Furthermore, combining three unique H2A.Z regions  was sufficient for expression of H2A.Z-dependent heterochromatin-proximal genes and GAL1 derepression. Surprisingly, hybrid constructs that restored the transcription of H2A.Z-dependent genes, did not fully recapitulate patterns of H2A.Z-specific enrichment at the tested loci. This suggested that H2A.Z function in transcription regulation may be at least partially independent of its specific localization in chromatin. Together, this work has identified three regions that can confer specific H2A.Z-identity to replicative H2A, furthering our understanding of what makes a histone variant a variant.Chromatin structure and underlying DNA accessibility is modulated by the incorporation of histone variants. H2A.Z, a variant of the H2A core histone family, plays a distinct and essential role in a diverse set of biological functions including gene regulation and maintenance of heterochromatin-euchromatin boundaries. Although it is currently unclear how the replacement of H2A with H2A.Z can regulate gene expression, the variance in their amino acid sequence likely contributes to their functional differences. To tease apart regions of H2A.Z that confer its unique identity, a set of plasmids expressing H2A-H2A.Z hybrids from the native H2A.Z promoter were examined for their ability to recapitulate H2A.Z function. First, we found that the H2A.Z M6 region was necessary and sufficient for interaction with the SWR1-C chromatin remodeler. Remarkably, the combination of only 9 amino acid changes, the H2A.Z M6 region, K79 and L81 (two amino acids in the α2-helix), were sufficient to fully rescue growth phenotypes of the  Chromatin structure in eukaryotes is integral to all aspects of genome function. Many essential biological processes depend on chromatin remodeling for regulating access to DNA such as transcription, replication, and DNA repair. One highly conserved method of chromatin remodeling is the replacement of the replicative histones with a histone variant. Unlike replicative histones, which are distributed equally in chromatin during DNA replication, histone variants can be transcribed and deposited throughout the cell cycle at specific locations. Once incorporated, small differences in a histone variant’s protein sequence and structure can result in unique chromatin neighbourhoods with distinct character and function from the replicative histone. Therefore, to better understand the role of a histone variant, it is important to define how its protein sequence contributes to its unique identity. Here, using a hybrid gene approach in budding yeast, we systematically determine which amino acids contribute to H2A.Z unique identity as a histone variant. In eukaryotic cells, chromatin structure is a major modulator of genomic processes that require DNA accessibility. The basic packaging unit of chromatin is the nucleosome, consisting of approximately 146 base pairs of DNA wrapped around a protein octamer composed of two copies of each of the core histones: H2A, H2B, H3, and H4  mutant and the H2A.Z mutant .As our H2A-H2A.Z hybrid constructs showed that no single H2A.Z region conferred upon a H2A background was able to fully rescue the nstructs . Growth nstructs  and S4B.Z mutant . In addiH2A-H2A.Z mutant were primarily driven by L81  which when placed in a htz1Δ background had similar growth phenotypes and protein abundance as the wild-type H2A.Z  to media with 2% galactose (which induces GAL1) and monitored GAL1 mRNA levels before and after changing media. Similar to our results for the heterochromatin-proximal genes, the H2A-H2A.Z mutant recapitulated the H2A.Z mutants GAL1 mRNA levels for every time point  were sufficient for expression of H2A.Z-dependent heterochromatin-proximal genes and GAL1 after derepression.Determining what makes a histone variant a variant is a fundamental question in chromatin biology. While characterizing the amino acids required for H2A.Z function has been the focus of several previous studies, determining which regions are actually sufficient to confer histone variant identity, and thus responsible for distinguishing H2A.Z from H2A, have remained unclear. In this study, we used hybrid constructs based on replicative histone H2A to systematically analyze all of the divergent regions between H2A.Z and H2A. Here we report that the M6 region, the C-terminal tail, and the G4 region in the α2-helix each contributed to H2A.Z’s unique identity from H2A . While cHere we report that while the M6 region was not sufficient to fully recapitulate H2A.Z identity, it was required for H2A.Z-specific function. We found that replacing the H2A.Z M6 region with the corresponding H2A residues resulted in growth defects in comparison to wild-type H2A.Z. Therefore, the seven amino acid differences between H2A and H2A.Z M6 regions must be required for H2A.Z-specific growth phenotypes . We confhtz1Δ growth phenotypes, an outcome not observed for any of the other regions when combined with M6  were diluted to 0.3 ODtic acid . Immunob600. Cells were then 10-fold serially diluted and spotted onto SC-TRP plates with or without the indicated amount of caffeine (Sigma), hydroxyurea (HU) (Sigma), and formamide (Sigma). Plates were incubated for three days at 30°C. Three biological replicates were performed for each culture.Overnight cultures grown in SC-TRP were diluted to 0.5 OD600 in 50 mL of SC-TRP and grown to 1.0 OD600. Cells were harvested and resuspended in TAP-IP buffer . Cells were mechanically lysed with glass beads for 5 minutes using a bead beater (BioSpec Products). Lysates were incubated with 40 μL of FLAG M2 agarose beads (Sigma) for 1.5 hours, or 20 μL of VSV agarose beads (MBL) for 16 hours. The beads were washed once with TAP-IP buffer containing protease inhibitors and twice with TAP-IP buffer lacking protease inhibitors. The beads were resuspended and boiled in SDS-PAGE sample buffer. Samples were analyzed by using SDS-PAGE followed by western blotting. Captured proteins were detected with anti-FLAG M2 (Sigma), anti-VSV (Bethyl Laboratories), and anti-Nap1 (Santa Cruz Biotechnology). Bands were visualized using Odyssey Infrared Imaging System (LI-COR).Co-immunoprecipitation (IP) of protein complexes were performed in triplicate as previously described . Briefly600 and then grown to 0.5 OD600. Cultures were diluted to 0.1 OD600 in 200 μL SC-TRP media plus 1.5% formamide (Sigma). OD600 readings were measured every 30 minutes over a period of 35 hours in a TECAN M200 plate reader. Plates were shaken for 10 minutes before each reading. Strains were tested in three biological replicates per plate and the area under the curve (AUC) was calculated for each replicate.Overnight cultures were diluted to 0.1 OD600 in 50 mL of SC-TRP and grown to 0.5 OD600. A total of 25 OD units were harvested, incubated in Pre-spheroplast Buffer  for 10 minutes at room temperature, and spheroplasted with 20 mg/mL Zymolyase-100T (Seikagaku Corporation) in Spheroplast Buffer  for 30 minutes at 37°C. The resulting spheroplasts were washed with Wash Buffer , resuspended in EB  and lysed on ice with 0.1% Triton X-100. A portion of the resulting whole cell extracts (WCE) were saved, while the remaining lysate was centrifuged through EBSX (EB + 0.25% Triton X-100 and 30% sucrose) to separate the chromatin pellet and supernatant fractions. The WCE, pellet, and supernatant fractions were analysed using SDS-PAGE followed by immunoblotting with anti-FLAG M2 (Sigma), anti-H4 (Abcam), and anti-PGK1 (Sigma) antibodies. Bands were visualized using Odyssey Infrared Imaging System (LI-COR).Chromatin association assays were performed in triplicate as previously described . Overnig600 in 250 mL of SC-TRP media and grown to 0.5 OD600. The procedure for ChIP was adapted from a previous report  and H2A-H2A.Z mutants in a htz1Δ background had improved growth in comparison to the swr1Δ or swr1Δhtz1Δ mutants and had similar growth phenotypes to the H2A.Z mutant. Cells expressing the indicated hybrid constructs were 10-fold serially diluted, spotted onto SC-TRP media with the indicated concentrations of formamide, caffeine, and hydroxyurea and grown for 3 days. (C) Decrease in mRNA levels in the htz1Δ mutant for heterochromatin-proximal genes was not caused by the presence of apo-SWR1-C. RT-qPCR analysis of heterochromatin-proximal genes mRNA levels from three replicates were normalized to ACT1 mRNA levels. Error bars indicate the standard deviation between the three replicates. Significant comparisons determined by unpaired two-tailed Student’s t-tests are indicated: ** = p-value <0.01.(A) The (TIF)Click here for additional data file.S8 FigCells expressing the indicated hybrid constructs were 10-fold serially diluted, spotted onto SC-TRP media containing either 2%-glucose or 2%-galactose with 0.02 mg/mL ethidium bromide and grown for 3 days.(TIF)Click here for additional data file.


