The objective of this study was to explore the clinical utility of the implementation of expanded carrier screening (ECS) in Chinese population of childbearing age.Based on capillary electrophoresis, a first-generation sequencing technology, a prospective screening study of carriers of 15 single-gene diseases was carried out in 327 subjects in Anhui Province, including 84 couples and 159 women of childbearing age, the disease carrier rate, types of screened pathogenic genes, and incidence of both partners carrying the same pathogenic genes were summarized and analyzed.In 320 people with normal phenotypes who underwent ECS for 15 genetic diseases and 7 spouses who underwent targeted gene sequencing, 65 carriers of at least one disease were detected, with a total carrier rate of 20.31% (65/320). Among the 65 carriers, 81.54% (53/65) carried one genetic variant, 16.92% (11/65) carried two genetic variants, and 1.54% (1/65) carried three genetic variants. In this study, the three diseases with the highest carrier rates were hereditary deafness , Wilson's disease , and phenylketonuria . One high-risk couple  was detected.It has certain clinical application value to implement ECS in the population of childbearing age in China. Single-gene disease is one of the major causes of malformation and disability in children. Although the incidence rates of single-gene diseases are relatively low, the overall incidence rate is more than 1%, and there are no effective drugs or treatment programs for most single-gene diseases. It is possible to reduce the number of children born with birth defects by conducting expanded carrier screening (ECS) before pregnancy or in early pregnancy, screening for autosomal recessive or X-linked genetic disease-related genes in the population with normal phenotypes, and providing genetic counseling and fertility guidance to these individuals [Initial single-gene disease carrier screening programs were carried out for specific populations, starting with the Tay‒Sachs Disease (TSD) carrier screening project launched in the 1970s . TSD is Currently, China's research on population carrier screening is in its infancy, and there is no relevant guidance or expert consensus. At present, carrier screening work in China is mainly focused on single diseases , 12. OccFrom January 2020 to November 2020, 327 subjects with normal phenotypes were recruited from the Maternity and Child Health Hospital of Anhui Province. Before examination, all subjects received professional genetic counseling regarding the scope, purpose, significance, limitations, and residual risks of ECS for the target disease; the subjects were fully informed and provided informed consent. This study included 320 patients undergoing ECS for 15 genetic diseases and 7 patients undergoing targeted gene sequencing, including 84 couples and 159 women of childbearing age. Among these patients, 84 were male  and 243 were female , with an average age of 31.43 years (21–49 years). This study was approved by the Ethics Committee of Anhui Maternal and Child Health Hospital. All subjects voluntarily underwent ECS and signed informed consent forms.In this study, ECS was conducted for 410 pathogenic variants in 21 genes associated with 15 diseases. The selection of these diseases followed the screening criteria recommended by the American College of Obstetricians and Gynecologists (ACOG) , 15. TheThis study mainly relied on the gold standard technology for clinical gene analysis, capillary electrophoresis, which is a first-generation sequencing technology. A total of 2 ml of peripheral blood was collected from the subjects; DNA was extracted with the DNA extraction kit produced by Beijing Tiangen Biochemical Technology, and then probe connection, multiple PCR, and gene sequencing were conducted. The ABI3730 and ABI3500 first-generation sequencing platforms were used for the precise sequencing and analysis of multiple mutation types  in the target disease genes. The detection technologies included single-nucleotide polymorphism (SNP) scan technology, copy number variation (CNV) plex technology, AQ-PLP technology, iMLDR technology, special fluorescent PCR, gap-PCR, etc., and the results of all detected gene mutations were verified.This study included two screening modes. The first mode was simultaneous screening, in which peripheral blood was collected from 67 couples at the same time for ECS of 15 single-gene diseases. The second mode was sequential screening. In this study, 176 women underwent ECS. Among 176 women, 38  were determined to be carriers. After outpatient genetic counseling, it was recommended that the spouses of these positive carriers be recalled for corresponding genetic testing. At present, 17  spouses have undergone follow-up genetic testing, of whom 10 have chosen to undergo ECS, and the other 7 have chosen to have the target gene sequenced only for the gene mutation carried by the woman.The data collected in this study were input into the computer and analyzed with SPSS 23.0 statistical software. This study included 320 patients undergoing ECS for 15 genetic diseases and 7 patients undergoing targeted gene sequencing. We mainly conducted descriptive statistical analysis on 320 subjects receiving ECS, calculated the total carrier rate of the target