Tick-borne viruses (TBVs) pose a significant risk to the health of humans and other vertebrates. A class of multisegmented flavi-like viruses, Jingmen tick virus (JMTV) was first discovered in Rhipicephalus microplus ticks collected from Jingmen of Hubei Province, China in 2010. JMTV has been confirmed to have a relatively wide distribution in vectors and hosts and is associated with human diseases.Parasitic and host-seeking ticks were collected in Wolong Nature Reserve, Sichuan Province. Total RNA was extracted and then enriched the viral RNA. The DNA library was constructed and then were sequenced with MGI High-throughput Sequencing Set (PE150). After the adaptor sequences,low-quality bases and host genome were removed, resulting reads classified as a virus were subsequently de novo assembled into contigs, which were then compared to the NT database. Those annotated under the kingdom virus were initially identified as potential virus-associated sequences. Phylogenetic and Reassortment analysis of sequences were performed using MEGA and SimPlot software, respectively.Two host-seeking ticks and 17 ticks that fed on giant pandas and goats were collected. Through high-throughput sequencing, whole virus genomes were attained from four tick samples  that shared 88.7–96.3% similarity with known JMTV. Phylogenetic tree showed that it was a novel JMTV-like virus, referred to as Sichuan tick virus, which also had the signals of reassortment with other JMTV strains, suggesting a cross-species transmission and co-infection of segmented flavi-like viruses among multiple tick hosts.We discovered and confirmed one new Jingmen tick virus, Sichuan tick virus. Further investigation is required to determine the pathogenicity of Sichuan tick virus to humans and animals, as well as its epidemiological characteristics in nature. Nairoviridae, Phenuiviridae, Flaviviridae, Orthom-yxoviridae and Reoviridae, and 15 species of TBVs that cause human diseases worldwide have been found  has been increasing and widely distributed all over the world. Since the confirmation of the first TBV, the Louping ill virus found in Scottish sheep, over 80 years ago , at leasen found . In Chinen found , severe en found , Crimeanen found , Jingmenen found , Alongshen found , and Sonen found . The TBVRhipicephalus microplus collected from the Jingmen region of Hubei Province, China in 2010  is a novel tick-borne RNA virus that was identified in a pool of  in 2010 . The vired virus . In 2014entified . Notablyentified . Subsequentified . More sientified .Rhipicephalus associated flavi-like virus also classified as jingmenviruses and share a high similarity with JMTV strain SY84, with 93.06–94.39%, 92.81–94.52%, 92.8–94.5%, and 93–99% identity respectively  in 2015 . This grectively . TherefoIn China, JMTV has been detected in various hosts, such as arthropods, humans, bats, rodents and mammals from Hubei, Fujian, Heilongjiang, Yunnan, Guizhou, Henan, Guangxi and Zhejiang provinces . HoweverParasitic and host-seeking ticks were collected in Wolong Nature Reserve, Sichuan Province using different methods in 2020 . The parThe RNA of collected live ticks was extracted using AllPrep DNA/RNA Mini Kit  according to the manufacturer’s instructions. The ticks were categorized into different groups, such as one engorged tick as a pool, 2–5 non-engorged ticks (around 20 mg) as one pool, and eggs of the engorged tick as one pool.A Nucleic Acid Microbes Purification kit  was also used to enrich the viral RNA from total RNA samples. The remaining RNA was fragmented to about 280 bp and then reverse-transcribed into cDNA. Next, a second strand synthesis was conducted by using a Reverse transcription kit . Through the synthetic double-stranded DNA, a DNA library was constructed through end-repair, UDI  adaptor-ligation, and PCR amplification.The constructed library was qualified with a Qubit® 4.0 Fluorometer . The library fragment size was analyzed with Qsep-100 . The Library Quant Kit  was used to accurately quantify the library for screening qualified libraries. The qualified library was pooled at 3 nmol each. After circularization and generating DNA nanoballs (DNBs), the resulting libraries were sequenced with MGI High-throughput Sequencing Set (PE150) on MGISEQ-2000 platforms .via the Kraken2 program (v2.0.9-beta) taxonomic classifier based on a custom extended RefSeq database. The outputs generated by Kraken2 were transferred to the Bracken program to acquire more accurate estimations using abundance and diversity.Adaptor sequences and low-quality bases were removed from raw sequencing reads by the fastp program (v0.23.1) to obtain clean data. The process of sequence mapping/alignment in the clean reads uses HISAT2 (v2.1.0) based on the hierarchical indexing of host genomes with default parameters to remove the host genome. The unaligned reads were used for microbial taxonomy de novo assembled into contigs using the Megahit program (v1.2.9) . There were more than 50 bases aligning the reference genome defined as reads of the target species. An initial scaffold extension and reassembly from those reads were built using the de novo assembler SPAdes (v3.15.3). Reads aligned to the reference genome were returned to the assembled scaffold using bwa (0.7.17-r1188), and the scaffold was calibrated according to the sequencing depth of the aligned reads to output a complete sequence.The resulting reads classified as a virus by Kraken2 were subsequently (v1.2.9)  with def(v1.2.9)  with an Complete sequences obtained in this study were compared with all sequences in GenBank through blastn of NCBI, and appropriate reference sequences were selected for Phylogenetic analysis. Subsequently, MEGA_11.0.13 was used to align the sequences and reference sequences using the MUSCLE package and default parameters. The maximum-likelihood method of MEGA_11.0.13 was used to process the phylogenetic analyses with 1,000 bootstrap replicates.Reassortment analysis of sequences for virus sequences was performed using SimPlot (v3.5.1). The schematic diagram of the genome structure was drawn on the basis of blastx comparison information of RNA sequence. Variation analysis was conducted through bwa, varscan, and snpeff bioinformatics software.Ixodes acutitarsus, 7 Haemaphysalis longicornis, and 8 Ixodes ovatus, of which 3 Ixodes ovatus ticks had laid eggs. Then 15 pools of tick samples, with 7 Ixodes ovatus from giant pandas (4 adult ticks and 3 groups of eggs), 2 Ixodes acutitarsus parasitizing on goats and 2 free Ixodes acutitarsus, and 4 Haemaphysalis longicornis from goats , 1 Ixodes ovatus from giant panda (PC-16) and 1 Ixodes acutitarsus from goats (PC-19), the relative abundance of which were more than 95%. Three other viruses were primarily distributed in 3 samples , 3 samples  and 3 sample  separately.The relative abundance of the first 30 viruses was shown in Through comparison with all nucleotide sequences in GenBank database, it was found that the assembled RNA sequences in the 4 tick samples  shared a higher similarity with JMTV, including four segments, which have been temporarily referred to as Sichuan tick virus. More significantly, the complete nucleotide sequences of the Sichuan tick viruses were obtained from the four tick samples . These sRhipicephalus associated flavi-like virus and JMTV strains from Lao PDR. These sequences were also found to be similar to JMTV found in Lao PDR, Brazil, Kenya and some provinces of China, with an 88.73–96.28% identity. Subsequently, the analyzed Sichuan tick viruses had an 88.77–95% nucleotide identity with Heilongjiang tick virus, Guangxi tick virus, Mogiana tick virus, Amblyomma virus and Rhipicephalus-fassociated flavi-like virus deposited in GenBank. Through the maximum-likelihood method based on four segments of Sichuan tick viruses, the phylogenetic analyses revealed that every segment of the Sichuan tick viruses clustered together, and then formed a separate clade  and had high similarity (93.96%–95.71%) with te clade –D. Segmeidentity . Yet segidentity –D. The sAmblyomma testudinarium (Japan and Laos), Bubalus bubalis (China), Myotis siligorensis (China), Haemaphysalis longicornis (China). Especially, the signals of recombination between the four segments of the Sichuan tick viruses and GXTV (Guangxi tick virus) from Amblyomma javanense in China, MGTV (Mogiana tick virus) from Amblyomma testudinarium in China, suggested cross-species transmission and co-infection of segmented flavi-like viruses among multiple tick hosts , JMTV and Jingmenviruses group have been found to be distributed across the world, while there is little research on them in Sichuan Province. In this study, we described the discovery of Sichuan tick virus (SCTV), a novel JMTV-like virus, in three tick samples collected from goats and one from a giant panda. This is the first record of a JMTV-like virus in Sichuan Province.Rhipicephalus, Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, and Ixodes  may increase the incidence of zoonosis related to SCTV and the hazard of human and wildlife infection with SCTV.Rhipicephalus associated flavi-like virus strain YNTV4 from Yunnan province and JMTV strain from Lao PDR, illustrating that these jingmenviruses in special geographical locations  manifested a consistent evolutionary direction under similar environmental factors , or that they share a common virus ancestor. JMTV appears to be highly conserved in terms of its evolution as a concerned virus. However, reassortment analysis results demonstrated the sequence differences between SCTV and Rhipicephalus associated flavi-like virus, JMTV, were associated with gene recombination. In addition, although SCTV, a JMTV-like virus, showed the highest similarity with JMTV strains ISK55, IM-OI96, and IM-OI108 from Japan, SCTV phylogenetically displayed the closest relationship with Rhipicephalus associated flavi-like virus from Yunnan and JMTV from Laos, manifesting that may be the evolutionary consequences of different JMTV lineages or extensive mutations of the genome. Furthermore, the sequences of SCTV were remotely different from other jingmenviruses, with ALSV, HLJTV, Amblyomma virus, and Guangxi tick virus from China and Russia, suggesting that the gene recombination between flavivirus and other segmented flavi-like viruses was very common.Phylogenetic analysis regarding Sichuan tick virus showed the four segments of SCTV had the closest evolutionary relationship with This study covered the discovery of Sichuan tick virus in Wolong Nature Reserve of Sichuan Province, which can increase our understanding of tick-borne viruses in the area, provide early warning and prediction of possible tick-borne diseases, reduce misdiagnosis