Backyard swine farming is critical to generating subsistence and food security in rural and peri-urban households in several developing countries. The objective of this systematic review was to analyze the molecular and serological prevalence of influenza A virus (IAV) in backyard swine populations globally.We identified 34 full-text research articles in NCBI-PubMed and Google Scholar databases that have reported IAV sero- and/or virological prevalence in backyard swine up to 11 July 2021. The highest number of studies were reported from Asia (n = 11) followed by North America (n = 10), South America (n = 6), Africa (n = 6), and Europe (n = 1). While the maximum number of studies (44.12%) reported human-to-swine transmission of IAV, swine-to-human (5.88%), poultry-to-swine (5.88%), and wild birds-to-swine (2.94%) transmissions were also reported. An overall higher IAV seroprevalence (18.28%) in backyard swine was detected compared to the virological prevalence (1.32%). The human-origin pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 was the more frequently detected IAV subtype in virological studies (27.27%) than serological studies (18.92%). In addition, the avian-origin highly pathogenic H5N1 and H5N8 viruses were also detected, which further substantiated the evidence of avian–swine interactions in the backyards.Human–swine and avian–swine interactions in backyards may transmit IAV between species. Monitoring the circulation and evolution of IAV in backyard swine would help stakeholders make informed decisions to ensure sustainable backyard swine farming and public safety. Sus scrofa domesticus), chicken, ducks, turkey, and cattle, raised for either household consumption or supplying within the local community for subsistence. One prominent challenge backyard swine farming faces is the lack of suitable biosecurity, which may facilitate the dissemination of zoonotic pathogens, including influenza A virus (IAV), endangering backyard farming and public health. Negligence of biosecurity at the backyard farms may provide a suitable environment for disseminating IAV within the backyard animals, resulting in IAV disease outbreaks causing economic losses to the swine growers [Swine farming is the largest meat-producing industry, with gross production of more than one-third of all the meats consumed globally , 2. Whil growers –10. Seve growers –15 that  growers . InteracFor the first time, we reported the serological and molecular prevalence and the transmission dynamics of IAV exclusively in backyard swine populations globally. This study describes the existing IAV circulation dynamics in backyard swine farms, which is essential to assess the current threat regarding the IAV disease outbreak. This study would also assist stakeholders in making an informed decision regarding minimizing the risk of IAV disease dissemination in backyard farms to ensure sustainable backyard swine farming.Our objective was to analyze the transmission of IAV to the swine populations raised in the backyards, which may occur through swine–swine, poultry–swine, human–swine, and wild birds–swine interactions. A comprehensive search of NCBI-PubMed and Google Scholar databases was conducted up to 11 July 2021 for identifying the available research articles reporting IAV disease in backyard swine populations globally. The search terms including “Influenza A virus in backyard swine,” “Influenza A virus in backyard pigs,” “Influenza A virus in backyard production systems,” “Influenza A virus in rural pigs,” “Influenza A virus in household pigs,” and “Swine backyard production systems” were entered in Google Scholar and NCBI-PubMed databases one by one for identifying relevant full-text research articles. The research articles from the database search investigating IAV serological and/or molecular prevalence in backyard animals, including swine, poultry, and cattle, were segregated into groups for analysis based on the interaction patterns. An overview of Preferred Reporting Items for systematic reviews and meta-analysis (PRISMA) flowchart  used to The full-text original research articles which investigated molecular  and/or serological prevalence of IAV exclusively in the backyard or household swine populations were included in this systematic review. The analysis did not include research articles that reported IAV sero- and virological prevalence in commercial, feral, exhibition swine, and wild boars. Conference abstracts, review articles, editorials, letters, commentaries, and opinions were not included in the analysis. We excluded the conference abstracts (n = 2) from the analysis because they did not provide sufficient information on the methodology as well as the sample size in one of the studies. Only English-language articles were included in the analysis. The relevant records were thoroughly screened through the titles, abstracts, and/or methodology, emphasizing the swine holding types for determining their relevance for inclusion in the study. The references of the identified relevant research articles were also screened to find other eligible research articles to be included in the analysis. All the relevant full-text research articles were downloaded for a detailed analysis.The first study that documented H1N1 virus antibodies in backyard swine sera was reported from a family backyard farm in Wisconsin, USA, in 1977 . After tThe studies included active and passive IAV surveillance in backyard swine from various countries, including clinically healthy and symptomatic swine. The clinical signs of IAV disease symptoms in backyard swine populations included nasal and ocular discharge, coughing, sneezing, anorexia, lethargy, incoordination, paralysis of the hindquarters, rapid weight loss, pneumonia, and mortality –24. The The highest number of studies that investigated IAV sero- and/or virological prevalence in backyard swine populations were reported from Asia (n = 11) followed by North America (n = 10), South America (n = 6), Africa (n = 6), and Europe (n = 1). We analyzed the sero- and virological prevalence of IAV in backyard swine in a continent-wise manner as presented below.Following a previous outbreak of highly pathogenic avian H5N1 virus in West Africa, a study was conducted to determine the prevalence of other IAV subtypes in backyard swine populations of Benin and Cote d’ Ivoire during 2009–2010. In this surveillance, backyard farms were preferred for monitoring because the previous H5N1 outbreak, during 2006–2008, predominantly occurred in backyard flocks. Sixty-two and 1548 nasal swab samples were collected from backyard pigs in Benin and Cote d’ Ivoire, respectively, and tested using real-time RT-PCR; none of them were IAV positive, resulting in a negative molecular prevalence. In addition, 457 blood samples obtained from backyard pigs in Cote d’ Ivoire were tested with ELISA and hemagglutinin inhibition (HI) assays, which resulted in a negative IAV seroprevalence .The second study in Africa, which investigated IAV in backyard swine, was conducted in rural Cameroon during December 2009 and August 2012. Here, nasal swabs and sera samples were collected from 325 backyard swine. Only two swine were found infected with pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 through real-time RT-PCR, while the HI assay detected pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 and H3N2 virus antibodies in one swine serum , suggestA total of 1491 nasal swab samples were collected from backyard swine in Kibera, Nairobi, during 2010–2012. Real-time RT-PCR and virus isolation identified 11 (0.7%) pigs infected with pandemic A(H1N1)pdm09 virus clade 1A.3.3.2. The complete genomes of three of the pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 viruses were sequenced in this study. Additionally, 13 (10.2%) out of 127 swine sera samples were found IAV positive using the ELISA assay, while the HI assay detected H1N1 and H3N2 virus-specific antibodies. The IAV specific antibodies were also detected in poultry raised simultaneously with swine. The HA genes of the isolated pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 viruses were closely related to the human A(H1N1)pdm09 virus clade 1A.3.3.2 viruses reported from Kenya in 2009, which suggested human-to-swine transmission of the pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 viruses in Kenya .There was only one study that investigated the serological prevalence of IAV in Ugandan backyard swine. This study reported that the IAV specific antibodies were detected in 26 (4.98%) of 522 clinically healthy swine sera samples which were collected from household swine in the Lira and Masaka districts in 2015 .Surveillance for IAV was conducted in Nigerian backyard swine during 2015–2016. Blood samples from 500 backyard pigs were collected for serological investigation, and 129 tracheal swabs and lung tissues were collected for virological investigation. The ELISA detected IAV-specific antibodies in 222 sera by HI assay, which confirmed pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 (n = 14) and highly pathogenic H5N1 virus (n = 6) antibodies in sera samples. Real-time RT-PCR detected 43 IAV positive samples suggesting active IAV infections. Sanger sequencing confirmed the presence of highly pathogenic H5N1 viruses (n = 5) in Nigerian backyard swine [A significantly low rate of past IAV infections  and active infections  were determined by various studies that were conducted in Asian countries, with a few exceptions, including China, India, Bangladesh, and Bhutan, where a relatively higher seroprevalence  was reported in the backyard swine. An overview of IAV sero- and virological prevalence in Asian backyard swine is represented in Fig. There was only one study that reported IAV sero-surveillance in backyard swine in Bangladesh. This study collected 180 sera samples from the backyard swine from July to December 2013. The ELISA assay detected IAV-specific antibodies in 22 (12.22%) swine sera  which suA cross-sectional study during October 2011–February 2012 was conducted to identify the seroprevalence of IAV in rural backyard swine in Bhutan. Sera samples were collected from 465 backyard pigs in 69 villages and subjected to an ELISA test which detected IAV antibodies in 55 sera samples. All the ELISA positive sera were confirmed with the HI assay, which identified that 36 sera had pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 antibodies while 35 sera had European swine H1N1 virus clade 1C.1 antibodies .Active IAV surveillance in rural Cambodia included 198 nasal swabs collected from backyard swine in 159 households located across 30 villages during May 2011–March 2013. Nasal swabs collected from a pig in 2011 and from two other pigs in 2012 were found IAV positive with real-time RT-PCR. The full-length genome sequencing of IAV positive swine identified H3N2 viruses in these three backyard swine .A sero-surveillance study detected H1N1 and H3N2 virus antibodies in 12 backyard swine sera samples collected during November 1993 in backyard farms in Nanchang, China. Households that reared swine and poultry in the backyards were selected for the study. Samples were also taken from the household members which determined the occurrence of H1N1 and H3N2 virus antibodies in human sera. Additionally, four H3N2 viruses were isolated from the human nasal swab samples. The data suggested transmission of H1N1 and H3N2 viruses from household members to the backyard swine . AdditioIn another study, a European avian-like swine H1N1 virus clade 1C.1 was isolated from the tracheal specimen of a 3-years and 8-months old deceased boy at a family backyard farm who developed fever and dyspnea, which led to his admission to a local hospital where he later died due to further complications. Further investigation at the family backyard farm detected European avian-like swine H1N1 virus clade 1C.1 antibodies in two swine sera samples. Another family member was also found to be H1N1 positive while being asymptomatic. The swine and poultry were raised free-range in this backyard farm; however, poultry was found seronegative for IAV infections. Several of the pigs raised on this backyard farm were reported to have developed fatigue and had been slaughtered before the commencement of the study . While tCo-circulation of pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 and seasonal human H1N1 influenza viruses were reported from Indian backyard swine during 2009- 2016. A total of 2632 sera and 1610 nasal swabs from backyard swine were either collected or received from ten Indian provinces. All the sera samples were analyzed for pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 seroprevalence. While no virus could be isolated from the