diseases and the carrier rate of pathogenic genes in the region, and conducted descriptive statistical analysis on the types of pathogenic genetic variants detected in this study.When a subject was determined to be a carrier of a target disease, their spouse was recalled for appropriate genetic testing. If a pregnant was a carrier of an X-linked genetic disease, the risk of her offspring being a male patient or a female carrier was discussed. If necessary, prenatal diagnosis was provided with full informed consent and at the request of high-risk couples to determine whether the fetus had the disease. In addition, for positive carriers, it was recommended that members of their immediate family who are of suitable age for pregnancy also be screened. In addition, follow-up was conducted for high-risk couples with subsequent pregnancy outcomes and their neonates, including physical examinations, hearing screening, and screening for any other clinical or genetic abnormalities. In the case of abortion, stillbirth or neonatal malformation during pregnancy, fetal tissue or neonatal peripheral blood was collected for detection whenever possible.Among the 320 subjects who underwent ECS, 65 were identified as carriers of the target diseases, with an overall carrier rate of 20.31% (65/320). Among the 65 carriers, 53  carried one genetic variant, 11  carried two genetic variants, and 1  carried three genetic variants, as shown in Fig. All genetic variants detected in the study were validated by other methods, such as Sanger sequencing, multiplex ligation-dependent probe amplification, gap-PCR, etc. The results showed that the mutation sites detected in the study were 100% consistent with the verification results.In this study, a total of 12 subjects were determined to have pathogenic genes for target diseases, of whom 8 had a carrier frequency of more than 1%. The carrier frequencies were as follows: 4.38% (14/320) for the GJB2 gene for hereditary deafness; 3.75% (12/320) for the SLC26A4 gene for hereditary deafness; 4.06% (13/320) for the ATP7B gene for Wilson’s disease; 2.81% (9/320) for the CYP21A2 gene for congenital adrenal hyperplasia; 2.50% (8/320) for the SMN1 gene for spinal muscular atrophy; 2.50% (8/320) for the PAH gene for phenylketonuria; 1.56% (5/320) for the HBA1/HBA2 gene for alpha-thalassemia; 1.25% (4/320) for the MMACHC gene for methylmalonic acidemia; and the remaining four carrier frequencies were less than 1%  of all detected variants  was found to be at high risk of having a child with autosomal recessive deafness. In this couple, the woman was first screened as an extensibility carrier for 15 genetic diseases, and the results showed that she was a carrier of the pathogenic genes ATP7B and GJB2 for Wilson’s disease and hereditary deafness, respectively. After genetic counseling, her spouse chose to undergo sequencing for the target genes (GJB2 and ATP7B), namely, GJB2: c.109G > A , was detected, which usually leads to autosomal recessive hereditary deafness. C.109G > A has incomplete dominance. According to the literature , 17, GJBSingle-gene recessive diseases include autosomal recessive and X-linked recessive genetic diseases, which usually have no family genetic history and cannot be detected in routine prenatal examination; these diseases are discovered after birth when symptoms slowly appear. Recessive genetic diseases easily cause blindness, deafness, body deformities, intellectual disability, growth retardation, the dysfunction or failure of tissues and organs, and even serious harm, such as death, which brings a heavy burden to families and society. In 2011, for the first time, Bell et al.  used nexThe total carrier rate of pathogenic gene variations in the population screened was 20.31% in this study, indicating the need for carrier screening. Lazarin et al.  performeWith the development of sequencing technology, next-generation sequencing (NGS) has been widely used in carrier screening for single-gene diseases with the advantages of high throughput and low cost . HoweverHowever, this study also has some limitations. First, a limited number of definite pathogenic sites were included, and pathogenic mutations and emerging variants outside the detection range could not be identified, which may increase residual risks. The pretest and posttest consultations to inform the subjects of the benefits and limitations of this study needs to be emphasized. Second, we observed 8 out of the 15 genetic diseases in the study population, which may be related to the small sample size and the types of diseases screened. We will include more subjects for screening in the future. Third, due to time constraints and insufficient follow-up, the follow-up of pregnancy outcomes and newborns will be carried out in the future. Finally, the results of this study were not evaluated by NGS. In the future, we will continue to conduct in-depth research to compare the results of ECS in this study with those of NGS and explore the consistency of the two methods in estimating overlapping genetic variants.In conclusion, this study provides an inexpensive and effective method for ECS, and the implementation of expanded carrier screening in China has certain clinical application value and is an important measure to prevent birth defects.