and delay in treatment of tick-borne diseases. It can also improve the diagnostic rate and contribute to the survival of local residents. At the same time, tick-borne viruses in the area were investigated to prevent the occurrence of viral tick-borne infectious diseases, which is beneficial to the safety of residents and is also of great public health significance. Furthermore, early discovery of tick-borne viruses, their vectors, and hosts can provide support for the prevention and control of relevant tick-borne diseases, thereby protecting humans and wildlife, which is significant for public health.Limitations of this study are as follows. Firstly, the study lacks PCR’s validation of SCTV and its serological evidence in humans or animals. Secondly, Sichuan tick virus has not yet been isolated from ticks. Thirdly, the small number of ticks collected in this study is insufficient to indicate the prevalence of JMTV in Sichuan Province, China. In the future, a large-scale epidemiological survey of JMTV needs to be conducted in Sichuan Province to understand more relevant details, such as distribution, hosts, and prevalence. In addition, further serological testing is needed to assess Sichuan tick virus pathogenicity.In summary, we discovered and confirmed one new Jingmen tick virus, Sichuan tick virus, in ticks parasitizing protected animals and livestock, in the Wolong Nature Reserve of Sichuan Province, and its complete nucleotide sequences have been obtained. However, further investigation is required to determine the pathogenicity of Sichuan tick virus to humans and animals, as well as its epidemiologi-cal characteristics in nature.The data presented in the study are deposited in the Genbank of National Center for Biotechnology Information, accession number OQ320755-OQ320766 and OQ158902-OQ158905.JJ and XR conceived and designed the experiments. LH, BJ, and LX performed the experiments. WC, LC, SL, and LH analyzed the data. JJ, FW, PY, HT, and QZ were implemented sample collections. LH and JJ drafted and reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the State Key Research Development Program of China (2019YFC1200501).LC is employed by Beijing Macro & Micro-test Bio-Tech Co., Ltd.; WC is employed by BGI PathoGenesis Pharmaceutical Technology.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Giardia intestinalis is a flagellated unicellular protozoan that colonizes the small intestine, causing the diarrheal disease called giardiasis. The production of extracellular vesicles (EVs) by G. intestinalis and the role of these EVs in the parasite’s interaction with the host have been described. According to biogenesis, EVs are grouped mainly into large (microvesicles—derived from the plasma membrane) and small (exosomes—derived from multivesicular bodies). Populations of EVs are heterogeneous, and improved methods to separate and study them are needed to understand their roles in cell physiology and pathologies. This work aimed to enrich the large extracellular vesicles (LEVs) of G. intestinalis in order to better understand the roles of these vesicles in the interaction of the parasite with the host. To achieve the enrichment of the LEVs, we have modified our previously described method and compared it by protein dosage and using Nano tracking analysis. Giardia intestinalis vesiculation was induced by incubation in a TYI-S-33 medium without serum, to which 1 mM of CaCl2 was added at 37 °C for 1 h. Then, the supernatant was centrifuged at 15,000× g for 1 h (15 K 1 h pellet), 15,000× g for 4 h (15 K 4 h pellet) and 100,000× g for 1.5 h (100 K 1h30 pellet). The pellet (containing EVs) was resuspended in 1× PBS and stored at 4 °C for later analysis. The EVs were quantified based on their protein concentrations using the Pierce BCA assay, and by nanoparticle tracking analysis (NTA), which reports the concentration and size distribution of the particles. The NTA showed that direct ultracentrifugation at 100,000× g for 1.5 h and centrifugation at 15,000× g for 4 h concentrated more EVs compared to centrifugation at 15,000× g for 1 h. Additionally, it revealed that centrifugation at 15,000× g 4 h was able to concentrate at the same particle concentration levels as a direct ultracentrifugation at 100,000× g for 1.5 h. As for the enrichment of LEVs, the NTA has shown a higher concentration of LEVs in direct ultracentrifugation at 100,000× g for 1.5 h, and in centrifugation at 15,000× g for 4 h, compared to centrifugation at 15,000× g for 1 h. Our results have shown that the most used method at 15,000× g for 1 h is not enough to obtain a representative population of large EVs, and we suggest that LEVs released by G. intestinalis can be better enriched by direct ultracentrifugation at 100,000× g for 1.5 h, or by centrifugation at 15,000× g for 4 h. Giardia intestinalis  is a flagellated, anaerobic unicellular protozoan capable of infecting the small intestine of humans [f humans . It is df humans . The parf humans ,2. A hosf humans . Trophozf humans . Most caf humans .G. intestinalis as the parasite constantly changes its variant surface proteins allowing it to evade the immune system, which makes vaccine development challenging [The treatment of giardiasis in humans is based on antiprotozoal drugs from the 5-nitroimidazole family . These dG. intestinalis has driven the search for new, important targets in the interaction of the parasite with host cells. This search may open the way for the development of new therapies. Among the components involved in the host–pathogen interaction that have been widely explored are the extracellular vesicles (EVs).The lack of an effective treatment and vaccine against In recent years, intercellular communication mediated by EVs has received attention due to its potential to carry biomolecules and trigger signals in adjacent cells, participating in physio and pathological processes . The terThe biogenesis of exosomes involves the formation of multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) formed by budding from the endosomal membrane. ILVs can be degraded by the fusion of MVBs with lysosomes, or secreted into the extracellular space by the fusion of MVBs with the plasma membrane; in the latter event, they are called “exosomes,” having a diameter that varies from 30 to 100 nm. On the other hand, MVs are released directly into the extracellular medium through the outer budding of the plasma membrane, and are heterogeneous in size, ranging from 100 nm to 1 μm ,9. Due tG. intestinalis and the role of Giardia EVs in the parasite-host interaction has been described. Kim et al. (2022) [G. intestinalis are able to increase the adhesion of trophozoites to the surface of Caco-2 cells [G. Intestinalis: the LEVs, mostly microvesicles, and the SEVs, consisting mainly of exosomes [G. intestinalis, altering its ability to adhere to the host cell, and we show that only LEVs were able to restore the ability of the parasite to adhere to the host cell after treatment with the inhibitor.The production of EVs by . (2022)  showed t-2 cells . In anotexosomes . We alsoThe separation and purification of extracellular vesicles is not easy to accomplish, and this has been indicated by differences in protocols with respect to differential centrifugation utilized by different groups ,17,18,19G. intestinalis with a host. In this study, we compared the different times of differential centrifugation required to achieve the enrichment of LEVs, using the parasite G. intestinalis as a study model.This work aimed to define a methodology based on differential centrifugation to enrich LEVs in order to better understand their role in the interaction of G. intestinalis (WB strain) were cultured in 13 mL of modified TYI-S-33 medium [2HPO4; 4.41 mM of KH2PO4; 3% yeast extract; 0.05% bovine bile; 55.55 mM of Glucose; 16.5 mM of L-Cysteine; 1.14 mM of Ascorbic acid) supplemented with 10% bovine adult serum (BAS) and 1% antibiotic (streptomycin), in a 15 mL Falcon tube at 37 °C.The trophozoite forms of 3 medium   and 1% antibiotic (streptomycin) in culture flasks at 37 °C inside an incubator with 5% COur group . The celG. intestinalis cultured in the logarithmic phase was placed on ice for 15 min to release it from the tube. To induce the release of EVs, 1 × 106/mL of the parasite was incubated in the TYI-S-33 medium without serum, to which1 mM of CaCl2 was added, at 37 °C for 1 h, in an incubator with 5% CO2. After the incubation time had elapsed, the tube supernatant was centrifuged at 425× g for 5 min to remove the cells. The supernatant from this first centrifugation was transferred to a new tube and centrifuged at 4000× g for 30 min to remove the cellular debris. Then, the supernatant was separated and used in different experiments; one part was centrifuged at 15,000× g for 1 h (15 K 1 h pellet), another part was centrifuged at 15,000× g for 4 h (15 K 4 h pellet) and the other part was ultracentrifuged at 100,000× g for 1 h and 30 min (100 K pellet). The pellet (containing the EVs) was resuspended in 1× PBS and stored at 4 °C for later analysis. 6/mL) were seeded in culture flasks and incubated for 24 h. After the incubation period, the cells were washed with RPMI medium without FBS, and then incubated in RPMI medium without FBS, plus 1 mM of CaCl2, at 37 °C for 1 h, in a 5% CO2 incubator. The EVs were isolated from the culture supernatant using the protocol described above.Caco-2 human intestinal epithelium cells , version NTA 3.4 Build 3.4.4, which reports the concentration and size distribution of the particles. For NTA, each sample was diluted 1:30 in PBS (1×) and subjected to a Nanosight, with readings taken in triplicate during 60 s videos with Detect Threshold 3. For analysis purposes, we considered large extracellular vesicles (LEVs) to be particles larger than 100 nm.Giardia intestinalis were centrifuged to generate a 15 K 1 h pellet and a 15 K 4 h pellet, and were then sonicated for 2 min in order to obtain a better aspect on the vesicles. Then, they were deposited on the grid for 10 min to achieve the adhesion of the sample. After this period, it was necessary to remove the excess of this sample, passing the base of the grid on a filter paper. Then, the process of fixation and negative contrast began. First, the grids containing the samples were fixed with 2.5% glutaraldehyde for 10 min and washed with 0.1 M sodium cacodylate 3 times through the deposition of the grid on a drop of this solution. It was necessary to dry the grid before proceeding to each subsequent wash. Finally, they were placed in 5% uranyl acetate for 3 min for the purpose of negative-contrast staining, washed quickly in Milli-Q water with a resistivity of 18.2 Ω, and dried. The