swine nasal swabs, 522 (19.83%) sera samples had antibodies for the pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 suggesting a past exposure of these swine to the pandemic A(H1N1)pdm09 virus clade 1A.3.3.2. Additionally, 14.3% of the tested samples also had antibodies only for seasonal human H1N1 influenza virus, suggesting a past co-circulation of these viruses in Indian backyard swine populations .Two IAV surveillance studies were conducted in Indonesian backyard swine. The first surveillance collected 344 sera and 304 nasal and throat swabs from backyard swine in Indonesian villages during 2005. The microneutralization (MN) assay could not detect IAV antibodies in the sera samples. Similarly, all the 304 nasal and throat swabs were also negative for IAV with virus isolation and reverse-transcription-PCR , suggestA single sero-surveillance for IAV was conducted in the Nepalese backyard swine from August 2016–February 2017. Total 68 sera samples were collected from free-range backyard swine and tested for IAV antibodies using ELISA assay. Only two sera samples were found positive for IAV antibodies .Only one sero-surveillance detected no IAV antibodies in 237 backyard swine sera samples collected from 74 households in Thailand during September 2016–February 2017 .A serological study collected 519 sera samples from backyard swine in 158 villages located in Northern Viet Nam during April 2005–August 2006 for determining IAV seroprevalence. ELISA tests detected IAV antibodies only in two swine sera samples , suggestOne highly pathogenic avian influenza H5N8 virus was detected in a backyard swine in France during an H5N8 outbreak among French poultry during 2016–2017. The HI assay confirmed that one backyard swine had antibodies against H5 clade 2.3.4.4b and identified that avian origin H5N8 virus would have been transmitted from domestic ducks raised on the backyard farm to the swine . No clinIn Costa Rica, nasal swabs were collected from 509 backyard swine from 25 observation units with clinical signs of influenza-like illness during October–November 2010. An observation unit was defined as a space that confined the pigs either within an entire backyard or the pigs physically confined within an individual barn. Real-time RT-PCR detected 11 (2.16%) pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 positive nasal swab samples, which indicated a low rate of active IAV infection in these backyard swine .Fifty-four sera samples collected from backyard swine in 36 premises in the Dominican Republic during August 2010 were tested for IAV seroprevalence using the HI assay, which detected H1N1 and H3N2 virus antibodies in 12 22.2%) and 17 (31.5%) samples, respectively. These results suggested a past exposure of these backyard swine to the IAV. In this study, the human-to-swine transmission of IAV was suspected [.2% and 1A study included collecting nasal swabs and sera samples from 426 backyard swine in 2010 and 2011 in Guatemala. Sera samples were tested with ELISA and HI assays, which identified 13 sera with A(H1N1)pdm09 virus clade 1A.3.3.2 and/or swine H1 and H3 virus antibodies. Interestingly, 52 nasal swabs were determined to be IAV positive, out of which four viruses were successfully isolated. While three isolates were confirmed to be pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 viruses, one isolate was a human-like H3N2 virus. This investigation suggested a reverse-zoonotic transmission of human A(H1N1)pdm09 virus clade 1A.3.3.2 and H3N2 viruses to the backyard swine in Guatemala .Sera samples were collected from 109 backyard swine from 10 regions in Haiti during April 2010. The HI assay identified H1N1 virus antibodies in 24 (22.01%) and H3N2 virus antibodies in 13 (11.92%) sera samples  which suAnas diazi), which emphasized the significance of active IAV surveillance in the region to monitor the possible future spillover [Three studies have been conducted to detect IAV in Mexican backyard swine populations. The first study retrospectively analyzed 2094 backyard swine sera samples for the IAV antibodies from 2000 to 2009. The HI assay identified the highest seroprevalence of swine H1N1 virus (74%), which was followed by swine H3N2 virus (24.2%), pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 virus (17.8%), and human H1N1 virus (1.3%) . Intrigupillover .In a sero-surveillance conducted during October 2013–February 2015, 139 swine sera were collected from small backyard farms located on the island of Trinidad. In addition, 45 swine sera were collected from the small backyard farms located on the island of Tobago. While the ELISA assay detected IAV antibodies in 14 (10.07%) sera obtained from Trinidad, the sera from Tobago were negative for IAV seroprevalence. Among the ELISA positive samples from Trinidad, H3N2 and pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 virus antibodies were detected by the HI assay [Following a respiratory disease outbreak in the swine on a family farm in Wisconsin in October 1975, characterized by sneezing, coughing, and anorexia in the swine, an 8-year-old boy who had close contact with the swine in the household became ill with fever, headache, chills, abdominal pain, and sore throat. The swine H1N1 virus antibodies were detected in the serum sample of the boy 3 weeks after the onset of the illness. As a result, an investigation was initiated to ascertain the source of infection to the boy. Interestingly, the antibodies for the swine H1N1 virus were also detected in eight of the ten swine sera samples collected from the boy’s family farm . These dIn a more recent study, after observing the symptoms of pneumonia and rapid weight loss in one-month-old piglets at a small backyard piggery during November 2010 in Colorado, intestine, and lung tissue samples were submitted to the laboratory for diagnosis. Molecular diagnostics followed by virus isolation and genome sequencing for the hemagglutinin (HA) gene identified that the two piglets were infected with pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 virus. Since the piggery was owned by a pharmacist who may have had occupational exposure to the A(H1N1)pdm09 virus clade 1A.3.3.2 virus, reverse-zoonotic transmission from the owner to the piglets was suspected . An overAn IAV serological surveillance during 2012 and 2014 consisted of 1667 backyard swine sera from 479 subsistence swine holdings in the Rio Grande do Sul province of Brazil. All the swine sera were initially screened for IAV antibodies using the ELISA assay. The ELISA positive sera  were subtyped using the HI assay, which detected pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 virus antibodies in 92 (5.51%) swine sera, while six sera had antibodies for H1N2 and five sera had antibodies for H3N2 viruses . This waChile reported the highest number of studies (n = 4) that conducted IAV surveillance in backyard swine populations. The first study investigating both IAV virological and serological prevalence was conducted in Central Chile during 2012–2014. A total of 67 nasal swabs and 127 sera were collected from backyard swine which were subjected to real time-PCR and ELISA tests, respectively. While all the nasal swabs were negative for IAV, only two sera had IAV antibodies suggesting a low circulation of IAV in backyard swine under investigation . The secA total of 1303 sera and 923 tracheal swab and lung tissue samples were collected at the time of slaughter from apparently healthy backyard swine in Tumbes, Peru, at four different times during March 2009–October 2011. While the HI assay detected pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 virus antibodies in 110 8.44%) sera, virus isolation and reverse transcription-PCR detected pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 viruses in one lung tissue sample and four tracheal swabs only. Despite a significantly low molecular prevalence, the phylogenetic analysis determined more than one human-to-swine transmission events for these pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 viruses in Tumbes . An over.44% seraIn summary, an overall significantly higher seroprevalence 18.28%; 2986/16328)  reported that swine were the only household animals in the backyards , 43, 45,.24% repoThe molecular investigations that utilized real-time RT-PCR and Sanger sequencing for detection and characterization of IAV reported that pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 was the more frequently present IAV subtype n = 6; 27.27%) , 27, 50 .27% , 27Serological studies that used HI assay reported the antibodies for H1N1 , pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 , H3N2 , H5N1 , H5N8 , and H1N2  viruses  swine farms globally during the twentieth century , the IAVSimilarly, South Africa, due to its unique geographic location, is a favourable destination for wild birds for over-wintering, and falls within the East Africa–West Asia flyway and East Atlantic flyway of wild migratory birds . In receWhile IAV active infection was detected only in a few backyard swine in Asia (Cambodia and Indonesia) , 36, serIn contrast, numerous studies have reported several IAV subtypes in Chinese commercial swine populations . The limMost intriguingly, the seroprevalence of pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 virus in backyard swine populations in Mexico during 2000–2009 suggested the occurrence and circulation of pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 viruses in Mexican backyard swine well before the emergence of the 2009 swine flu pandemic in Mexico . This suIt has been established that the IAV infected swine may start virus shedding one day post-infection (dpi) while the virus can be shed up to 5 dpi  and hencIt is evident that widespread prevalence and circulation of various avian influenza viruses in wild birds and domestic poultry poses a constant threat to backyard swine farming, given the inadequate biosecurity measures, and therefore needs continuous monitoring of IAV disease. Therefore, adequate biosecurity on the backyard swine farms is recommended, with minimized direct human–swine interactions to reduce the possibility of zoonotic and reverse-zoonotic IAV transmission, thereby safeguarding public health. These findings reiterate the need for ongoing surveillance to track IAV circulation and evolution in backyard swine populations.Backyard swine farms rearing both swine and poultry remain at a high risk of IAV interspecies transmission from domestic poultry to swine. In addition, migratory wild birds pose a significant threat and may introduce exotic IAV subtypes to the backyard swine. The possibility of zoonotic and reverse-zoonotic transmission between swine and humans also persists within the backyard farms. The occurrence of pandemic A(H1N1)pdm09 virus clade 1A.3.3.2, highly pathogenic avian H5N1, and several other IAV subtypes in backyard swine populations should be of concern as this may cause disease outbreaks in swine as well as in the exposed human populations. The occurrence of both human and avian IAV subtypes in backyard swine may facilitate their evolution, representing public health risk. A policy of active IAV surveillance in backyard swine populations should be implemented to track their molecular evolution.