Osteoarthritis (OA) progression involves multiple factors, including cartilage erosion as the basic pathological mechanism of degeneration, and is closely related to chondrocyte apoptosis. To analyze the correlation between apoptosis and OA development, we selected apoptosis genes from the differentially expressed genes (DEGs) between OA and normal samples from the Gene Expression Omnibus (GEO) database, used lasso regression analysis to identify characteristic genes, and performed consensus cluster analysis to further explore the pathogenesis of this disease.The Gene expression profile datasets of OA samples, GSE12021 and GSE55235, were downloaded from GEO. The datasets were combined and analyzed for DEGs. Apoptosis-related genes (ARGs) were collected from the GeneCards database and intersected with DEGs for apoptosis-related DEGs (ARDEGs). Least absolute shrinkage and selection operator (LASSO) regression analysis was performed to obtain characteristic genes, and a nomogram was constructed based on these genes. A consensus cluster analysis was performed to divide the patients into clusters. The immune characteristics, functional enrichment, and immune infiltration statuses of the clusters were compared. In addition, a protein–protein interaction network of mRNA drugs, mRNA-transcription factors (TFs), and mRNA-miRNAs was constructed.GDF15), NAMPT, TLR7, CXCL2, KLF2, REV3L, KLF9, THBD, and MTHFD2. Clusters A and B were identified, and neutrophil activation and neutrophil activation involved in the immune response were highly enriched in Cluster B, whereas protein repair and purine salvage signal pathways were enriched in Cluster A. The number of activated natural killer cells in Cluster B was significantly higher than that in Cluster A. GDF15 and KLF9 interacted with 193 and 32 TFs, respectively, and CXCL2 and REV3L interacted with 48 and 82 miRNAs, respectively.A total of 95 DEGs were identified, of which 47 were upregulated and 48 were downregulated, and 31 hub genes were selected as ARDEGs. LASSO regression analysis revealed nine characteristic genes: growth differentiation factor 15 (ARGs could predict the occurrence of OA and may be related to different degrees of OA progression. It is characterized by joint pain, stiffness, hypertrophy, and limited activity. It is most common in weight-bearing joints, such as the knee, hip, cervical, and lumbar spine, as well as the proximal and distal finger joints . It is tCurrently, most researchers believe that OA is the result of a combination of mechanical and biological factors leading to an imbalance in the degradation and anabolism of articular chondrocytes, extracellular matrix (ECM), and subchondral bone . ApoptosTo comprehensively understand the role of apoptosis and immune mechanisms in the occurrence and development of OA, we analyzed transcriptome data from the GEO database, selected characteristic apoptosis-related genes (ARGs) that could predict OA occurrence, and attempted to cluster patients with OA based on these genes. We analyzed the immune characteristics, biological pathways, and immune infiltration between different clusters and searched for drugs and molecules that inhibit OA occurrence.22.1Homo sapiens and the two dataset sequencing platforms were GPL96 and GPL97. The data set GSE12021 contains 33 samples in total, including 13 normal samples and 20 samples from patients with OA; the data set GSE55235 contains 20 samples in total, including 10 normal samples and 10 samples from patients with OA. We integrated the above sets of data expression into one data set, which was named as the combined data or combined gene expression. Then, the R package “sva”    is an R/2.8https://www.dgidb.org)   with ARD2.9MiRNAs  are a cl2.10All data were processed and analyzed using the R software (version 4.1.1). To compare two continuous variables that were distributed normally, an independent Student’s t-test was conducted. The Mann–Whitney U test was performed to determine the difference between non-normally distributed variables. Pearson’s correlation analysis was used to calculate the correlation coefficient (r) between the different genes, the Pearson’s correlation coefficient between two variables is defined as the quotient of the covariance and standard deviation between two variables:P values of all statistical processes were bilateral, with P values< 0.05 considered significant.The above equation defines the overall correlation coefficient, and the Greek lowercase letters are commonly used as representative symbols. To estimate the covariance and standard deviation of the sample, the Pearson correlation coefficient (r). 33.1The workflow is depicted in 3.2In this study, the two data sets GSE12021 and GSE55235 were combined, and the batch effect between the data sets was removed Figure 23.3GDF15), NAMPT, TLR7, CXCL2, KLF2, REV3L, KLF9, THBD, and MTHFD2, which occurred 413 times in 1,000 cycles , also CXCL2 and THBD   were identified using the consistency clustering method  cells in ClusterB was significantly higher than that in ClusterA. The M2 macrophage content in patients in ClusterA was significantly higher than that in ClusterB , whereas the gene expression of KLF9 was negatively correlated with activated NK cells  , whereas the gene expression of KLF9 was positively correlated with M2 macrophages  . Nevertheless, the contents of some immune cells in ClusterA were lower than those in ClusterB, such as regulatory T cell, the natural killer cell, the gamma delta T cell, Type 2 T helper cell, the neutrophil, and nature killer T cell . At this stage, the patient’s immune response is dominated by adaptive immunity, manifested by the disappearance of the ECM immune barrier and the release of specific surface antigens from chondrocytes, which stimulate the corresponding T cells in the lymphocyte pool of the immune system, triggering the