properly prepared samples were submitted to analysis under the transmission electron microscope.G. intestinalis EVs were isolated following the protocol described above, with the exception of centrifugation at 100,000× g for 1 h and 30 min. Also, the supernatant from the 15,000× g 1 h and the 15,000× g 4 h centrifugations were ultracentrifuged at 100,000× g for 1.5 h to generate a pellet containing small extracellular vesicles .For the uptake assay, G. intestinalis EVs (LEVs or SEVs) were incubated with 2 µL of DiI lipophilic membrane dye  in 1× PBS for 20 min in a dark at room temperature. After incubation, the dye was removed, 500 μL of bovine serum albumin (BSA) was added, and then it was centrifuged at 15,000× g for 1 h at 10 °C to wash the EV pellet. To demonstrate that the labeling of EVs was specific and not just for soluble DiI, the same amount of DiI was added to PBS, and the same centrifugations and treatments with EVs were maintained. Then, the cells were incubated with LEVs or SEVs (7 μg protein/mL) DiI-labeled for 6 h. Subsequently, the cells were washed 3 times with PBS, trypsinized and fixated in 0.2% paraformaldehyde. The samples were subjected to flow cytometric analysis on a FACSCanto (BD Biosciences). The data were analyzed with FlowJo.For the flow cytometric analysis, Caco-2 cells were seeded at a density of 100,000 cells/well in 24-well plates for 24 h. For the confocal microscopy analysis, Caco-2 cells  were incubated on sterile coverslips with LEVs or SEVs (7 μg protein/mL) that were DiI-labeled for 6 h. Caco-2 cell monolayers were also labeled for nuclei . After incubation, the cells were washed three times in cold PBS (1×), and fixed with 4% paraformaldehyde. The coverslips were washed with PBS (1×) and mounted with 10 μL of a Permount solution. Internalized EVs were detected using confocal microscopy . The images were processed using FIJI Image J software 1.56. p < 0.05. For statistical analysis, the GraphPad Prism Inc. version 6.1 program was used.To analyze the data, we used one-way ANOVA with Tukey’s post-test. The results were considered significant when G. intestinalis were isolated by differential centrifugation at 15,000× g for 1 h (15 K 1 h pellet) or 4 h (15 K 4 h pellet), and by differential ultracentrifugation at 100,000× g for 1 h 30 (100 K 1 h 30 pellet) in order to separate EVs subpopulations   b. The 15K pellet b, althouK pellet c. The thK pellet d. We anaK pellet e. Our data showed a good enrichment of LEVs in the condition of 15 K 4 h centrifugation, without altering the characteristics of the total population, and with the presence of only less than 5% of SEVs f. FigureG. intestinalis. The highest particle concentration peaks correspond mostly to LEVs  and those enriched for 4 h (15 K 4 h LEVs) and the internalization of isolated SEVs from the 1 h and 4 h 15 K centrifugation supernatant  (We completed an uptake assay of ctively) f,g. Flowctively) g, suggesctively) h. Confocctively) h.g for 35 min [g for 30 min [g for 90 min [g for 1 h [G. intestinalis can be better enriched by differential centrifugation at 15,000× g for 4 h. However, regarding purity, differential centrifugation at 15,000× g for 1 h contains less contaminating SEVs compared to differential centrifugation at 15,000× g for 4 h. With this protocol, LEVs can be enriched without the need for an ultracentrifuge, which is expensive equipment, and can be a limiting factor for many research groups that want to study extracellular vesicles.There are a variety of methods that have been employed to enrich EVs. Some are based on size , density gradient (sucrose and iodixanol gradient), and immunoaffinity, among others . Protocor 35 min ; 10,000×r 30 min ; 100,000r 90 min  and 15,0 for 1 h . In thisGiardia intestinalis. The stimulation of EVS production by environmental factors has been studied in several models. Shao et al. (2018) [G. intestinalis is capable of producing microvesicles (mostly LEVs) under different environmental conditions, including at pH 5. Based on these findings, we stimulated the enrichment of LEVs by incubating G. intestinalis in a pH 5 medium. Our results suggest that at pH 5, G. intestinalis produces a greater quantity of large extracellular vesicles (LEVs-> 100 nm) compared to a normal pH. This result suggests an effect of acidic pH on the plasma membrane that results in the budding of larger EVs. Certainly, the mechanisms that control biogenesis and different specializations of vesicle subpopulations are still uncertain; however, a simple method for achieving the enrichment of LEVs opens the door to further investigations of the dynamics of vesicle release in different biological models.This protocol may also help to further our understanding of factors involved with the secretion of the larger EV population, as we demonstrated with the acidic pH in . (2018)  observed. (2018) . Low pH . (2018) . Evans-O. (2018)  observedT. cruzi trypomastigotes with dendritic cells conferred protection to animals challenged with lethal infection by T. cruzi. Furthermore, the potential of EVs present in the serum of S. aureus osteomyelitis patients to facilitate the diagnosis has already been discussed [Understanding the processes of biogenesis, release and uptake of EVs goes beyond basic science. Recently, EVs have gained attention in translational applications, from the discovery of biomarkers for diseases, to their roles in drug delivery systems. For example, the work by Gutierrez et al. (2022)  showed tiscussed . Borghetiscussed  showed tiscussed .


There are 13 globally recognized rubella virus genotypes of which only 2 (1E and 2B) have been detected recently. The largest percentage of all reported rubella virus sequences come from China and Japan with Africa reporting limited data. In a bid to address the lack of rubella genotype data in Uganda and the World Health Organization Africa region, we sought to characterize rubella viruses retrospectively using sera collected from suspected measles patients that turned out rubella IgM positive.Seven sequences belonging to genotype 2B sub-lineage 2B-L2c were obtained. These sequences clustered with other genotype 2B sequences previously reported from Uganda. None of the other genotypes (1E and 1G) reported from Uganda in the earlier years were detected. In addition, none of the sequences were obtained after the introduction of the measles-rubella containing vaccine. The above highlight the need for continuous rubella virological surveillance to confirm interruption of endemic rubella genotype circulation. Rubivirus in the family Matonaviridae, is a positive-sense single stranded RNA virus that causes mild disease among humans. Although humans are the only known reservoir, the rubella virus has recently been shown to be related to the Rubiviruses Ruhugu and Rustrela, found in bats (Hipposideros cyclops) and mice (Apodemus flavicollis) respectively [The rubella virus, a member of Genus ectively , suggestectively , represeIn Africa, rubella surveillance is leveraged on measles case-based surveillance and utilizes sera that tests negative for active measles infection while CRS sentinel surveillance was initiated in selected countries (including Uganda) between 2011 and 2016 . BecauseWe previously reported on the utility of sera as an alternative specimen for molecular characterization of rubella viruses  through From January 2019 to April 2022, serum specimens collected from suspected measles patients through the measles case-based surveillance programme were tested for both measles and rubella IgM at Uganda Virus Research Institute. Different commercial enzyme-linked immunosorbent (ELISA) kits were used; the Enzygnost® Anti-Measles Virus/IgM and Enzygnost® Anti-Rubella Virus/IgM kits  in 2019 and the Anti-Measles Virus ELISA and the Anti-Rubella Virus Glycoprotein ELISA IgM kits  from 2020 to 2022. The measles IgM negative but rubella IgM positive sera collected within two days of rash onset, with volumes greater than 300 µl, were selected for molecular characterization. RNA extraction and genotyping assays were done as described previously . The resThe 739-nt sequence windows required for rubella genotype determination were obtained following analysis using Geneious prime version 2022.0.2 (Biomatters Ltd) and genotypes determined as previously described . The seqA total of 2,974 sera were received from suspected measles patients through the measles case-based surveillance programme. Of these, 337 11.3%) were rubella IgM positive and negative for measles IgM (Table 1.3% wereThree of the 7 sequences were from the Eastern region while the remaining sequences were from the Western region of the country. Kapchorwa district (Eastern region) had two cases that occurred within two months of each other, from which sequences were obtained Table . The seqWe retrieved a total of 1,261 genotype 2B sequences from GenBank and of these, 581 unique sequences were retained for analysis. Approximately 67% of all the sequences reported were from the Western Pacific region (WPRO) with the Africa region (AFRO) reporting only 44 (7.6%) sequences.The sustained circulation of the rubella genotype 2B in the pre-vaccination era signifies established endemicity in Uganda. This is an important benchmark against which to monitor progress towards disease elimination. Uganda introduced a measles-rubella (MR) containing vaccine in October 2019 through a nationwide campaign that targeted children aged 9 months to under 15 years . The couFirst reported in Uganda in 2014 , rubella− 3 subs/site/year [The two sequences from Kapchorwa district (Eastern region) were identical and possibly represent a contact transmission event since they were collected within two months of each other, and the rubella virus has an estimated overall substitution rate of 10ite/year . The levSub-Saharan Africa (SSA) shoulders 25% of the overall all-cause global disease burden  but onlyWe recovered few rubella sequences because archived sera, through multiple freeze-thaw cycles, might have suffered viral RNA degradation. It will hence be important to utilize this serum-based approach in real time specimens after collection.Since we relied on unvalidated historical data, we could have included sera collected later than two days from illness onset resulting into low recovery of the viral genomes. Hence, late detection of cases outside of the eligible inclusion period post onset could have eliminated opportunities of candidates for genotype(s) detection.We did not have sequence data for 2020 and 2021 which represents a molecular surveillance gap. This could be due to the COVID-19 pandemic, however, it may not undermine our findings given the sufficient genomic data (Fig.