Amaranthaceae (incl. Chenopodiaceae) shows an immense diversity of C4 syndromes. More than 15 independent origins of C4 photosynthesis, and the largest number of C4 species in eudicots signify the importance of this angiosperm lineage in C4 evolution. Here, we conduct RNA-Seq followed by comparative transcriptome analysis of three species from Camphorosmeae representing related clades with different photosynthetic types: Threlkeldia diffusa (C3), Sedobassia sedoides (C2), and Bassia prostrata (C4). Results show that B. prostrata belongs to the NADP-ME type and core genes encoding for C4 cycle are significantly upregulated when compared with Sed. sedoides and T. diffusa. Sedobassia sedoides and B. prostrata share a number of upregulated C4-related genes; however, two C4 transporters (DIT and TPT) are found significantly upregulated only in Sed. sedoides. Combined analysis of transcription factors (TFs) of the closely related lineages  revealed that no C3-specific TFs are higher in C2 species compared with C4 species; instead, the C2 species show their own set of upregulated TFs. Taken together, our study indicates that the hypothesis of the C2 photosynthesis as a proxy towards C4 photosynthesis is questionable in Sed. sedoides and more in favour of an independent evolutionary stable state. This mechanism leads to a decrease in the oxygenation reaction of RuBisCO, which in turn decreases photorespiration, because fewer toxic compounds resulting from the RuBisCO oxygenation reaction need to be recycled  and a CARBONIC ANHYDRASE isoform [CA (Bv8_194450_rkme.t1)] were significantly upregulated in Sed. sedoides (C2) when compared with B. prostrata (C4) (T. diffusa (C3) as compared with Sed. sedoides (C2) (As observed in other Cusa (C3) . Out of ata (C4) . Interesdes (C2) .4-related enzymes except ASPARAGINE SYNTHETASE (Asn Synthetase), SODIUM:HYDROGEN ANTIPORT (NHD) and a CARBONIC ANHYDRASE (CA) isoform (Bv6_148840_uffy.t1) were significantly upregulated in Sed. sedoides (C2) compared with T. diffusa (C3). These enzymes include typical C4 enzymes such as PEPC, NADP-ME, PPdK, PHT4, Ala-AT, and ASPARTATE AMINOTRANSFERASE (Asp-AT), as well as C4-associated transporters such as BASS2 and DIT.Twelve of the CSed. sedoides (C2) as compared with B. prostrata (C4), including the core photorespiratory enzymes GLYCINE DECARBOXYLASE , GLUTAMATE:GLYOXYLATE AMINOTRANSFERASE (GGT) and SERINE HYDROXYMETHYLTRANSFERASE (SHMT). In Sed. sedoides (C2), GGT, two SHMTs, GDC-T, GLYCOLATE OXIDASE (GOX), GDC-H, GLYCERATE 3-KINASE (GLYK), PHOSPHOGLYCOLATE PHOSPHATASE (PGP) were significantly upregulated when compared with T. diffusa (C3). Only one SHMT isoform (Bv6_143730_mggd.t1) was significantly upregulated in B. prostrata (C4) when compared with Sed. sedoides (C2) (T. diffusa (C3), only one gene GDC-P was significantly upregulated as compared with Sed. sedoides (C2) (Sed. sedoides (C2), compared with B. prostrata (C4), were significantly upregulated in T. diffusa (C3) when compared with B. prostrata (C4).Out of 14 transcripts associated with photorespiratory enzymes, 12 were annotated and assigned . All 12 des (C2) . In T. ddes (C2) . All pho2 and C4 photosynthesis, expression patterns of TFs from The Plant Transcription Factor Database v.5.0 , Sed. sedoides (C2), T. diffusa (C3); five species from Salsoleae: H. soparia (C4), Sal. divaricata Pop-184 (C2), Sal. divaricata Pop-198 (C2), Sal. oppositifolia (C4), Sal. soda (C3/C4), Sal. webbii (C3)) and were further analysed using Clust. Based on the expression pattern, from the initial 494 TFs, 431 TFs were grouped into 11 clusters with between 22 and 71 genes per cluster  usinantTFDB; ) were inp-value ≤ 0.01) more abundant in all C4 species compared with the two C3 species  and C3  pathways were assessed in the following comparisons: C2 species vs. C3 species and C2 species vs. C4 species. Then, specific TFs of the C2 pathway  were estimated in the pairwise comparison of C3 species vs. C4 species. Among the four TFs common to all C4 species, only one TF  was significantly upregulated in C2 species when compared with C3 species  was significantly higher in the C species . To asse species . Convers2, C4) and the ancestral C3 photosynthesis [4 and C2 photosynthesis was achieved by comparing differentially expressed genes of closely related species in the genus Flaveria (Asteraceae) considered as a model organism to study the evolution of C4 photosynthesis [Chenopodiaceae) has a large number of C2 and C4 species that differ anatomically and ecologically from Flaveria. This family therefore represents a good supplementary alternative to decipher the convergent evolution of C4 photosynthesis. With PCA based on gene expression, it was possible to clearly distinguish between T. diffusa (C3), representing the ancestral condition, and Sed. sedoides (C2) and B. prostrata (C4), representing derived conditions. The physiologically C3-C4 intermediate Sed. sedoides (C2) was positioned in a triangle with T. diffusa (C3) and B. prostrata (C4) in terms of transcript variation. This result was similar to what was found in Salsoleae [Salsoleae [Salsoleae, in Camphorosmeae, the three different photosynthesis types predominantly structure the gene expression pattern in assimilating tissue. Indeed, species with C3, C2, and C4 photosynthesis differ in leaf anatomical structure. T. diffusa (C3) exhibits the Neokochia type characterised by an undifferentiated chlorenchyma of several layers. Sed. sedoides (C2) has the Sedobassia type consisting of kranz-like cells near peripheral vascular bundles. B. prostrata (C4) depicts the Bassia prostrata type with the chlorenchyma differentiated in an outer mesophyll and inner kranz-layer [Flaveria explained only 27% [Flaveria study as suggested by Lauterbach et al. [Gene expression analysis predominantly paved the way to understand the difference between derived photosynthetic types . Similar results were found in Cleome [Flaveria [Salsoleae [4 photosynthesis are classified according to the predominant type of decarboxylation releasing CO2 around RUBisCo in the BSCs: NAD-ME, NADP-ME, and PEP-CK. However, PEPCK should be considered as a supplemental subtype to either NAD-ME or NADP-ME [B. prostrata (C4) uses a NADP-ME type C4 cycle. Asparagine synthetase (ASN) and NHD were found significantly expressed and upregulated only in B. prostrata (C4) as compared with T. diffusa (C3) and Sed. sedoides (C2). ASN was reported upregulated in C4 species Gynandropsis gynandra when compared with closely related C3 species Tarenaya hassleriana (Cleomaceae), as well as in C4 leaves of Sal. soda when compared with its C3 cotyledones [4 species compared with C3 and C2 species of Flaveria [4 enzymes in B. prostrata (C4) as compared with T. diffusa (C3) were Ala-AT, PPDK, and BASS2. ASN is involved in ammonium metabolism and asparagine in nitrogen transport [4 cycle requires the transport of pyruvate to the mesophyll cell (MC) for regeneration of PEP. While Ala-AT plays an important role in pyruvate generation, PPDK intervenes in the regeneration of PEP. Pyruvate transport is mediated by the BASS2/NHD transport system [2 acceptor (PEP), and therefore maintains the C4 pathway.Analyses of differential gene expression between Cn Cleome , FlaveriFlaveria ,35 and Salsoleae . Traditi NADP-ME . Signifiyledones . On the ransport . Achievet system . Taken at system  and Mall4-related genes were found significantly upregulated in Sed. sedoides (C2) compared with T. diffusa (C3), including, for example, PEPC, NADP-ME, PPdK, and PHT4. Upregulation of C4 typical enzymes such as PEPC, NADP-ME, PPdK, PPT was also reported in the C2 species when compared with the C3 species in studies of Flaveria and Salsoleae [4 cycle are present in Sed. sedoides (C2) and play an important role in C2 metabolism. A DIT isoform (Bv4_072630_xjai.t1), NADP-ME and Ala-AT were the three most upregulated C4 enzymes in Sed. sedoides (C2) as compared with T. diffusa (C3). Moreover, we found two transporters (TPT and DIT) upregulated in Sed. sedoides (C2) when compared with B. prostrata (C4). These transporters were found highly expressed in some C2 species when compared with C4 species in Flaveria [2 leakage from BSCs to MCs [2 leakage from the Kranz-like cells back to the MCs due to the presence of RuBisCo, which is not the case for C4 plants. This explains the low CO2 compensation observed in C2 species [4 enzymes to handle constraints related to the C2 pathway and not an entirely congruent set. This does not support their interpretation as an intermediate state towards C4 photosynthesis, but is more in line with their interpretation as an independent evolutionarily stable state  and T. diffusa (C3) compared with B. prostrata (C4). Likewise, we found key photorespiration enzymes were differentially expressed and upregulated in the C2 species (Sed. sedoides) when compared with T. diffusa (C3) and B. prostrata (C4). This corroborates the expression patterns reported in the C2 species of Flaveria [Salsoleae [2 photosynthesis in Sed. sedoides (C2). Our transcript data showed that GDC-P and a SHMT isoform (Bv6_143730_mggd.t1) were downregulated in Sed. sedoides (C2) when compared with T. diffusa (C3) and B. prostrata (C4), respectively. Similar results were obtained in the C2 species of the genus Flaveria [2 pathway in Flaveria. Since GDC-P and a SHMT isoform are known to be involved in glycine decarboxylation, their downregulation in Sed. sedoides (C2) might have similar consequences. It is worth noticing that the number of significantly upregulated photorespiratory genes in Sed. sedoides (C2) was equal to T. diffusa (C3) when compared with B. prostrata (C4).Transcripts associated with photorespiration were about twice as abundant in CFlaveria ,35 and Salsoleae , implyinFlaveria  showed tB. prostrata (C4). All photorespiratory genes except GLYK were downregulated in B. prostrata (C4) as compared with T. diffusa (C3). Mallmann et al. [4Flaveria except the transport proteins DIT1 and DIT2 and one isoform of GLDH. On the other hand, GLYK was expressed in the M of C4Sorghum bicolor [B. prostrata (C4) could clarify its high expression and role.A significant reduction in almost all photorespiratory genes was observed in n et al.  reported bicolor . GLYK ca3, C2 and C4 species of Salsoleae and Camphorosmeae are widely spread in desert, semi-desert, saline, and arid regions [Chenopodiaceae, C4 photosynthesis evolved as an adaptation to hot, dry, or saline areas from the C3 ancestor which was already preadapted to grow in these harsh environments [3, C2, and C4 species/states irrespective of the lineage, to further reduce the amount of differentially expressed TFs to a small subset of actually C4-, C2-, and C3-related changes. Indeed, a small number of TFs were found differentially expressed between C3, C2, and C4 species/states.Transcription factors are proteins that bind to the DNA promoter or enhancer regions of specific genes and regulate their expression. They have a crucial role on plant growth, development and adaptation under various stress conditions, and therefore are excellent candidates for modifying complex traits in plants . C3, C2  regions ,19. In fronments . We focu4 species irrespective of the lineage when compared with C3 species. The families to which these TF families belong play an important role in regulatory networks controlling plant growth and development, and plant adaptive responses to various environmental stress conditions [4 M and Kranz cell identities [4 species and co-regulated, our data suggest a critical role of these TFs in the development of any C4 Kranz anatomy in the Amaranthaceae/Chenopodiaceae complex.Cluster analysis showed that BBX15, SHR, SCZ, and LBD41 were co-regulated and significantly more abundant in all Cnditions ,48,49,50entities ,53,54,55entities . Since m3 species enhanced different TFs compared with C4 species. Three TFs , HSFA6B (HSF TF family), and NAC083 (NAC TF family), in which two TFs (HSF6B and NAC083) are co-regulated, were significantly higher in all C3 species when compared with C4 species. As C4 TFs, they are involved in plant growth, development, and stress tolerance. The NAC TF family was shown to contribute to root and shoot apical meristems formation in Arabidopsis [4 photosynthesis in maize and rice [4 maize and rice plants. This may imply higher activity of these TF families in C3 species. Nevertheless, different transcripts of these TFs families were involved when compared with the present study. Thus, a significant expression of these TFs in C3 species could indicate a potential function of these TFs in the C3 pathway.We found that Cbidopsis ,58, orgabidopsis , salt anbidopsis , leaf sebidopsis , and secbidopsis . The HD-bidopsis ,64. Inteand rice ,65. Howe2 species, one transcript of the BASIC HELIX-LOOP-HELIX (bHLH106) protein from the bHLH TF family was found to be upregulated compared with C4 and C3 species. Two TFs of the bHLH TF family were shown to regulate a C4 photosynthesis gene in maize [2 species may suggest its possible role in the development and establishment of the C2 photosynthesis specificities relative to other photosynthesis types. Interestingly, one C4-specific TF  was significantly higher in C2 species when compared with C3 species. Thus, this TF could be responsible for similarities of C4 photosynthesis found in C2 species such as the Kranz-like anatomy. Surprisingly, no C3-specific TF was significantly expressed in C2 species when compared with C4 species. This indicates that C2 and C4 photosynthesis represent more derived types of photosynthesis compared with C3 photosynthesis. Nonetheless, this seems to be inconsistent with the current model of C4 evolution which relies heavily on the interpretation of the physiological intermediacy of C2 photosynthesis as an evolutionary stepping stone to C4 [3-specific TFs to be higher in C2 species when compared with C4 species if the C2 photosynthetic type represents an intermediate step along the evolution of C3-to-C4 photosynthesis as revealed by differential expression analysis of core photorespiratory genes in C2 and C3 species of Camphorosmeae (this study) and Salsoleae [In Cin maize . This upne to C4 . One woualsoleae .2 intermediate species suggests an evolutionarily stable state in its own right. Similarities with C4 relatives might result from a hybrid origin involving C3 and C4 parental lineages, parallel recruitment of a number of TFs in C4 and C2 lineages or common ancestry, and later divergent evolution. The position of Sedobassia as sister to Bassia  allows all of these three scenarios [2 species in Salsoleae, however, phylogenomic evidence points to a hybrid origin of the Sal. divaricata agg. . Further phylogenomic analyses are needed to discern if an early hybridisation event of a C4  lineage and a C3 lineage led to the origin of the Sedobassia lineage which then evolved towards stable C2 photosynthesis.Taking the results of this study together, the unique derived TF profile of the Ccenarios . For C2 Camphorosmeae species (Bassia prostrata (L.) Beck (C4), Sedobassia sedoides (Schrad.) Freitag and G. Kadereit (C2), and Threlkeldia diffusa R.Br. (C3) (Plants of three Br. (C3)  were groB. prostrata, Sed. sedoides, and T. diffusa. Sequencing of 101 bp single-end reads was performed on an Illumina HiSeq2000 platform. For each species, three individuals were sequenced . Sequencing reads of these three species are available under study accession PRJEB36559.Total RNA extraction, library preparation, and mRNA sequencing were performed as described by Lauterbach et al. ,24. TotaSalsoleae species were retrieved from Lauterbach et al.  , cotyledons and leaves of the Salsola divaricata population 184 , Salsola divaricata population 198 , and Salsola oppositifolia (C4); leaves of Salsola webbii (C3); and assimilating shoots of Hammada scoparia (C4). For all of these samples, triplicates per species and organ were available [RNA-Seq data of the five EB22023) . These dvailable .www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 15 March 2021), and filtered and trimmed using Trimmomatic v.0.38 [Beta vulgaris, contigs were annotated using Arabidopsis (TAIR10). Reads of each of the replicates were separately mapped against these reduced data sets via bowtie2 v.2.3.4.1 [Single-end sequencing reads were checked for quality using the FASTQC tool  using tc v.0.38 . Number c v.0.38 ,72 and oc v.0.38  run locac v.0.38 ) as a re.2.3.4.1 . Re-form.2.3.4.1 .2 transformed read counts were carried out with Multiexperiment Viewer (MeV) v.4.9 (http://mev.tm4.org/ accessed on 5 February 2020). Co-expressed gene clusters of (1) all expressed transcripts and (2) transcripts annotated as transcription factors were carried out with Clust v.1.10.7 [4′. To identify TFs putatively involved in the formation/regulation of C2 and C4 photosynthesis, the 1163 annotated TFs from Beta vulgaris , Sed. sedoides (C2), and B. prostrata (C4); 2: leaf transcriptome data of the five Salsoleae species Sal. webbii (C3), Sal. divaricata Pop-184 (C2), Sal. divaricata Pop-198 (C2), H. scoparia (C4), Sal. oppositifolia (C4), and Sal. soda (C4); for study accession numbers see above) and transcribed TFs grouped using Clust v.1.10.7 [3, C2, and C4. VENNY v. 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/ accessed on 12 July 2021) were deployed to find intersected TFs across all pairwise comparisons.Read counts were normalised into transcripts per million (TPM) and used for differential gene expression analysis. Here, pairwise comparison between all eight species was statistically evaluated using edgeR  in R  from ThtSet-2′, ,31) werev.1.10.7  and grouChenopodiaceae family provided new insight into C4 evolution. Proteins encoding for C4 transporters (DIT and TPT) were found significantly upregulated in Sed. sedoides (C2) when compared with B. prostrata (C4). Upregulation of those transporters reduces CO2 leakage from BSCs to MC, which could otherwise be detrimental to C2 photosynthesis due to the presence of RuBisCo in the MC. This suggests evolution of a stable C2 photosynthesis independent of C4 photosynthesis. Combined analysis of TFs of the sister lineages provides further support of this result. Indeed, while one C4-specific TF (BBX15) was significantly higher in C2 species when compared with C3 species, no C3-specific TFs were higher in C2 species compared with C4 species. Finally, apart from well-known TFs involved in the development of C4 Kranz anatomy such as SHR, BBX15, SCZ, and LBD41 may also be associated with its development and physiology. Furthermore, bHLH106 could be related to specific C2 anatomy and BBX15 to a characteristic C4-like expression pattern found in species with C2 photosynthesis. This study sheds light on the differentiated regulation and evolution of transcription factors in C2 and C4 photosynthesis.The transcriptome data of the


Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels  is required. Macrophage-localized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1β (pro-IL-1β)-degrading caspase-1 to lead to IL-1β release. Moreover, inflammatory diseases  are generated downstream of the P2X7R-induced upregulation of intracellular second messengers . In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral  and central tissue (microglia). They occur as M1  or M2  phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na Whereas P2XRs respond only to ATP, P2YRs respond to ATP/ADP, UTP/UDP, or UDP-glucose. Signaling through P2/P1Rs is rapidly terminated by the conversion of ATP to adenosine and eventually to the inactive inosine within the extracellular space by the activity of ecto-nucleotidases . Th. Th31]. and Ca2+ ,14. Whiland Ca2+ ,32 . It has been shown that intracellular Ca2+ in murine peritoneal macrophages [+ and Ca2+ is generated after the stimulation of all P2XRs (including P2X7Rs), the degree of current desensitization is quite diverse [2+.As already mentioned, the activation of P2X7Rs has an ability to open a typical ion channel for small cations, including Carophages  or humanrophages  is eleva diverse . NoteworMycobacterium tuberculosis [Repetitive activation of P2X7Rs with ATP evoked a biphasic membrane current in human macrophages, which is supposed to reflect large plasma membrane pore generation for the polyatomic cationic dye YO-PRO-1 . Upon strculosis ,65 were rev) measurements with the whole-cell patch clamp technique, that the ion conducting pathway undergoes progressive dilation [rev in a medium in which the counter ion of intracellular K+ was NMDG+ instead of Na+, emerged due to time-dependent alterations in the concentration of intracellular ions rather than channel dilation [A particularly intensively discussed issue is whether the initially opened cationic channel dilates and, thereby, establishes the larger diameter pore permeable to cationic dyes or whether two different channels are involved in this effect . Originadilation . Howeverdilation . Moreovedilation .The P2X7R C-terminal tail constitutes about 40% of the whole protein; it distinguishes P2X7Rs from the other P2XR types and plays a key role in P2X7R ion channel function . The delPannexins are a family of vertebrate proteins identified by their homology to the invertebrate innexins . While iHowever, pannexin-1-induced dye uptake is also observed independent of P2X7R occupation by ATP . Moreove− channels appears to be necessary for ATP-induced dye uptake in mouse and human macrophages, and the activity of caspase-1, which is essential for interleukin release, is also blocked by Cl− channel antagonists [2+-activated Cl− and non-selective anionic channel, contributes to P2X7R pore formation, and YO-PRO-1 uptake produced by P2X7R activation is attenuated by knockdown of ANO6 [However, the activation of Clagonists . Anoctam of ANO6 . ANO6 ma of ANO6 .These rather conflicting results regarding the P2X7R pore formation suggest that more than one channel is probably responsible for dye uptake, and pannexin-1 and/or ANO6 associated with P2X7Rs participate to a certain extent in this process. Nevertheless, a possibility that pannexin-1 allows dye uptake by altering the function of plasma membrane lipids and membrane transporters cannot be ruled out either. In fact, the panda P2X7R, when purified and reconstituted into liposomes, forms a dye permeable pore in the absence of other cellular components . P2X7R c2+-activated ion channel/scramblases [+, K+, and Ca2+ [Along this line of thinking, it was recently suggested that current facilitation and macropore formation involve functional complexes comprised of P2X7R and TMEM16, a family of Caamblases . Macropoand Ca2+ .+ efflux and Ca2+ influx is also involved in macrophage membrane blebbing.Membrane blebbing is a characteristic feature of injured cells . ExtensiThe macrophage exocytosis, proliferation and cell morphology are regulated by P2X7Rs as well. Convincing evidence proves that P2X7Rs play a crucial role in macrophage exocytosis induced by single-walled carbon nanotubes . The pro+ after P2X7R stimulation initiates P2X7R-dependent NLRP3 inflammasome activation [The P2X7R pore is believed to be an essential component of regulating inflammasome activation in macrophages ,89. Infltivation ,96.2+ overload as a macrophage death trigger [Indeed, brief stimulation of P2X7Rs is sufficient to initiate the processing of IL-1β in macrophages ,98. Ther trigger . The rel trigger .P2X7R signaling also protects against bacterial infections through enhancing bacterial killing by macrophages, which is independent of the inflammasome. ATP release through connexin channels is instrumental in inhibiting inflammation and bacterial burden, and ATP protects against sepsis through activation of P2X7Rs in macrophages by enhancing intracellular bacterial killing .+ efflux [In addition to the classic inflammatory pathways, the interaction between the P2X7R and other proteins located on macrophages is also essential for the inflammatory process. The P2X7R cross-talk with the extracellular matrix (ECM) can be exerted via paxillin. Paxillin is a multi-domain protein that localizes at the ECM; ATP triggers a P2X7R-paxillin interaction in murine and human macrophages to promote NLRP3 deubiquitination and K+ efflux .2+ influx in response to BzATP, partly inhibited by pharmacological blockers of TRPA1 [Additional inflammasome-independent but P2X7R-dependent pro-inflammatory mechanisms have also been described. (1) A recent study suggested that transient receptor potential ankyrin 1 (TRPA1) was considered to co-localize with P2X7Rs in human cultured macrophages; it mediates Caof TRPA1 . This prof TRPA1 . (3) Theof TRPA1 .In contrast to their role in pro-inflammation, P2X7Rs might play an anti-inflammatory role in M2 macrophages . The P2XMultiple studies indicate that the activation of P2X7Rs in macrophages is involved in peripheral inflammatory diseases, including rheumatoid arthritis , Crohn’sIn rheumatoid arthritis patients, ATP release and P2X7R activation are significantly increased by anti-citrullinated protein antibodies (ACPAs) which are targeted against citrullinated proteins/peptides utilized as rheumatoid arthritis biomarkers . ACPAs pThe involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1β production in mouse macrophages supplemented with human hepatic stellate cells might contribute to extracellular matrix deposition and suggests that blockade of the P2X7R-NLRP3 inflammasome axis represents a potential therapeutic target for liver fibrosis ,110. P2XHowever, P2X7Rs play a totally different role in sepsis. P2X7R activation on mice peritoneal macrophages suppresses sepsis-induced inflammation and augments killing of intracellular bacteria; connexin hemichannels may have a role in this process . HoweverCytokine storm, defined as macrophage activation syndrome (MAS), is characterized by the release of multiple pro-inflammatory cytokines  and chemokines  from macrophages . MAS is In the meantime, it is a well-known fact that P2X7Rs amplify CNS damage in neurodegenerative diseases, such as Alzheimer’s Disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, post-ischemic conditions, and neurodegeneration as a cause or consequence of epilepsy . It is eThe expression of microglial P2X7Rs has been enhanced in a rat model of Parkinson’s disease, induced by the intranigral injection of LPS; the application of the P2X7R antagonist Brilliant Blue G (BBG) reduced the activation of microglia and the loss of nigral dopamine neurons by decreasing the phosphorylation level of p38 MAPK . Under tP2X7Rs play a two-fold role in the course of amyotrophic lateral sclerosis (ALS) depending on the disease state . BBG decSimilar changes in microglial polarization were observed at the later stage of multiple sclerosis or its rodent model, experimental autoimmune encephalomyelitis (EAP) . In addiP2X7R expression on microglia was increased during epilepsy induced by the intra-amygdala injection of kainic acid; P2X7R