds of asthma than the control group . The increased odds of asthma in the herpes zoster group persisted in all the age and sex subgroups.The odds for asthma were higher in the herpes zoster group. Herpes zoster is defined as an infectious diseases caused by varicella zoster virus reactivation . ApproxiAsthma is a lower airway disease involving reversible airflow obstruction accompanied by airway hyperresponsiveness and remodeling. The global prevalence of asthma has been reported to be approximately 4.3–4.5% . In KoreHowever, few previous studies matched case and control groups by socioeconomic factors and past medical history. Because asthma patients have demonstrated a higher incidence of past medical history than those in control groups, these potential confounders need to be matched for between asthma and control groups in studies of adult populations . In addiThe Ethics Committee of Hallym University (2017-I102) approved the use of these data. Written informed consent was waived by the institutional review board.This national cohort study relies on data from the Korean Health Insurance Review and Assessment Service—National Sample Cohort (HIRA-NSC). The Korean National Health Insurance Service (NHIS) selects samples directly from the entire population database to prevent nonsampling errors. The details of the methods used to perform these procedures are provided by the National Health Insurance Sharing Service .Out of 1,125,691 cases with 114,369,638 medical claim codes, we included participants who were diagnosed with herpes zoster (ICD-10: B02). Among them, we only included participants who were treated ≥ 2 times or who were treated with antiviral medication ≥ 1 time. From 2002 through 2013, 64,152 participants with herpes zoster were selected.Asthma was defined by the ICD-10 codes for asthma (ICD-10: J45) and status asthmaticus (J46). We selected participants who were treated for asthma ≥ 2 times and who were treated with corticosteroids, a steroid inhaler, long-acting muscarinic antagonists (LAMA), leukotriene receptor antagonists (LTRA), and xanthine . This method has been modified from a previous study .The herpes zoster participants were matched 1:4 with participants (control group) who were never diagnosed with herpes zoster from 2002 through 2013 among this cohort. The control group was selected from the mother population . The characteristics of the matched participants were identified including age, group, sex, income group, region of residence, and past medical history  . To prevThe age groups were classified using 5-year intervals: 20–24, 25–29, 30–34…, and 85+ years old. A total of 14 age groups were designated. The income groups were initially divided into 41 classes . These groups were recategorized into 11 classes (class 1 [lowest income]-11 [highest income]). Participants’ region of residence was divided into 16 areas according to administrative districts. These regions were regrouped into urban  and rural  areas.The participants’ past medical history was evaluated using ICD-10 codes. For the accuracy of diagnosis, participants were considered to have hypertension (I10 and I15), diabetes (E10-E14), and dyslipidemia (E78) if they were treated ≥ 2 times. They were considered to have ischemic heart disease (I24 and I25) and cerebral stroke (I60–I66) if they were treated ≥ 1 time. Depression was defined using the ICD-10 codes F31 (bipolar affective disorder) through F39 (unspecified mood disorder) if these codes were indicated by a psychiatrist ≥ 2 times.To analyze the odds ratio (OR) of asthma for herpes zoster, unconditional logistic regression analysis was used. In this analysis, crude (simple) and adjusted  models were used, and 95% CIs was calculated.For the subgroup analyses, we divided the participants by age  and sex .Two-tailed analyses were conducted, and P values less than 0.05 were considered to indicate significance. The results were statistically analyzed using SPSS v. 22.0 .The rate of asthma was higher in the herpes zoster group  than in the control group (12.8% , P < 0.001, Table The herpes zoster participants have the 1.32 times higher chance of previous asthma histories in adjusted model (95% CI 1.28–1.35, P < 0.001, Table In each subgroup according to age and sex, the herpes zoster participants have the higher OR of previous asthma histories in crude and adjusted model (each P < 0.05, Table Patients with herpes zoster showed a higher odds of asthma than those in the control group that was independent of age, sex, income, region of residence, and past medical history of diabetes, hypertension, dyslipidemia, ischemic heart disease, and stroke. Few previous studies have used control groups matched by socioeconomic status and past medical history. The odds of asthma in individuals with herpes zoster were evaluated according to age and sex. In addition, this is the first study to evaluate the association between asthma and herpes zoster in a Korean population.Several previous studies have demonstrated an association between asthma and herpes zoster –17. ThreImpaired immune responses in asthma patients could increase their susceptibility to herpes zoster infection. Asthma is associated with skewed Th1/Th2 immunity, which leads to Th2 predominant conditions. On the other hand, reciprocal Th1 immune insufficiency could occur in asthma patients. The varicella zoster virus infection is predominantly related to T cell-mediated cellular immunity . The impAsthma can indirectly increase the risk of herpes zoster infection via unidentified comorbidities. Asthma is associated with numerous comorbid conditions including chronic obstructive pulmonary disease, diabetes, and depression –31. ThesThe increased risk of herpes zoster infection in asthmatic patients was consistent in all age and sex subgroups in the present study. Although the risk of herpes zoster infection is high in the elderly population, an association between asthma and herpes zoster infection was present in all the adult age groups. Similar to the present results, a previous study also demonstrated higher hazard ratios of herpes zoster infection in an asthmatic adult population > 20 years old . BecauseOne of the advantages of this study compared to the abovementioned previous studies is that the control group was matched for socioeconomic status and past medical history. In addition, this study used a representative, nationwide population and objective inclusion criteria for both asthma and herpes zoster. The NHIS data includes all the medical records of the Korean population without exception. The unified national health care system using the NHIS allows tracing of the medical records of all Koreans including medical claim codes and prescriptions. Based on this mother population, a specialized statistician extracted a representative sample cohort including data regarding age, sex, income, and region of residence. Cohort studies using medical claim codes have merit because of the relatively objective disease classification compared to questionnaire-based survey studies. However, comparable medical accessibility and availability between the study and control groups must be attained to minimize selection bias because the NHIS data collects medical records that are dependent on health care visits. Thus, matching and adjustment for socioeconomic status are important in analysis of NHIS day.The objective inclusion criteria of ICD-10 codes and medication history improved the fidelity of the current study. However, the severity and management of both asthma and herpes zoster infection could not be determined using the NHIS data. The heterogeneity in the severity or type of disease leaves the exact pathophysiologic link between asthma and herpes zoster to be further elucidated. For instance, asthma has various endotypes with respectively different pathophysiological mechanisms. However, a previous study demonstrated that the risk of herpes zoster infection in asthma patients persisted independent of the definition of asthma that was used . In addiAsthma increased the risk of herpes zoster infection in an adult population compared to the risk of herpes zoster infection in a control group matched for socioeconomic status and past medical history. This increased risk was consistent according to age and sex.