Ecophysiology and herpetology share a close historical relationship, but earlier work at the interface of these disciplines was carried out in temperate regions. Tropical regions like the Neotropics exhibit the highest species richness for amphibians and reptiles, but the pace for ecophysiological research on tropical herpetofauna has been slower relative to temperate counterparts. We are a group of early-career, Latin American researchers interested in the physiological diversity exhibited by neotropical herpetofauna. As such, we have engaged in the organization of the Symposium on the Ecophysiology of Neotropical Amphibians and Reptiles (ECOPHYSHERP) to integrate the scientific community interested on these topics. ECOPHYSHERP has been held three times already within the Colombian Congress of Herpetology, and collectively it has hosted >60 contributions from researchers at 26 institutions and eight countries. Participation has been diverse in terms of gender, age, and career stage, but most participants have been young undergraduate biology students. This generation of early-career researchers is producing excellent research in a broad range of topics, but difficulties to convert this research into scientific publications may exist. Identifying and contributing in order to solve such problems are priorities for this organizing committee, and also our endeavours towards ECOPHYSHERP 4.0 in Santa Marta in 2025. Summary: We present progress in the ecological physiology of neotropical amphibians and reptiles through the lenses of our specialized symposium held in Colombia, and highlight the fundamental role of early-career herpetologists. Ecological physiology (or ecophysiology), the study of physiological diversity in relation to the environments in which organisms live, arose at the interface of comparative physiology and natural history and consolidated as a separate field around the 1940s . Since iAs for many organisms, the herpetofauna species richness is highest in the Neotropics , where sKey events favored the interaction between herpetology and ecophysiology in Colombia during the 20th century. Based on the influence of such events on the establishment of a research program at this disciplinary interface, we have identified three periods. The first period – the exploratory phase (late 1950s – early 1970s) – was marked by field expeditions carried out during this time. Hyla labialis at that moment, now Dendropsophus molitor; Hutchison's pioneer work opened the door to the second period of the ecophysiology-herpetology interaction in Colombia, the developing phase (∼early 1970s – late 1990s). At first, ecophysiological research was mainly species-specific, so that certain species were studied in depth , our passion for ecophysiological research, and our Colombian roots, we have engaged in the organization of the Symposium on the Ecophysiology of Neotropical Amphibians and Reptiles (ECOPHYSHERP). This symposium has been held within the Colombian Congress of Herpetology (main conference), organized by the Colombian Association of Herpetology, three times : the firSupplementary Material). Nevertheless, eight research themes accounted for 81% of all presentations, and thermal biology alone represented 45% , a much less studied topic in the country. After seeing that 47% of presentations at ECOPHYSHERP 1.0 dealt with aspects of thermal biology, we knew that the next editions of the symposium should contemplate at least one keynote talk on this topic. Then, Prof. Patricia Burrowes  participated on ECOPHYSHERP 2.0 and presented her work across distinct tropical ecosystems to illustrate different responses of amphibians and reptiles to climate warming. During ECOPHYSHERP 3.0, Dr. Agustín Camacho  questioned the use of distinct thermal traits to infer aspects of the vulnerability of reptiles to climate warming.Leptodactylus lithonaetes. The thermal dependency of physiology, a classic topic in the field, was also well represented throughout all symposium editions, and here we highlight Juliana Poveda Cantini's talk  at ECOPHYSHERP 2.0 on the effect of environmental temperature on the metabolism of the frog Dendropsophus molitor through its ontogeny. Individual physiology may also affect thermal biology, and that possibility was illustrated by Dr. Jesús E. Ortega-Chinchilla  in his talk at ECOPHYSHERP 2.0 on the effects of gestation on the body temperature of the neotropical rattlesnake Crotalus durissus. The utility of thermal traits to infer aspects of the herpetofauna's vulnerability to warming was shown by Nathalia Suárez Ayala's talk , in which she mechanistically predicted the impact of several warming scenarios on activity time of the lizard Anolis tolimensis across distinct populations. Other addressed topics included thermoregulation in the field or laboratory conditions, impacts of the thermal environment on life history traits, and behavioral fever.The enthusiasm of ECOPHYSHERP participants for thermal biology was evident by the broad scope of their investigations. The question of how some species thrive in extreme thermal environments has intrigued ecophysiologists for decades, and Valeria Ramírez Castañeda  addressed it nicely at ECOPHYSHERP 1.0 in her oral presentation on the heat tolerance of tadpoles and adults of the frog Anolis antonii display morphological variations across elevation that relate to both thermal and hydric gradients.Other themes were comparatively less represented relative to thermal biology, but some emerging patterns are worth mentioning. In contrast with the recent literature, research on water balance and hydro-thermal regulation has been important throughout all ECOPHYSHERP editions. These areas have so much room to grow, as shown by Prof. Carlos A. Navas  in his keynote talk during ECOPHYSHERP 1.0. Although the importance of water is considered obvious for amphibians (eight out of nine presentations on this topic used anurans or urodeles), the same is not for reptiles. Interestingly, Yelenny López Aguirre  showed at ECOPHYSHERP 3.0 that males and females of Crocodilus crocodilus. At ECOPHYSHERP 2.0, Dr. Faride Lamadrid Feris  showed that experimentally-induced chronic stress probably has species-specific effects on anuran immunity, pointing out to the complexity of the relationship between stress and immunocompetence in anurans. To further stimulate this emerging theme, we invited Dr. Carla B. Madelaire  to participate in ECOPHYSHERP 3.0, where she presented a keynote talk on her work on the seasonality of stress hormones, metabolism, and reproductive activity of anurans from the Brazilian semiarid Caatinga.Another research theme that has been important at ECOPHYSHERP is Ecoimmunology. Interest in this area among Colombian ecophysiologists is likely very recent, considering it is still been assimilated in the literature. In this context, Leidy A. Barragán-Contreras  presented a talk during ECOPHYSHERP 1.0 about the effects of testosterone on morphometry and reproductive success of the caiman Bolitoglossa ramosi) is being used as a model organism to study aspects of water balance and hydro-thermal regulation in urodeles . For snakes, three out of six presentations used the thickhead ground snake (Dipsadidae: Atractus crassicaudatus) as model organism. Although common and abundant species may be good models for ecophysiological research, disentangling aspects of physiological diversity in both salamanders and snakes will require to expand efforts to other species. Regarding empirical research conducted within the Colombian territory and presented at ECOPHYSHERP  in eight countries A: ColombInformation on diversity is available only for the third edition of ECOPHYSHERP. Participants and attendees were asked to fill out a form (in Spanish) to obtain their consent to collect, store, and disseminate data and/or photographs in connection with their participation at the symposium. Thirty-seven people, including all participants, from ten institutions answered the form and consented to share their data, which are summarized in In the context of the main conference, the best oral presentations by undergraduate students have been recognized in each ECOPHYSHERP edition. These presentations stood out for the excellence of the investigation as a process, from the identification of a problem or gap, the hypothesis/aims of the study, to the collection and interpretation of the resulting data. The speaker's domain of the study topic and the clarity of the presentation were also considered in our performance assessments. Besides the recognitions, issued by the Colombian Association of Herpetology, students also received relevant textbooks granted by the organization of the main conference.B. ramosi. Raiza N. Castañeda  – ECOPHYSHERP 2.0 awardee – studied the influence of the hydric state on the locomotor performance of three treefrog species from the Colombian Caribbean. Laura A. Calvache-Naranjo  – ECOPHYSHERP 3.0 awardee – analyzed the impact of the hydric state on the locomotor performance of the Ramos' mushroom-tongue salamander.Interestingly, all awardees were young women herpetologists working on aspects of amphibian water balance. Erika X. Cruz-Rodríguez  – ECOPHYSHERP 1.0 awardee, ECOPHYSHERP 3.0 co-organizer, and co-author of this Meeting Review – investigated the influence of relative humidity on the thermal preferences of Funding acquired for ECOPHYSHERP 3.0 was key to foster the development of early-career researchers. In coordination with the Society for the Study of Evolution – SSE (USA), we were able to grant 1-year SSE memberships to 19 participants and attendees  from ten institutions. Moreover, we recognized the excellence of four presentations according to the career stages represented among symposium participants, i.e. undergraduate student, academic professional, master's student, and PhD student. Awardees  received a copy of the book ‘Evolution, 5th ed. ’ by Douglas Futuyma and Mark Kirkpatrick.Ecophysiology and herpetology have a close historical relationship, and young Latin American biologists are finding an attractive niche at the intersection of these fields since early stages of their careers. Our analyses of the recent literature and our symposium indicate that the interest in the ecophysiology of amphibians and reptiles is growing in Colombia. Yet, the disparity between the number of research themes represented at ECOPHYSHERP and those in the literature indicates that early-career researchers likely face difficulties to convert symposium presentations into scientific publications. For instance, only seven works presented at ECOPHYSHERP were published from 2017–2022 . We do nResearch themes: conservation physiology of the herpetofauna; neurophysiology of thermoregulation; physiological diversity of hydro-thermal regulation strategies; microclimate landscapes; temperature – disease interfaces.Taxonomic groups: anurans (embryos), salamanders, caecilians, snakes, turtles.Colombian regions: Pacific, Orinoco, Amazon.About scientific innovation and creativity, George A. Bartholomew once said that “…one can sometimes enhance the probability of making an innovative scientific contribution by transferring ideas and questions from one field of specialization to another, or from one taxonomic group to another…” . Both th10.1242/biolopen.059959_sup1Supplementary informationClick here for additional data file.