antagonists reduced the accompanying microgliosis and produced enduring suppression of epileptic seizures in mice injected with intra-accumbal kainic acid . The typMicroglial P2X7Rs participate also in post-ischemic neurodegeneration. P2X7Rs are widely expressed on microglial cells in both hemispheres after a monolateral brain infarction; in this case, transition from the M1 to the M2 state occurs and a partial protective effect against the post-ischemic neurological impact develops in rats .hP2RX7 [A number of P2X7R isoforms derived from alternative splicing were identified both in humans and rodents ,132. SomhP2RX7 . MacrophP2RX7 coding for the E496A-P2X7R impaired the ATP-induced IL-1β release from human monocytes [Early investigations characterized various polymorphisms, but did not couple their identification with population genetic studies in order to define possible illnesses linked to these mutations. The rs3751143 polymorphic mutant of onocytes . It was onocytes . Apparenonocytes . rs17181onocytes . The SNPonocytes .Toxoplasma gondii-infected human macrophages showed that the loss-of-function rs3751143 SNP (E496A) significantly reduced P2X7R-mediated parasite killing [More recently, some of these SNPs were suggested to be involved in various illnesses. Especially, the killing of intracellular bacteria surviving in phagosomes within macrophages depended on undisturbed P2X7R function. It was found that the 1513C allele  is a risk factor in the development of extrapulmonary and pulmonary tuberculosis in many ethnic populations ,138,139.P2RX4 and P2RX7 genes in macrophages [The risk of age-related macular degeneration was increased by the existence of a rare functional haplotype of the rophages . WhereasP2RX7 polymorphism in osteoclast pathophysiology [P2RX7 SNPs in the development of bone loss, osteoporosis, and risk of fractures [A particularly interesting issue is the role of ysiology . Osteoclysiology . Geneticractures . Bone maractures .P2RX7 are associated with diverse psychiatric and neurological illnesses. In this case, a microglia-based neuro-inflammatory reaction might be causally involved, e.g., in mood disorders. It has been suggested that the SNP rs2230912 coding for the Q460R-P2X7R indicates a predisposition for major depressive disorder (MDD) [P2RX7 SNPs rather than a single receptor-polymorphism was thought to be responsible for MDD predisposition [Linkage studies also suggested that SNPs of er (MDD) ,147,148.er (MDD) ,150. In er (MDD) . Howeverer (MDD) . Similarer (MDD) . In concposition .P2RX7 polymorphism (rs1718119 coding for A348T) was reported to be involved in this disease in contrast to that responsible for MDD [MDD and bipolar disorder are aggregated in families, and epidemiological studies have found in both cases evidence for genetic susceptibility. However, in the case of bipolar disorder a different  for MDD ,155.With respect to neurological illnesses the rare SNP rs28360457 (R307Q) with absent pore formation was suggested to protect against neuroinflammation in multiple sclerosis . The R30− channels to inflammasome activation, and consequently, (neuro)inflammation (P2X7Rs are highly expressed on macrophages, appear to be crucial for proliferation, differentiation, and apoptosis of this cell type, and are involved in the macrophage immune response to pathogens and peripheral/central disorders of diverse origin. The activation of P2X7Rs initiates pore formation in the plasma membrane of macrophages allowing the entry of large cations into the intracellular space; this leads through interaction with pannexin-1, TRPA1, P2X4, and ANO6 Clammation . The ass


S100A4 is a member of the S100 calcium-binding protein family and is increased in patients with chronic obstructive pulmonary disease (COPD). Sphingosine-1-phosphate (S1P) is a naturally occurring bioactive sphingolipid, which regulates the adhesion between the cells and the extracellular matrix and affects cell migration and differentiation. The goal of this study was to analyze the correlations among S100A4, S1P, and pulmonary function among COPD patients.  All 139 serum samples and 15 lung specimens were collected in COPD patients and control subjects. S100A4 and S1P were detected in two groups. The markers of fibrosis and epithelial-mesenchymal transition (EMT) were measured in the lungs of COPD patients and control subjects.  The protein expression of S100A4 was higher in the lungs and serum of COPD patients than control cases. Additionally, serum S100A4 was inversely associated with pulmonary function among COPD patients. Meanwhile, collagen deposition and EMT nuclear transcription factors were elevated in the lungs of COPD patients. Moreover, the protein expression of S1P was increased in the serum of COPD patients. Serum S1P was gradually increased along with pulmonary function decline in COPD patients. Further correlation analysis revealed that serum S1P was negatively associated with pulmonary function in COPD patients. Furthermore, there was a positive correlation between S1P and S100A4 in COPD patients.  These results provide evidence that the elevation of S100A4 and S1P may be involved in the onset and progression of COPD. Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow limitation and chronic airway inflammation, which evokes a heavy burden on the whole society and individuals . As the S100A4 (fibroblast-specific protein 1) is a member of the S100 calcium-binding protein family. It is generally known that S100A4 exerts central roles in the pathophysiology of tumor metastasis, fibrotic diseases, and autoimmune disorders –8. RecenThe purpose of this research was to evaluate the associations among S100A4, S1P, and pulmonary fibrosis in COPD patients based on a case-control study. The current research found that S100A4 and S1P were increased in lung tissues and serum samples of COPD patients. The levels of S100A4 and S1P were inversely correlated with pulmonary function in COPD patients. These results have demonstrated that S1P is positively correlated with S100A4 and pulmonary function decline in COPD patients.β-actin (8H10D10) and S100A4 (ab197896) were purchased from Abcam . Antibodies against Twist (10E4E6) and α-SMA (ab119952) were obtained from Cell Signaling Technology . ECL detection kits were bought from Advansta .The primary antibodies against All 139 COPD patients were randomly enrolled from the Anhui COPD Cohort (AHCC) study which was a hospital-based prospective cohort study –24. COPDRIPA buffer and PMSF were added to lung tissue to prepare the lung homogenate. All protein samples were separated on a 15% SDS-PAGE gel, and then the samples were transferred to the PVDF membrane. The membrane was washed three times with phosphate-buffered saline after being blocked in the 5% skim milk for 1.5 h at room temperature. The membrane was incubated with different primary antibodies overnight at 4°C in the refrigerator overnight. After being washed three times with phosphate-buffered saline, the secondary antibodies whose source derived from the primary antibodies were continued to incubate for 2 h at 37°C. An ECL kit was used for chemiluminescence detection as previously described , 30. Denhttps://www.cusabio.com/). The levels of S100A4 and S1P were measured using ELISA according to the manufacturer's protocol [S1P ELISA kits were purchased from Wuhan Colorful Gene Biological Technology Co., Ltd. S100A4 ELISA kits were bought from Cusabio, Wuhan, China  and Twist (1 : 300) for 36 h at 4°C and 2 h at room temperature. Then, the pulmonary slides were washed in phosphate-buffered saline three times. Next, the pulmonary slides were incubated with the secondary antibodies for 45 min at room temperature. Pulmonary slides were counterstained with PBS after being stained with DAB and hematoxylin. Finally, α-SMA-positive cells and Twist-positive nuclei were observed and calculated under a light microscope after dehydration and sealing [After lung tissue dewaxing, hydration, antigen retrieval, and serum blocking, slides were incubated with primary antibodies  sealing , 34.The paraffin sections were dip-stained in a mordant solution and then stained with lapis lazuli blue and Mayer's hematoxylin. Pulmonary slides were stained with ponceau magenta dye after being differentiated with acid alcohol differentiation solution. Slides were washed with tap water and distilled water in turn and then treated with phosphomolybdic acid solution and aniline blue. Finally, the slides were rinsed, dehydrated, and observed .t-test or the Mann-Whitney U test between two groups. All categorical variables were compared using the chi-squared test between two groups. Correlation analysis was accessed through linear regression among COPD patients. P < 0.05 was considered statistically significant.Statistical analysis was carried out using SPSS version 19.0 software . All continuous variables were expressed with mean and median. All categorical variables were shown using frequencies and percentages. The difference of continuous variables was evaluated via the Demographic data and clinical characteristics are presented in The protein expression of S100A4 was detected in lung tissues of COPD patients and control cases. As shown in Figures R2 = −0.405, P < 0.01). Moreover, forced vital capacity (FVC) was inversely correlated with serum S100A4 in COPD patients   and FEV1%  in COPD patients , FEV1/FVC% , FEV1 , and FVC  in COPD patients. In order to control confounding factors, age, gender, and smoking were adjusted. Multivariable linear regression found that serum S100A4 was inversely associated with FEV1% , FEV1/FVC% , FEV1 , and FVC  in COPD patients  . Besides Figures . Moreovepatients .α-smooth muscle actin (α-SMA), the hallmark of mature myofibroblasts, was measured through IHC between COPD patients and control cases. As shown in Figures α-SMA-positive cells were obviously elevated in lung tissues of COPD patients. In addition, pulmonary Twist-positive nuclei, one of the EMT transcription factors, were higher in COPD patients than in control subjects , FVC , FEV1/FVC% , and FEV1%  in COPD patients.Serum S1P was detected with ELISA in COPD patients and control subjects. As shown in R2 = 0.228, P = 0.034)  and COPD patients    . Then, t~14.518) . After a~12.365) .In the present study, the levels of S100A4, S1P, and related markers of pulmonary fibrosis were detected between COPD patients and control subjects. The correlations among S100A4, S1P, and pulmonary function were analyzed in COPD patients. The present study primarily revealed the following. (1) Pulmonary S100A4 and serum S100A4 were increased in patients with COPD. (2) Serum S1P was elevated in patients with COPD. (3) The level of pulmonary fibrosis was increased in patients with COPD. (4) Serum S100A4 was positively associated with S1P and inversely associated with pulmonary function in patients with COPD.The high incidence of COPD seriously affects the life quality of patients and brings a heavy burden to society . SmokingSeveral studies from our team and other researchers have demonstrated that EMT is involved in the process of COPD , 28. Theα-SMA expression and collagen deposition and activates pulmonary fibroblasts through S1P [S1P is a biologically active lipid synthesized intracellularly by sphingosine kinase and degraded by S1P lyase and various phosphatases. Mainly, S1P is generated in mammalian cells and released by endothelial cells, red blood cells, and monocytes . S1P hasough S1P . Besidesough S1P . In the There are several limitations to this research. First, this was a single-center research with a small sample size, and a multicenter study with a larger sample size will further prove these results. Second, this is only a case-control study, and the causal link between the elevation of S100A4 and S1P and the decline of pulmonary function was unclear. Further animal experiments may clarify this problem. Third, the mechanism of S1P-mediated EMT was unclear in this case-control study, and only experiments in vivo and in vitro can resolve this confusion.In summary, this research primarily evaluated the associations among S100A4, S1P, and pulmonary function in patients with COPD based on a case-control study. Our results revealed that S100A4 is increased in the lungs and serum of COPD patients. Meanwhile, the markers of fibrosis and EMT are elevated in the lungs of COPD patients. Moreover, serum S1P has risen in COPD patients. Correlative analysis revealed that pulmonary function is inversely associated with S1P and S100A4 in COPD patients. Additionally, serum S1P is positively associated with S100A4 in COPD patients. These results provide evidence that the elevation of S100A4 and S1P may be involved in the onset and progression of COPD patients.