Fm) locus on the 20th chromosome results in increased endothelin-3 (EDN3) gene expression and is responsible for melanin hyperpigmentation in BBC. We use public long-read sequencing data of the Silkie breed to resolve high-confidence haplotypes at the Fm locus spanning both Dup1 and Dup2 regions and establish that the Fm_2 scenario is correct of the three possible scenarios of the complex chromosomal rearrangement. The relationship between Chinese and Korean BBC breeds with Kadaknath native to India is underexplored. Our data from whole-genome re-sequencing establish that all BBC breeds, including Kadaknath, share the complex chromosomal rearrangement junctions at the fibromelanosis (Fm) locus. We also identify two Fm locus proximal regions (∼70 Kb and ∼300 Kb) with signatures of selection unique to Kadaknath. These regions harbor several genes with protein-coding changes, with the bactericidal/permeability-increasing-protein-like gene having two Kadaknath-specific changes within protein domains. Our results indicate that protein-coding changes in the bactericidal/permeability-increasing-protein-like gene hitchhiked with the Fm locus in Kadaknath due to close physical linkage. Identifying this Fm locus proximal selective sweep sheds light on the genetic distinctiveness of Kadaknath compared to other BBC.Black-bone chicken (BBC) meat is popular for its distinctive taste and texture. A complex chromosomal rearrangement at the fibromelanosis ( Fm allele  gene located within the Fm locus is responsible for hyperpigmentation seen in the BBC  Evaluating how the BBC breeds dispersed to various parts of Asia. (4) Using population genetic statistics to identify signatures of selection in Kadaknath compared to other BBC breeds.Despite its immense popularity and commercial value, the genomics of the Kadaknath chicken breed has received limited attention. Therefore, further research and genomic analyses are required to understand adaptations in this breed and its genetic history. The aims of this study are as follows: (1) Performing whole-genome re-sequencing of Kadaknath and evaluating its relationship with other black-bone and non-black-bone chicken breeds. (2) Assessing whether Kadaknath and other BBC breeds share a common origin for the The study was approved by the Institutional Ethics Committee (IEC) of the Indian Institute of Science Education and Research, Bhopal, vide reference number IISERB/IEC/Certificate/2018-11/03 dated 8th June 2018. We purchased the meat of six individuals  of Kadahttps://nabic.rda.go.kr/). Out of 88 individuals, 23 are BBCs, which we considered for further analysis. Hence, we analyze a dataset of 101 individuals (88 public +13 sequenced as part of this study) from different breeds  and Korean National Agricultural Biotechnology Information Center (KNABIC) (t breeds . Transcrt breeds . We onlyhttps://github.com/broadinstitute/picard). We performed the variant calling using FreeBayes  using the BWA   using the STAR   and Dup2 (∼170 Kb)) separated by an intermediate (Int) region. Dup1 and Dup2 regions are both duplicated and are involved in a complex rearrangement consisting of two junctions: (A) Dup1 + (inverted Dup2) and (B) (inverted Dup1) + Dup2. To identify the base-pair level positions of Dup1, Dup2, and Int regions, we compared the short-read coverage of black and non-black chicken in 1 Kb windows along chromosome 20. We used the makewindows command of bedtools  generated by Fm locus is partially assembled at Chr 20 in Yeonsan Ogye (Fm locus occur on chr20 and unplaced scaffold (JAJMOI010001544.1) (Fm locus in BBC breeds. While the junction sequences have been reported earlier (Fm locus junction in the Korean BBC PacBio data from ENA (SRR6189090). Based on our search of PacBio reads that mapped to the Gallus_gallus.GRCg6a genome, we shortlisted reads that simultaneously aligned to two of the five  genomic regions we have defined. We found several reads spanning Flank1-Dup1, Dup1-Int, Int-Dup2, and Dup2-Flank2, which is unsurprising because these regions are adjacent.The junction between the rearranged regions in the BBC does not occur in the Gallus_gallus.GRCg6a genome . In searsan Ogye . In the 01544.1) . However earlier  for the We found five reads  that span across both Dup1 and Dup2. Dup1 and Dup2 are far apart in their genomic location in Gallus_gallus.GRCg6a assembly. Hence, these reads support a rearrangement that leads to junctions between these two regions. Three of these five reads  support the junction Dup1 + (Inverted Dup2) , and the other two reads (SRR6189090.54824 and SRR6189090.56386) support the junction (inverted Dup1) + Dup2 . The read coverage at these junctions consistently differed between all black-bone and non-black-bone chickens. Based on the read coverage across the Fm locus junctions, we identified 34 out of 101 samples analyzed as BBC.Fm locus and identify the exact order of rearrangement. For mapping the PacBio and Nanopore long-read data of this Silkie individual, we used the Gallus_gallus.GRCg6a  genome with the BWA  v0.7.17-r1188 bwasw read mapper with the flags -t 24, -a2, -b3, -q2, -r2, and -z1 to obtain high-quality read mapping.Recently, a new high-coverage multi-platform genomic public dataset for the Silkie BBC became available on the European Nucleotide Archive (ENA) as part of the Bioproject# PRJNA805080 . With a ∼65X (Nanopore) and >660X coverage (PacBio) of the same Silkie individual, this dataset is well suited to resolve the haplotypes at the We used the bam-readcount  tool to Fm locus region-wise  for the 34 black-bone and all 101 chicken individuals. We used several flags in ANGSD for population structure analysis as follows: -GL 2, -doMaf 1, -minMapQ 30, -minQ 20, -doGlf 2, and -SNP_pval < 1e-6. Genotype likelihood values from ANGSD were used to identify principal components using PCAngsd and genome-wide admixture proportions using NGSadmix  was performed using PCAngsd  based onNGSadmix . NGSadmiNGSadmix  method iNGSadmix  web servFm locus region-wise  using 123 individuals (BROL1 and BROL2 are excluded).Using the variant calls of 125 individuals for 67,617 SNPs, we performed the admixture analysis through AdmixPipe v3  for K1– Chinese black-bone breeds have diverged to differing extents from Kadaknath and have limited sample sizes. Hence, we combined the individuals from closely related XBBC (Xichuan black-bone chicken), LCEM (Emei black fowl), and LCMY (Miyi black fowl) breeds into a single population representative of Chinese black-bone  chicken and JETB, PENC, and GOLD into another population representative of Kadaknath  . We calcST) and divergence (Dxy) using ANGSD and popgenWindows python script  for each population pair. The FST estimates from the two methods were highly correlated  (We used the folded site frequency spectrum (SFS) approach implemented in ANGSD to calculate genome-wide population-specific estimates of π (the average pairwise differences), Watterson’s θ (the average number of segregating sites), τ (Tajima’s D), and Fu and Li’s D. We estimated inter-population genomic differentiation (F.2 e−16) . We defi.2 e−16)  R packag.2 e−16) .n = 3), and pencil (PENC n = 3). We also sampled one individual with a golden–pencil-like phenotype (GOPE n = 1), one with complete white plumage with black bone (CROS n = 1), one non-black-bone individual where plumage was black (NONB), and one individual of broiler breed , golden (GOLD er breed . We comper breed .n = 17) (see The major axes of genetic variation in the BBC (PC1:18.9% and PC2:13%) separated the Kadaknath, Yeonsan Ogye (YOSK n = 4), and Chinese BBC ( 17) see . We obse 17) see . Similar 17) see .π = 0.002, θ = 0.002) in YOSK (n = 4), while Kadaknath (n = 9) and CHIN (n = 9) BBC have comparable genetic diversity   . Geneticin both) . Crossbrin both) . Howeverin both) .Fm locus region on chromosome 20, which codes for the black-bone phenotype, is the defining feature of all BBCs. Even after excluding chromosome 20, the PCA of the remaining chromosomes finds that Kadaknath is genetically distinct from other BBC breeds , the Dup2 region probably consists of only long-non-coding RNA genes. All non-BBCs have a single copy of this region, referred to as *N locus  that form a complex chromosomal rearrangement in which both Dup1 and Dup2 regions are duplicated, giving rise to two junctions: (A) Dup1 + (inverted Dup2) and (B) (inverted Dup1) + Dup2. Although the exact ordering of these regions in the rearrangement is not conclusively established, both Dup1 and Dup2 regions are known to be duplicated due to these regions having a sequencing coverage that is twice the genomic average , Int (∼412 Kb), and Dup2 (∼170 Kb) regions occur in a single copy, are arranged sequentially, and are flanked by Flank1 (∼500 Kb) and Flank2 (∼500 Kb) regions . While t*N locus . The cor average . The pre average . Based o average . The *Fm chicken . Howeverscenario .Fm_1, *Fm_2, and *Fm_3) scenarios requires long-range connectivity information such as long-read sequencing data , Hi-C (high-resolution chromosome conformation capture)  vs. *Fm_3. However, to differentiate between *Fm_1 and *Fm_2 scenarios, we must span the entire ∼170 Kb Dup2 region in a single read. Spanning Dup1 or Dup2 regions with PacBio (average read lengths of 10–25 Kb) or Nanopore (average read lengths of 10–30 Kb) technologies with a single read is challenging but not impossible as reads longer than 1 Mb are possible (Fm_2 scenario).Distinguishing between these three (*capture) , or opticapture) . Both *Fpossible . Withoutpossible . Using aMethods). To ensure the robustness of our read-backed phasing approach, we required that more than 10 sequencing reads support haplotype-specific alleles at each pair of adjacent sites  were recognized at the Dup1 start region potentially separating the two haplotypes of Dup1 . We extent sites . Dup1 hant sites . The Flant sites  along thnt sites  spans Dunt sites  spans DuFm_1 and *Fm_3 scenarios, suggesting that the *Fm_2 scenario is correct  junction. The Dup2 + Flank2 junction containing the haplotype is Dup2 haplotype-1 (D2H1) and contains the allele A at position 11476819. The Dup2 + (inverted Dup1) junction containing the haplotype is Dup2 haplotype-2 (D2H2) and contains the allele G at position 11476819 . To veri correct .Fm locus. The normalized short-read coverage in BBCs (Fm locus junctions Dup1 + (inverted Dup2) and (inverted Dup1) + Dup2 using published PacBio data  size and complexity of the rearrangement. However, in the case of Kadaknath, it has not even been established whether Dup1 + (inverted Dup2) and (inverted Dup1) + Dup2 junctions identified in other BBCs are present. We have compared the normalized short-read coverage at these junctions to evaluate whether all BBC breeds share the same rearrangement junctions at the  in BBCs  abruptly in BBCs . The dra in BBCs . In the  in BBCs . We also non-BBC . We can Fm locus to investigate the history of the BBC breeds. The FST between the BBC and non-BBC along chromosome 20 is elevated at Dup1 and Dup2 regions (see π and θ) and divergence (Dxy), lack any prominent signatures  in Dup1 and Dup2 regions separates the BBC from non-BBCs (ST) landscape between BBC breeds found reduced FST at Dup1 and Dup2 regions compared to the genomic background (ST at Dup1 and Dup2 regions (ST), local PCA, and phylogenetic tree also support a common origin of the Fm locus in all BBC breeds.Crossbreeding with native and commercial breeds affects the genome-wide patterns of genetic variation in black-bone breeds . Hence, ions see . Howevergnatures . As evidnon-BBCs . A phylonon-BBCs . Our evackground . However regions . Hence, Fm locus. An independent origin for the Fm locus would mean separate rearrangement events have created identical junction sequences. Given the lack of repeat sequences at the junctions, such independent origins seem highly unlikely and are not consistent with the genetic relatedness of BBC and non-BBC breeds at the Fm locus. Hence, the current distribution of BBC breeds across Asia needs an explanation. We hypothesized . The IBD pattern persisted even after we repeated the analysis using ANGSD/variant-call-based estimates of nucleotide differentiation (FST) after excluding various populations to avoid errors due to auto-correlation and biased estimates from isolated populations (ST∼0.17). The other black-bone breeds that are geographically more distant from Jhabua  occur at increasing genetic distances from Kadaknath  is prevalent. The IBD plot shows a consistent increase in genetic distance with an increase in the geographic distance  and integrated haplotype score (iHS) should be strikingly different between KADK and CHIN. Two prominent signatures from these shortlisted regions indicative of selective sweeps in the KADK breed are evident on the 20th chromosome in the vicinity of the Dup1 region. The first region (R1) of ∼300 Kb is ∼160 Kb before Dup1, and the second region (R2) of ∼70 Kb is ∼0.7 Mb before Dup1. In both regions, the genetic diversity (π and θ) and Tajima’s D (τ) are strongly reduced in KADK compared to CHIN (xy) windows  with the Korean Yeonsan Ogye to identify population-specific signatures. Despite the sizeable geographic separation, the genome-wide mean Fnatures  . A pairwwindows  . We furt to CHIN . CompareDNTTIP1), acyl-CoA thioesterase 8 (ACOT8), and solute carrier family 12 member 5 (SLC12A5) genes contain non-synonymous changes. The TELO2-interacting protein 1 homolog (TTI1), which occurs near the first sweep region (R1), also has a non-synonymous change (HM13), bactericidal/permeability-increasing protein-like (BPIL), and targeting protein for Xklp2 (TPX2). The most striking differences were in the BPIL gene, which has three fixed sites with non-synonymous changes. Of these changes, two alleles are unique to the KADK population  occurs at position 69 in exon 2. The second non-synonymous change  is within the first BPI superfamily protein domain at position 159 in exon 4. The third and last non-synonymous change  is in the second domain at position 405 in exon 11. The impact of non-synonymous changes  in BPIL specific to Kadaknath is annotated as a moderate effect in the snpEff database. The PolyPhen-2 (Polymorphism Phenotyping v2) tool found that the non-synonymous changes R69Q and E159K are possibly damaging, while R405H is probably damaging , deoxynucleotidyltransferase terminal interacting protein 1  than to Kadaknath (mean FST = 0.3). However, the genome-wide mean FST is still low enough to distinguish selection signatures. A comparison of YOSK with KADK confirmed that R1 and R2 regions are specific to Kadaknath  the high-sequence identity of the rearranged junctions across the BBC breeds, (3) the local and phylogenetic relationship between chicken breeds at the Fm locus, and (4) the patterns of pairwise genetic differentiation between BBC and non-BBC breeds. Independent structural variants can produce the same phenotypes, as seen in the case of blue eggshell  (260 Km) and Lothal (300 Km) on the west coast of India. Given the proximity to port cities and the prevalence of BBC only in Western India (and the spread of BBC to Africa), we suspected that Kadaknath may have traveled through a marine route. The ancient sea trade between India, Korea, and other parts of Asia are well documented . MoreoveHistorical records are patchy, prone to error/obfuscation, and fail to provide conclusive information about the spread of BBC across Asia . Hence, The BBC chicken from Tibet is genetically closest to the Kadaknath breed and suggests that the old Tibet–Nepal salt trade route or the maritime silk route may have facilitated the spread of BBC. Interestingly, the Tibetan BBC is genetically more similar to Kadaknath than some Chinese BBC breeds. Unfortunately, we lack BBC samples from Nepal. However, we believe BBC poultry in Nepal is Kadaknath chicken, recently imported from India. Hence, the ancestral stock of the BBC spread across Asia may not be currently available. Changes in trade routes and the introduction of commercial poultry breeds limit our ability to trace the historical prevalence of BBC breeds.ST of 0.11 between Jhabua and Bhopal is comparable to the differentiation between some Chinese BBC breeds. The presence of three distinct morphs within Kadaknath chicken suggests phenotypic diversity derived either from commercial breeds, native Indian breeds such as Ankleshwar, or existing variations within Kadaknath. The extent of phenotypic diversity within BBC breeds from China and Korea is not documented to allow a fair comparison. The high genetic diversity in Kadaknath supports the potential origin of all BBC in Jhabua, India. The export of black chicken from India to Africa in ∼1600 AD also supports that BBC was present in ancient India  resembles the *N arrangement found in non-BBC breeds. However, the proximal region (Flank1+Dup1+(inverted Dup2)) has a very different arrangement than the non-BBC *N arrangement. In contrast to the *Fm_2 scenario, the proximal region would be similar to the *N arrangement in *Fm_3 scenario. In the *Fm_1 scenario, only the first occurrence of Dup1 and the last occurrence of Dup2 are similar to the *N arrangement. Hence, in the *Fm_2 and *Fm_3 scenarios, recombination with *N arrangement may be easier  or 170 Kb (Dup2) region) compared to the * Fm_1 scenario ) region). The ease of recombination with *N arrangement could explain the prevalence of the *Fm_2 scenario, which requires two rearrangement events. Ongoing improvements to genome assembly methods such as haplotype-aware de novo assembly  from the outer membrane of Gram-negative bacteria  in the vicinity of the bacteria . BPIL isn breeds . The Fayn breeds  and poten breeds . Earliern breeds . Along wn breeds . The nonBPI-like genes occur near BPI (C9 gene required to effectively eliminate pathogens through the membrane attack complex (MAC) formation  between Kadaknath and Chinese BBCs is strongly suggestive of hitchhiking. Moreover, the sweep signature is in the Chinese BBC in chr 4 and chr 9 cases. Hence, the proximity of R1 and R2 regions to the Fm locus indicates that selective sweep may be due to close physical linkage with the Fm locus. Such co-selection of traits due to linkage and pleiotropy has occurred during animal domestication, including in chicken (Several other genes with non-synonymous changes occur in R1 and R2 genomic regions. Although we cannot identify the functional consequences of these changes, the strong signature of selection and KADK-specific protein-coding alterations suggest phenotype-altering ability. Predictions from snpEff and PolyPhen-2 also suggest a major effect of these protein-coding changes located within the domain regions. The genomic region of high differentiation is close to the  chicken . Hitchhi chicken . WhetherThe genomic co-occurrence of economically important traits in domesticated plants and animals is also known . The allFm locus. We also identify specific genes with selection signatures that are likely responsible for the Kadaknath-specific phenotypes. The co-selection of genes that are in linkage, as shown in Kadaknath, is widespread in domestic species (Genome-wide data generated as part of this study and comparative analysis with other BBC breeds establish the genetic uniqueness of Kadaknath that extends beyond the  species . Such cl


We compare photoaligning properties of polymer layers fabricated from the same constituents: polymethyl-methacrylate (PMMA) and azo-dye Disperse Red 1 (DR1), either chemically attached to the PMMA main-chain, or physically mixed with it. Photoaligning properties depend on the preparation method drastically. Photoalignment was found to be far more efficient when PMMA is functionalized with DR1 compared to the case of physically mixing the constituents. This finding is supported by atomic force microscope (AFM) scans monitoring the light-induced changes at the polymer–air interface, and revealing a photoinduced mass transfer, especially in the case of functionalized PMMA. Photoalignment of liquid crystals (LCs) ,2,3—discThe above-described advantages, combined with the relative ease of patterned photoalignment make the application potential of photoalignment very high in display and photonic devices . Those aCommand surfaces are usually produced as monolayers of dye derivatives anchored on a substrate ,14,15,16E/Z) isomerization  , as we cn is parallel with the surface of the plate). The photosensitive plates were prepared by spin-coating on the glass substrate either the For the experiments on photoalignment, liquid crystal cells of typical thickness ye pDR1, a, or thePMMA+DR1 b in toluPMMA+DR1 : for 10  on PMMA . The refMixtures of PMMA+DR1 were prepared with various PMMA polymers having different number-average degrees of polymerization from  of PMMA ,53. On the layer . For thevely see b, while vely see a a Tg=11measured . In the The choice of the LC mixture E7 for further measurements on photoalignment is based on our previous studies. Namely, besides the conveniently wide temperature range of the nematic liquid crystal phase  photoalignment angle,  in Ref. . The onl in Ref. ,40,41 isin Refs. ,40,41). in Refs. . ConsequAtomic force microscopy (AFM) scans on the photosensitive substrates prior to and after the polarized laser illumination were carried out with an Agilent 5500 AFM system equipped with PicoView 1.14.3 control software. The images were acquired in the semi-contact (tapping) mode using medium soft silicon cantilevers  with the resonant frequency of 70 kHz (typ.), and spring constant of 2 N/m (typ.). The measurements were performed at ambient relative humidity of MA, USA) .n at the reference plate.In Results on the azimuthal photoalignment in the LC cell with pDR1 are in agreement with the previous results ,40: at lIn contrast, in the LC cell with the polymer layer from the PMMA1+DR1 mixture, upon the light excitation (despite the much higher light power), a much smaller change in the azimuthal photoalignment angle was observed , while the polarization of the pump beam was set perpendicular to n, which ensures the absence of the azimuthal photoalignment and its influence on the results. When significant zenithal photoalignment occurs, due to the change in the birefingence, oscillations appear in the transmitted light intensity of the probe beam when the pump beam is switched on/off. The results on zenithal photoalignment are shown in For the investigations on the zenithal photoalignment, the polarization of the probe beam has enclosed Again, results on the zenithal photoalignment in the LC cell with pDR1 a are in n, or eventually, from a slight zenithal photoalignment as it was discussed in Ref. [In contrast, in the LC cell with the polymer layer from the PMMA1+DR1 mixture, upon the light excitation (despite the much higher light power), only a slight change in the transmitted light intensity of the probe beam was observed in the whole temperature range  the orientation of the DR1 molecules, and (ii.) the rigidity of the PMMA matrix.For the very weak photo-response measured in the LC cells with PMMA+DR1 substrates, one can anticipate two possible reasons: (i.) comes from both theoretical considerations and experimental data, evidencing that rod-like molecules often have a tendency to orient perpendicular to the free (air contacting) surface of the film [(i.) can be, however, tested by a