MET gene remain understood incompletely. In the present study, Xp11.2 translocation/NONO-TFE3 fusion renal cell carcinoma (NONO-TFE3 tRCC) with up-regulated CircMET was employed to investigate its mechanism in cancer progression and post-transcriptional regulation.Functions of CircMET (hsa_circ_0082002) which is a circular RNA and derived from NONO-TFE3 tRCC tissues and cells. The functions of circMET in tRCC were investigated by proliferation analysis, EdU staining, colony and sphere formation assay. The regulatory mechanisms among circMET, CDKN2A and SMAD3 were investigated by luciferase assay, RNA immunoprecipitation, RNA pulldown and targeted RNA demethylation system.FISH and real-time PCR were performed to explore the expression and localization circMET in NONO-TFE3 tRCC tissues and cells, and overexpression of circMET significantly promoted the growth of NONO-TFE3 tRCC. Mechanistic studies revealed that circMET was delivered to cytosol by YTHDC1 in N6-methyladenosine (m6A)-depend manner. CircMET enhances mRNA decay of CDKN2A by direct interaction and recruitment of YTHDF2. Meanwhile, circMET competitively absorbed miR-1197 and prevented those from SMAD3 mRNA.The expression of circMET was upregulated by NONO-TFE3 fusion in NONO-TFE3 tRCC, and the regulation to both CDKN2A and SMAD3 of circMET was revealed. CircMET has the potential to serve as a novel target for the molecular therapy of NONO-TFE3 tRCC as well as the other cancer with high-expressing circMET.CircMET promotes the development of The online version contains supplementary material available at 10.1186/s12943-022-01497-w. TFE3) fusion gene. Some strictly expressed genes, including ASPL, PRCC, NONO and CLTC, fused to TFE3 gene /2).The 6-week-old BALB/c nude mice were chosen for xenografts experiments and maintained in specific pathogen-free conditions. All procedures were approved by the Animal Care and Use Committee of Nanjing University under the animal protocol number SYXK (Su) 2009–0017. The mice were subcutaneously inoculated with 786-O cells stably transfected with lentiviruses carrying sh-NC/sh-circMET, respectively  were used to assess the significance of differences. P < 0.05 was considered statistically significance . All values are expressed as the means ± standard deviation.Statistical analyses were performed using SPSS 22.0 software . GraphPad Prism 8.0  was applied to plot the data. Student’s MET was identified as a potential target gene of NONO-TFE3 by ChIP-seq. To explore if the expression of circRNAs derived from MET gene is correlated with NONO-TFE3 tRCC, real-time PCR was performed to determine the expression of them. The results showed high level expression of circMET in NONO-TFE3 Fig. In our previous study, To verify that circMET is produced by the head to tail splicing, the convergent and divergent primers were used to amplify circMET and linear MET, respectively. Real-time PCR revealed that circMET could only be detected in cDNA but not in gDNA Fig. B, C, wheNONO-TFE3 tRCC progression, we first evaluated circMET expression levels in various RCC cell lines and normal cell. The lowest expression of circMET was observed in HEK293T cell line, whereas the highest level was detected in UOK109 cell line which was derived from tumor tissue of patient with NONO-TFE3 tRCC , pGL3- METpro2 (− 1000 ~  + 48) and pGL3- METpro3 (− 400 ~  + 48), were cloned into luciferase reporter plasmid to identify the binding sites of NONO-TFE3 fusion. Dual-luciferase reporter assays revealed that NONO-TFE3 fusion could bind to the region of − 400 ~  + 48  [6A reader proteins, YTHDC1 is known to be involved in RNA splicing [https://whistle-epitranscriptome.com/) [http://m6avar.renlab.org/) [6A modification site in circMET  , 27 to psplicing  and RNA splicing . Accordime.com/) and m6AVaab.org/) , we founMET Fig. C. The reMET Fig. D. MoreovRIP Fig. D. The reion Fig. D, E. To MET Fig. F. Besideays Fig. G-I.6A-binding defective YTHDC1 (YTHDC1N367D) [A270/831T was detained in the nucleus  fused to dCas13 was used to remove the m6A modification on circMET, and nuclear localization signal (NLS) was added to this fusion protein for the nuclear localization of dCas13-NLS-ALKBH5 fusions [To verify that circMET was exported to cytosolic in the YTHDC1-depend manner, nuclear/cytosol fractions were isolated prior to RNA extraction. YTHDC1 knockdown caused the retention of circMET in nucleus Fig. E, F, andC1N367D) , rescuedC1N367D) , and theeus Fig. J, K. To  fusions . Cytopla6A modification of circMET in NONO-TFE3 tRCC, CCK-8, colony formation and tumor sphere formation assays were performed  [AAAAAA, Fig. Since it has been established that circMET accelerates proliferation of NONO-TFE3 tRCC, we want to know whether circMET can regulate the expression of genes associated with cell cycle. AREsite analysis (REsite2)  and BLASREsite2) –37. AddiThen, the validation of real-time PCR showed that the expression of CDKN2A were increased in UOK109 cells upon circMET knockdown Fig. D. Simila. 6A modification could enhance the stability of CDKN2A mRNA  decreased the protein and mRNA level of CDKN2A in UOK109 cells transfected with gRNA-circMET, and YTHDF11–350 (ΔYTHDF1) increased the protein level of CDKN2A  [6A modification of circMET located in the cytosol enhanced the stability of CDKN2A mRNA  , cannot RNA Fig. G, H, ind it Fig. I-L.NONO-TFE3 tRCC by recognizing circMET and facilitating the CDKN2A mRNA decay.CCK-8, colony formation and tumor sphere formation assays were performed to illuminate the impact of YTHDF2 to the behavior of UOK109 cells. The results revealed that cell proliferation and growth were inhibited by transfection with ΔYTHDF2 and gRNA-CDKN2A compared with the negative control in UOK109 cells Fig. N-P. CollNONO-TFE3 tRCC. Then, we predicted potential miRNA targeting sites on circMET using ENCORI and Circular RNA Interactome (https://circinteractome.nia.nih.gov/) [Numerous studies have confirmed that circRNAs exert critical roles in regulating gene expression by function as a biological sponge for miRNAs. The intracellular distribution of circMET suggested that circMET might also have a post-transcriptional regulation function that contributes to the proliferation of ih.gov/)  databaseih.gov/) , while mDual-luciferase assay was processed to confirm the interaction between miR-1197 and circMET. Luciferase activities were repressed by co-transfection with miR-1197 compared with the control  [Interestingly, through miRWalk, ENCORI and TargetScan (an.org/)  analysisNONO-TFE3 tRCC.Overexpression or downregulation of miR-1197 caused a significant decrease or increase in SMAD3 expression at mRNA and protein levels Fig. H-K. AddiNONO-TFE3 tRCC. Importantly, circMET regulated the expression of CDKN2A, a critical tumor suppressor gene, and SMAD3 which could maintain the stem cell properties of cancer stem-like cells [NONO-TFE3 tRCC, which might provide novel insights for better tailoring treatment and identification of new therapeutic targets, such as targeted therapy using small RNA and detection kit based on CRISPR/Cas12. Due to the small sample size of NONO-TFE3 tRCC in the validation cohort, we were limited to firmly establish the link between circMET upregulation and prognosis. Moreover, we propose to continuously carry out a prospective cohort study to detailly explore the clinical outcome with the expression of circMET, which helps the development of NONO-TFE3 tRCC diagnosis and treatment. Otherwise, we predicted the possibility of encoding protein of circMET, the possibility of encoding protein is relatively low  mRNA and then inhibit the decay of HMGA2 mRNA, and the stability of HMGA2 mRNA was enhanced via Insulin-Like Growth Factor 2 mRNA-Binding Protein 2 (IGF2BP2) . Besides6A modification is the most widespread base modification in mRNA, which has a broad biological function, while any perturbation in m6A levels can result in malfunction or diseases. An increasing number of studies have confirmed that m6A writers , erasers (FTO and ALKBH5) and readers  have distinct functions within different types of cancer cells [6A modifications may play important roles in RNA production, stability, subcellular localization and interactions in cancers [6A reader proteins reflect their divergent biological functions. YTHDC1 facilitates mRNA binding to both the canonical export receptor NXF1 and splicing factors SRSF3 mediating export and metabolism of m6A-modified mRNAs [6A-modified circRNA [6A labeling promoted the cytoplasmic localization of circMET through recruiting YTHDC1. Surprisingly, the m6A modification on circMET could even recruit the YTHDF2 and lead to the decay of CDKN2A mRNA interacted with circMET.Methyltransferase 3 (METTL3)-mediated mer cells , 47. M6A circRNA . Here, iThere has been substantial interest in the ceRNA network mechanism in recent years, with many studies in the area revolving around how dysregulation of ceRNA, including long non-coding RNAs (lncRNAs), circRNA, mRNA and miRNA, can influence tumor initiation and progression. In this study, we found that circMET could function as a biological sponge and modulate the expression of SMAD3 through post-transcriptional regulation of miR-1197. Increasing studies revealed the important functions of ceRNA network in tumor proliferation, antiapoptosis, metastasis and other tumor development processes. For example, circ3823 contributes to growth, metastasis and angiogenesis of colorectal cancer by absorbing miR-30c-5p and mediating TCF7 . In addiNONO-TFE3 tRCC. YTHDC1 promotes cytoplasmic export of circMET via binding to m6A modification. CircMET markedly weakened CDKN2A expression by binding to its mRNA directly to recruit YTHDF2 which led to mRNA decay of CDKN2A, and enhanced the expression of SMAD3 through absorbing miR-1197  and 786-O (B) after transfected with indicated lentivirus. Figure S3. ChIP assay was performed after UOK109 cells transfected indicated lentivirus. Figure S4. The transcription of MET gene is enhanced by NONO-TFE3. Figure S5. The m6A methylation is involved in the export of circMET from the nucleus. Figure S6. The function of m6A modification on exon 2 of MET mRNA. Figure S7. The m6A modification on circMET promotes tumor proliferation. Figure S8. The stability of GAPDH mRNA in cells transfected with indicated lentivirus after treatment with α-amanitin. Figure S9. CircMET mediates NONO-TFE3 tRCC proliferation through CDKN2A. Figure S10. CircMET mediates the RNA level of CDKN2A through YTHDF2. Figure S11. CircMET recruits YTHDF2 to CDKN2A mRNA via m6A methylation. Figure S12. The function of potential peptide encoded by circMET. Figure S13. Relationship between circMET and CDKN2A and SMAD3 mRNA in NONO-TFE3 tRCC (A) and ccRCC (B). Figure S14. The function of m6A modification on exon 2 of MET mRNA. Figure S15. Relationship between circMET and AKT1, CCNB1 and CCND1 mRNA. Table S1. Primers used for real-time PCR. Table S2. Primers used for ChIP assay and MeRIP. Table S3. Probes used for RNA FISH. Table S4. ShRNA used for silencing target genes. Table S5. Guide RNA used for dCas9-ChIP system and targeted RNA methylation system. Table S6. Primary antibodies used in this study.