slantwise illumination [Reason the film . Such anmination . Namely,mination  we have (ii.), i.e., the rigidity of the PMMA matrix in the glassy state stays behind the poor photoalignment performance of the polymer layers made from PMMA+DR1 mixtures. Presumably, the rigid matrix hinders the cooperative motion (induced by the trans-cis isomerization of the DR1 molecules) necessary for an efficient photoalignment. In contrast to that, recently we have shown for a polymer segment of pDR1 that the trans-isomer of the azo-benzene moiety can take any direction at an energy expense of few Therefore, we assume that reason er chain .The results obtained from AFM scans on polymer reliefs in contact with the air are in line with the photoalignment measurements. The pDR1 surface evidently becomes smoother after the illumination, and the photoinduced changes in surface relief are accompanied by a significant photoinduced mass transfer. In contrast, the relief of the PMMA1+DR1 surface does not change noticeably upon the illumination, and the photoinduced mass transfer was found very close to the resolution of the AFM.The results of the specific system presented in this work give a definite answer to the question posed in the title: the photoalignment is far more efficient when the azo-dye DR1 is chemically attached to the PMMA backbone, compared to the case when PMMA and DR1 are physically mixed. We attribute the poor photoalignment performance of the polymer layers prepared from PMMA+DR1 mixtures to the rigidity of the PMMA matrix.The long-lasting  photoinduced changes in the surface relief of pDR1, accompanied with significant photoinduced mass transfer support most of the results on photoalignment measurements at temperatures close to the room temperature. Namely, when in those measurements liquid crystals with phenylcyclohexane or bicyclohexane rigid core were contacting the pDR1 layer, extremely slow, or no back-relaxation occurred upon switching off the pump beam ,41. On t


Costs associated with biologic switching and discontinuation can be high in chronic inflammatory diseases. Inappropriate use of medications may have cost implications for both payers and patients. Understanding of biologic utilization and switching rates is lacking among patients with ankylosing spondylitis (AS).To compare direct costs of treatment switchers, nonswitchers, and discontinuers among patients with AS who newly initiated a biologic.Patients aged ≥ 18 years with an AS diagnosis and ≥ 1 pharmacy claim for an FDA-approved subcutaneous biologic for AS between January 1, 2016, and December 31, 2016 (identification period) were identified from the Truven Health MarketScan Databases. At the time of biologic initiation (index date), eligible patients were continuously enrolled with medical and pharmacy claims for ≥ 1 year before (baseline period) and ≥ 1 year after the index date (follow-up period). Patients with AS were categorized into 3 mutually exclusive groups of nonswitchers (patients who remained on their index biologic), switchers (patients who had a prescription for a biologic therapy other than their index biologic), and discontinuers (patients who had gaps in prescription claims [> 120 days]) based on their biologic utilization pattern during the 1-year follow-up period. Health care costs (inflated to year 2017 costs) during follow-up were described across the 3 groups separately and by disease. Adjusted health care resource utilization and costs during 1-year follow-up were estimated, controlling for age, sex, fulltime work status, and Charlson Comorbidity Index (Deyo’s modification) during the baseline period.P < 0.001). Switchers had increased outpatient visits and costs versus nonswitchers in the adjusted analysis (P < 0.001); small sample sizes in the switcher group may have limited the ability to detect significant trends in the adjusted analysis.A total of 791 patients with AS who were newly initiating a biologic were categorized as switchers (15.4%), nonswitchers (53.1%), and discontinuers (31.5%). Switchers and discontinuers had higher all-cause health care utilization than nonswitchers during the 1-year follow-up period. Switchers had higher mean total health care costs than nonswitchers  due to increased medical  and pharmacy costs . Discontinuers had the lowest total health care costs  but had the highest medical costs . Adjusted analyses for covariates confirmed similar trends as the unadjusted analysis, where discontinuers had significantly lower total health care and pharmacy costs but had higher medical costs (with increased emergency department and outpatient visits) than nonswitchers (all Biologic switching resulted in higher total health care costs than remaining on the same biologic in patients with AS. These findings highlight the clinical and economic impact of discontinuing or switching biologic therapies in patients with AS, which may inform treatment and/or formulary decision making. What is already known about this subjectCurrently, the available biologics approved for the treatment of ankylosing spondylitis (AS) in the United States include tumor necrosis factor inhibitors  and interleukin-17A inhibitors (ixekizumab and secukinumab).Patients may discontinue or switch biologic therapies in clinical practice due to adverse events, lack of efficacy, or formulary changes.A few studies have retrospectively identified treatment patterns in patients with AS, but there is limited real-world evidence comparing health care costs and resource utilization associated with switching or discontinuing between approved biologics.What this study addsThis retrospective analysis of U.S. administrative claims data shows that within 1 year of initiating a biologic , 53.1% of patients with AS remained on their index biologic; 15.4% switched from their index biologic to a new therapy; and 31.5% discontinued their index biologic.Switchers and discontinuers had higher health care resource utilization (emergency and outpatient visits) than nonswitchers.Switchers had the highest total health care costs, which were driven by increased pharmacy and medical costs, while discontinuers had the lowest total health care costs due to decreased pharmacy costs from early discontinuation of medications but also had the highest medical costs.1 The main clinical feature of AS is inflammatory back pain, which is caused by inflammation of the vertebrae and joints of the spine and entheses.2 The prevalence of AS is estimated at 0.35% to 0.67% in the United States.6 However, given that AS is often underdiagnosed or diagnosed late,7 some believe it may actually affect approximately 1% in the United States.3 The usual age of symptom onset is 15-35 years, with a delay in diagnosis of up to 14 years.9 Factors that likely contribute to delayed diagnosis of AS include the broad prevalence of chronic lower back pain in the population , gradual onset of the disease, and lack of specific symptoms or biomarkers unique to AS.9 Several lines of evidence suggest that severe deformities, disabilities, and functional limitations often develop within the first 10 years of AS.11Ankylosing spondylitis (AS) is a chronic, immune-mediated rheumatic disease that primarily affects the spine and manifests as progressive spinal stiffness and fusion.13 In a study of U.S. administrative claims data, patients with AS had approximately 4-fold higher mean total all-cause health care costs than a matched control population without AS , driven by increased use of medical outpatient services  and pharmacy costs .12 The majority of AS-specific total health care costs were related to AS medication costs. In a systematic review of the indirect costs relating to work absenteeism and presenteeism associated with AS, the mean annual indirect cost was $6,455 per patient per year, with a range of $661 to $45,954 depending on the components of indirect costs and their definitions.14 Other studies, mostly from outside the United States, have found that indirect health care costs associated with AS were higher than direct costs.15Along with severely affecting physical function, AS poses a substantial economic burden to patients, payers, and society due not only to health care utilization and costs related to delayed diagnosis but also to lifelong, continuous treatment.17 Biologic therapies approved for the treatment of AS include the tumor necrosis factor inhibitors (TNFis) adalimumab, etanercept, certolizumab pegol, and golimumab and the interleukin (IL)-17A inhibitors secukinumab and ixekizumab.26 Current practice suggests the use of a TNFi as first-line biologic therapy.16 The 2019 update of the ACR/SAA/SPARTAN guidelines for the treatment of AS and nonradiographic axial spondyloarthritis conditionally recommends IL-17 inhibitors over treatment with a different TNFi among primary nonresponders to TNFis and treatment with a second TNFi over a non-TNFi biologic if the initial TNFi loses efficacy over time.17 Lack of primary response or loss of effectiveness over time is a common reason for biologic switching.27 Previous studies have also shown that switching biologics may be effective if the initial biologic fails29; however, switching biologics may lead to increased health care utilization. Adherence to and persistence with biologic therapy in patients with AS is vital to achieve optimal outcomes of pain relief, improved physical function, and minimal joint damage.30 Therefore, appropriate selection of medications is important for optimizing patient outcomes and minimizing costs.The Assessment of SpondyloArthritis international Society (ASAS), the European League Against Rheumatism (EULAR), the American College of Rheumatology (ACR)/Spondylitis Association of America (SAA), and the Spondyloarthritis Research and Treatment Network (SPARTAN) all recommend nonsteroidal anti-inflammatory drugs (NSAIDs) as first-line therapy in patients with active disease who have pain and stiffness; in patients with active disease despite NSAID treatment, use of biologic therapy is recommended.32 Therefore, there is a need for more studies evaluating the real-world health care costs and utilization associated with newer biologic treatments for AS. This study aimed to compare health care utilization and costs of switching and discontinuing biologics versus remaining on index biologic during 1 year of follow-up among patients with AS.Because of the array of biologic treatment options, it is important to assess the cost of switching or discontinuing biologics among patients with AS. Previous retrospective studies have predominantly focused on treatment patterns and costs of TNFis.A retrospective, observational cohort study was conducted using the Truven Analytics MarketScan Commercial Claims and Encounters Database (CCAE) and the Medicare Supplemental and Coordination of Benefits Database (MDCR) from January 1, 2015, to December 31, 2017. The Truven Analytics MarketScan data source is widely used for analyzing epidemiologic and economic outcomes in the U.S. health care system. CCAE contains administrative claims data from a privately insured population, while the MDCR contains administrative claims data from retirees with Medicare supplemental insurance paid by employers, employer-paid portion, services provided under Medicare-covered payment, and any out-of-pocket expenses. The CCAE and MDCR include fully adjudicated deidentified medical claims, outpatient prescription drug claims, and person-level enrollment data that are linked with unique patient identifiers. Demographic variables  and detailed health care costs and utilization for health care services performed in both inpatient and outpatient settings are captured.International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] 720.0 and Tenth Revision [ICD-10-CM] M45.x) during the baseline period. Patients who had > 1 different biologic of interest at the index date were excluded. The treatment washout period of 1 year allowed for a reduction in bias introduced by prevalent biologic use.Patients aged ≥ 18 years with ≥ 1 pharmacy claim for a subcutaneously administered biologic that was approved by the U.S. Food and Drug Administration for AS by 2017  from January 1, 2016, to December 31, 2016 (identification period), were identified in the Truven Health Analytics MarketScan Commercial and Medicare Supplemental Databases. Eligible patients at the time of biologic initiation (index date) were continuously enrolled, had medical and pharmacy claims for ≥ 1 year before (baseline period) and ≥ 1 year after the index date (follow-up period), and had ≥ 1 AS diagnosis  and federal guidance on Public Welfare and the Protection of Human Subjects.Patients were categorized into 3 mutually exclusive groups based on their biologic treatment pattern groups during the 1-year follow-up period.Nonswitchers: Patients who remained on the index biologic with no gaps in treatment (> 120 days) and no other claims for biologic therapies during the 1-year follow-up periodSwitchers: Patients who had a prescription claim for a different biologic therapy  than the index biologic and who switched therapies before permissible treatment gaps (> 120 days) were reached during the 1-year follow-up periodDiscontinuers: Patients who had prescription claim gaps (time from exhaustion of previous days supply) during the 1-year follow-up period (gap > 120 days)Patient demographics  were assessed at the index date; all-cause health care resource utilization  and all-cause health care costs  were measured both during the 1-year baseline period and the 1-year follow-up period. All-cause health care resource utilization and health care costs were calculated per patient per year; health care costs were inflated to year 2017 costs.2 tests for categorical variables and Wilcoxon rank-sum tests or unequal variance 2-sample t-tests for continuous variables.Descriptive statistics were tabulated for patient characteristics and outcome variables  comparing nonswitchers versus switchers and nonswitchers versus discontinuers. Continuous variables were summarized as means and standard deviations, while categorical variables were presented as counts and percentages, with missing data considered a separate category. Comparisons between treatment pattern groups were made using χMultivariable models were fitted to compare all-cause health care resource utilization and costs between switchers versus nonswitchers and discontinuers versus nonswitchers using a negative binomial distribution with log link function for all outcomes. Adjusted health care utilization and costs were estimated for nonswitchers, switchers, and discontinuers based on the models. All models were adjusted to control for baseline covariates, including age, sex, full-time work status (yes or no), and baseline Charlson Comorbidity Index (Deyo’s modification). All analyses were done using SAS software version 9.4  in the EVICO platform.This study included 87,748 patients with ≥ 1 claim for adalimumab, certolizumab pegol, etanercept, golimumab, or secukinumab during the identification period between January 1, 2016, and December 31, 2016 . After iP < 0.05).Patient demographics were mostly similar across treatment groups regarding age and employment status. Overall, the mean (SD) age was 46.5 (12.4) years, 51.2% were female, and most were from the southern region of the United States (44.2%) . The proP < 0.001). Both switchers and discontinuers had significantly higher mean (SD) number of ED visits per patient per year compared with nonswitchers .Across all treatment groups, the mean (SD) number of patient visits per year was 0.13 (0.42) for hospitalizations, 0.51 (1.19) for ED visits, and 31.31 (22.69) for outpatient visits . SwitcheP < 0.0001). Switchers also had significantly higher medical costs , mainly due to higher outpatient costs , as well as higher pharmacy costs . The medical and pharmacy costs of discontinuers were comparable to those of nonswitchers .At baseline, the overall mean (SD) total all-cause health care cost across all treatment pattern groups was $25,679 , and medical and pharmacy costs were significantly different between switchers and nonswitchers . SwitcheP = 0.027) and outpatient  visits per patient per year compared with nonswitchers. Discontinuers had a statistically significantly higher mean (SD) number of ED  and outpatient  visits per patient per year compared with nonswitchers; however, differences in the frequencies of hospitalizations were not statistically significant.In the 1-year follow-up period, discontinuers and switchers had higher all-cause health care utilization than nonswitchers . SwitcheP = 0.002), which was contributed to by increased medical costs . The mean total all-cause health care cost for discontinuers  was significantly much lower than that for the other treatment pattern groups due to reduced pharmacy costs . However, discontinuers had the highest medical costs  among all 3 treatment pattern groups.In the 1-year follow-up period, switchers had a higher all-cause total health care cost compared with nonswitchers, while discontinuers had a lower all-cause total health care cost . SwitcheP < 0.001) and discontinuers . Discontinuers also had more frequent ED visits than nonswitchers . Adjusted analyses of health care costs showed similar trends as the unadjusted analysis  further demonstrated higher health care utilization for switchers and discontinuers compared with nonswitchers . Compareanalysis .P = 0.003). Similar to the unadjusted analysis, discontinuers had significantly lower total all-cause health care costs than nonswitchers , which was driven by significantly lower pharmacy costs. However, discontinuers still had significantly higher medical costs than nonswitchers , despite controlling for covariates that may contribute to worse clinical state, such as age and comorbidities.Compared with nonswitchers, switchers had significantly higher outpatient costs  revealed that discontinuers did have higher medical costs than nonswitchers due to higher outpatient costs from early discontinuation, further supporting that these patients may still have active disease.38 Of note, higher disease activity is a predictor of discontinuation35 or switch,39 as shown by higher Bath Ankylosing Spondylitis Disease Activity Index and Health Assessment Questionnaire scores, respectively.Although the reasons for discontinuation or switch were not evaluated in this study, prior studies in patients who discontinued or switched biologics found that the most common reasons were adverse events, lack of treatment effect, or disease progression.32 To account for potential confounding of baseline health status, we adjusted for baseline covariates that may represent a patient’s need for health care services as a proxy for health status or predictors of future health care utilization .In our study, at baseline, switchers had significantly higher health care resource utilization and medical costs compared with nonswitchers, which may suggest that these patients had more complicated disease conditions at baseline. A previous study using a claims database in China reported that patients with AS receiving biologics have the highest direct medical costs compared with those on other conventional therapies.Few studies in the United States have reported on health care utilization and costs of switching or discontinuing biologics among patients with AS; therefore, this study provides valuable insight into the financial implications of switching treatments, which is important for payers to make informed formulary decisions. From a policy perspective, this study informs health care administrators on the costs of switching and/or discontinuing therapies, allowing them to better weigh the option of incurring costs of forcing patients and physicians to switch therapies or potentially saving costs by remaining on the current therapy. Furthermore, these findings provide physicians with essential information on the financial burden of switching, suggesting proper treatment decisions and appropriate treatment selection as a means to alleviate both the clinical and economic burdens of AS.37 more than 40% of patients with AS treated with TNFis may initiate add-on therapy with NSAIDs, opioids, corticosteroids, conventional synthetic disease-modifying anti-rheumatic drugs, antidepressants, anxiolytics, sleeping aids, and topical analgesics.30 This results in an increase in medication burden and treatment costs; thus, shared decision making between physicians and patients is important to optimize treatment and outcomes. Future studies exploring reasons for switching or discontinuing are warranted, as these may provide a better perspective on selecting more durable treatments.Given that patients’ reasons for switching among TNFis were primarily due to a lack of efficacy or adverse events,Despite having several strengths, due to its nature, this study has a few limitations. There is potential for misclassification of clinical characteristics or events, as all data were dependent on claims information. The validity of the analysis related to switching is only sensitive to the accuracy of the data recorded from the claims database. The measurement error around claims for expensive medication is likely low; however, uncertainty around the cost of switching may remain. For the medical claims, the data on drugs administered by a medical procedure do not contain information on the number of days of supply. As the data are limited to those patients who have commercial and Medicare supplemental insurance, the current findings may not be generalizable to the general population; furthermore, costs may have been underestimated for patients who received supplemental health care or those who did not have insurance coverage and were not included in the claims database.Based on the definition of a permissible treatment gap of > 120 days, patients may be misclassified as discontinuers, and this may bias differences in cost estimates toward null. Our study did not capture data for patients who restarted therapy or their reasons for switch or discontinuation. Additional long-term analyses of real-world data, as well as analyses linking claims to electronic medical records data, are warranted to corroborate these findings, with the goal of identifying treatments with sustained safety and efficacy to alleviate the clinical and economic burden of switching or discontinuing biologic therapies.This analysis of U.S. administrative claims data suggests that patients with AS who switch or discontinue biologics within the first year of initiating their biologic have higher health care utilization and higher medical costs than those who remain on their index biologic. For payers, biologic discontinuation may impose economic burden due to medication wastage. These fndings reflect current real-world treatment patterns and emphasize the clinical and economic impact of discontinuing or switching biologic therapies in patients with AS.This analysis may encourage payers to weigh the additional costs associated with switching therapies or potential savings associated with remaining on current therapies during formulary decision making. Moreover, these results suggest a need to identify and appropriately select treatments with sustained safety and efficacy to lessen the clinical and economic burden of discontinuing or switching biologic therapies.