Neuropilin-1(NRP1) is a cofactor that enhances SARS-CoV-2 coronavirus cell infectivity when co-expressed with angiotensin-converting enzyme 2(ACE2). The Renin-Angiotensin System (RAS) is activated in type 2 diabetes (T2D); therefore, the aim of this study was to determine if hypoglycaemia-induced stress in T2D would potentiate serum NRP1(sNRP1) levels, reflecting an increased risk for SARS-CoV-2 infection.A case-control study of aged-matched T2D (n = 23) and control (n = 23) subjects who underwent a hyperinsulinemic clamp over 1-hour to hypoglycemia(<40mg/dl) with subsequent timecourse of 4-hours and 24-hours. Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement determined RAS-related proteins: renin (REN), angiotensinogen (AGT), ACE2, soluble NRP1(sNRP1), NRP1 ligands  and NRP1 proteolytic enzyme .Baseline RAS overactivity was present with REN elevated and AGT decreased in T2D (p<0.05); ACE2 was unchanged. Baseline sNRP1, VEGF and ADAM9 did not differ between T2D and controls and remained unchanged in response to hypoglycaemia. However, 4-hours post-hypoglycemia, sNRP1, VEGF and ADAM9 were elevated in T2D(p<0.05). SEMA3A was not different at baseline; at hypoglycemia, SEMA3A decreased in controls only. Post-hypoglycemia, SEMA3A levels were higher in T2D versus controls. sNRP1 did not correlate with ACE2, REN or AGT. T2D subjects stratified according to ACE inhibitor (ACEi) therapies showed no difference in sNRP1 levels at either glucose normalization or hypoglycaemia.Hypoglycemia potentiated both plasma sNRP1 level elevation and its ligands VEGF and SEMA3A, likely through an ADAM9-mediated mechanism that was not associated with RAS overactivity or ACEi therapy; however, whether this is protective or promotes increased risk for SARS-CoV-2 infection in T2D is unclear.https://clinicaltrials.gov, identifier NCT03102801. The gen ADAM10) . NRP1 in ADAM10) , contrib ADAM10) .T2D is associated with high risk for acquiring SARS-Cov-2 infection, severe disease, acute respiratory distress syndrome and increased mortality . DiabetiHere, we hypothesized that underlying RAS activation may potentiate the levels of sNRP1 and its ligands (VEGF and SEMA3A) in hypoglycaemia-induced stress in type 2 diabetes (T2D), reflecting an increased risk for SARS-CoV-2 infection.www.clinicaltrials.gov (NCT03102801) and conducted according to the Declaration of Helsinki.This prospective parallel study was performed in 46 subjects, adult T2D (n=23) and control (n=23). The duration of diabetes was <10 years and all T2D subjects were on a stable dose of medication  over the prior 3 months. For those with T2D, no medications for glycemic control except metformin were allowed. The study was performed at the Diabetes Centre at Hull Royal Infirmary. All participants provided written informed consent. The trial was approved by the North West-Greater Manchester East Research Ethics Committee (REC number:16/NW/0518), registered at The method for performing the insulin clamp has been published previously . A schemVenous blood samples collected during the screening visit were analysed for serum insulin, total cholesterol, triglycerides, HDL cholesterol, C-reactive protein (CRP) and glycated haemoglobin (HbA1C).Blood samples were separated immediately by centrifugation at 3500g for 15 minutes at 4°C, and the aliquots were stored at –80°C, within 30-minutes of blood collection, until batch analysis. Serum insulin was assayed using a competitive chemiluminescent immunoassay performed on the manufacturer’s DPC Immulite 2000 analyser , with a coefficient of variation of 6 and no stated cross-reactivity with proinsulin. Fasting plasma glucose (FPG), total serum cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol levels were measured enzymatically using a Beckman AU 5800 analyser . LDL cholesterol was calculated using the Friedewald equation. Plasma whole blood samples were analysed for HbA1C on a Menarini Diagnostics HB9210 premier .The SOMAscan assay used to quantify proteins was performed on an in-house Tecan Freedom EVO liquid handling system  utilizing buffers and SOMAmers from the SOMAscan HTS Assay 1.3K plasma kit  according to manufacturer’s instructions and as described previously –17. Init2 RFU values using R version 3.5.2  including base R package. Data handling and differential protein expression were analyzed using the autonomics and limma  and control (n=23) subjects were matched for age (p=ns); T2D had higher BMI (p=0.0012); duration of disease was 4.5 ± 2.9 years Table 1.vs 2279 ± 488 RFU, T2D vs control, p=ns); normalization of glucose in T2D did not alter sNRP1   (Baseline levels of sNRP1 did not differ between T2D and controls (2298 ± 385  p=0.04) Figure 3vs 1255 ± 81 RFU of ADAM9, T2D vs control, p=ns) but were decreased following hypoglycaemia in T2D (1h and 2h post-hypoglycemia)  or baseline   has been reported to be involved in proteolysis of NRP1 in human endothelial cells and that this process was stimulated by vascular endothelial growth factor (VEGF) . Therefovs 9113 ± 212 RFU of VEGF, T2D vs control, p=ns; 970 ± 43 vs 935 ± 45 RFU of SEMA3A, T2D vs control, p=ns). Normalization of hyperglycemia did not alter plasma VEGF or SEMA3A levels in T2D . While insulin-induced hypoglycemia did not alter plasma VEGF levels in either control or T2D subjects   (Soluble isoforms of NRP1 (sNRP1) also exist without transmembrane or cytoplasmic domains, expressing only the extracellular domains which allow them to bind NRP1 ligands ; therefoTo determine whether hyperglycemia affects the levels of RAS or sNRP1 associated proteins, we performed correlation analysis between the change in blood glucose from hyperglycemia (baseline) to normoglycemia with basal levels of plasma RAS-related proteins  , NRP1 proteolytic enzyme ADAM9 and NRP1 ligands, VEGF and SEMA3A, in response to insulin-induced hypoglycemia in obese patients with T2D. Our data demonstrate that soluble sNRP1, VEGF or SEMA3A and ADAM9 levels did not differ from control subjects in the basal condition in obese T2D; however, an alteration of their levels was observed in response to insulin-induced hypoglycemia. An elevation of sNRP1 levels in association with its proteolytic enzyme as well as ligands post-hypoglycemia suggests the possible connection of glycemic control with NRP1 cleavage in obese T2D subjects.165, and enhances its activity in functions such as endothelial cell migration , which are negative mediators of neuronal guidance , 22. NRPigration , 24. NRPigration . In addiigration  and thisd SEMA3A . Therefod SEMA3A ; therefoThere is evidence to suggest that increased sNRP1, seen here following insulin-induced transient hypoglycaemia but also as a consequence of strict glycemic control, might offer protection against COVID-19 disease severity. Optimizing glycemic control during hospitalization has been associated with a reduction in the risk of severe disease and death in patients with COVID-19 . MoreoveThe mechanism increasing sNRP1 levels in obese T2D in response to hypoglycemia is unclear; however, post-hypoglycemic ADAM9 levels were also higher at similar timepoints as the elevated sNRP1 levels in T2D cases. ADAM9 is a disintegrin and metalloproteinase involved in a wide array of cellular processes, especially those involving cell to cell interactions, adhesion, cell-matrix interactions, growth factor and cytokine signalling –32. Membvia NRP1. Serum SEMA3A levels have also correlated with the phenotypes of diabetic retinopathy (Our data also demonstrate post-hypoglycemic elevation of plasma SEMA3A in T2D. SEMA3A levels have been reported to be significantly elevated in the vitreous fluid of patients with diabetic macular edema  and prolinopathy . Since sinopathy , it is lThis study further showed that, whilst plasma renin was elevated, and angiotensinogen suppressed in T2D , that thWhilst hyperglycemia may lead to more severe COVID-19 disease, the risk of hypoglycemia has also been shown to be increased during the COVID-19 pandemic, especially for those patients on the hypoglycemic agents sulphonylureas and insulin  and, parStudy strengths include that these T2D subjects had a short disease duration and were relatively medication naïve. Study limitations include the small population under study and that measurement of circulating sNRP1, ADAM9, VEGF and SEMA3A may not reflect tissue levels; also, correlation of sNRP1 was with renin proteins rather than renin activity. In addition, the SOMAscan assay is designed as a discovery platform and measures relative protein concentrations using only external controls. Without internal controls and standard curves, it remains unclear which measurements are within the linear dynamic range ; therefovia a mechanism involving ADAM9-mediated proteolytic cleavage of NRP1 that may continue to enhance sNRP1 levels following the hypoglycemic episode, independent of RAS activation or ACEi therapy. This post-hypoglycemia elevation of plasma sNRP1 may place patients with pre-existing obesity and T2D at increased risk for severe COVID19 disease; however, studies to clarify the role of sNRP1 in COVID19 patients are needed to determine if it promotes risk or affords protection for SARS-CoV-2 infection in T2D.In conclusion, this data shows that strict blood glucose control associated with increased hypoglycemia potentiates sNRP1 levels The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by North West-Greater Manchester East Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.AM and AB analyzed the data and wrote the manuscript. AA-Q contributed to study design, performed experiments, collected, analyzed, and interpreted data and edited the manuscript. TS supervised clinical studies and edited the manuscript. SA contributed to study design, data interpretation and the writing of the manuscript. AB is the guarantor of this work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.