process of cell-mediated immunity. In this process, T cells release lymphatic factors to clear antigens, causing damage to the cartilage cells and initiating BPs, such as protein repair and purine rescue. However, the high level of NK cells is related to the clearance of inflammatory factors, and the high concentration of neutrophil-related pathways in cluster A suggests that the patients have progressed to the stage of inflammatory circulation, and its immune response is dominated by innate immunity, with an increase in the level of NK cells as a significant feature.CXCL2, GDF15, KLF2, and MTHFD2 genes were positively correlated with the number of activated NK cells and negatively correlated with the number of M2 macrophages; however, the reverse was true for KLF9. Therefore, we speculate that these five genes are involved in regulating the immune response. Evidence for this can be found in various literature. CXCL2 is a member of the chemokine superfamily that encodes secreted proteins involved in immune regulation and inflammatory processes. This chemokine is a member of the CXC subfamily, expressed at inflammatory sites, and may inhibit the proliferation of hematopoietic progenitor cells  . which iTreg cells have been found to participate in changes in subchondral bone remodeling, an important process in the pathogenesis of OA. Treg cells secret various of cytokines and further promote osteoclast maturation . HoweverT helper (Th) cells interact with other immune cells, such as B lymphocytes, through cytokine signaling and participate in immune regulation . B lymphIn drug sensitivity analysis, we compared the differential genes of two modes of cell apoptosis (Cluster A and Cluster B) and attempted to find targeted therapeutic drugs. We found 29 molecular compounds targeting key genes of Cluster A and 38 molecular compounds targeting Cluster B. Although these drugs were rarely used in the clinical treatment of osteoarthritis, they may become potential treatment agents, which remain to be discovered in the future studies.This study has some limitations. First, although there are significant differences in the immune characteristics and functional enrichment between Cluster A and Cluster B, we have not yet found an exact grade of OA to correspond to the clusters; second, our data were from GEO. The total sample size for OA was only 30 cases, which is relatively small, and a data bias may have occurred during the research process. Third, we did not directly identify specific drugs or molecules that regulate the nine characteristic genes that affect OA progression, which should be further explored in future studies.GDF15, NAMPT, TLR7, CXCL2, KLF2, REV3L, KLF9, THBD, and MTHFD2 could independently and accurately predict OA occurrence. Patients with OA can be divided into two clusters with significant differences in immune characteristics, functional enrichment, and immune infiltration, which may be related to different degrees of OA progression. However, more clinical evidence is needed to confirm this. CXCL2, GDF15, KLF2, MTHFD2, and KLF9 are strongly correlated with immune infiltration in patients with OA and could become novel therapeutic targets that affect OA progression.Apoptotic genes play a key role in the development and occurrence of OA. The ARGs https://www.jianguoyun.com/p/DbKXe40Q1ujHCxig64IFIAA.The original contributions presented in the study are publicly available. This data can be found here: EY and JY conceived and designed the research; EY, MZ, GX and XL performed the research, analyzed the data, and prepared figures; JY wrote the paper. Al authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Stentor amethystinus, while the effects of WD on copepods may have been mainly induced by the responses of Calanus sinicus and Paracyclopina nana. By inhibiting Stentor amethystinus and Vorticella lutea, pH significantly affected protozoa. In addition, Rotifera and copepods were affected by SAL mainly through the responses of Brachionus calyciflorus, Calanus sinicus, and Ectocyclops phaleratus. Importantly, fundamental alternations in the variation trends of zooplankton abundance and biomass along the salinity gradient were found when the salinity was approximately 4–5. By combining these results with the findings on phytoplankton responses to salinity in previous studies, it can be concluded that salinity may influence the river ecosystem by influencing zooplankton abundance and biomass rather than phytoplankton.As the primary consumers in aquatic organisms, zooplankton play an important role in aquatic ecosystems. It is valuable for management and researchers to have an insight into the responses of zooplankton community patterns to environmental factors. In this study, RDA and variation partitioning analysis were adopted to determine the important environmental factors affecting zooplankton abundance and biomass, as well as the relative importance of different environmental factors. The findings reveal that TN , WD (water depth), pH, and SAL  were all important abiotic factors shaping the zooplankton community pattern in the study area. TN affected protozoa by influencing  As the primary consumers in aquatic food chains, zooplankton feed on bacteria, debris, and phytoplankton, which can change the community structure of phytoplankton and further affect the function of the aquatic ecosystem through the trophic relationship of phytoplankton–zooplankton–fish . Thus, zZooplankton play important parts in transforming energy from lower-trophic organisms to higher ones, regulating phytoplankton growth, and shaping planktonic ecosystems ,4. NumerIn recent years, the effects of salinity on zooplankton have received much attention. Previous studies have revealed that salinity is an important environmental filter that may control the species composition and biomass of zooplankton communities in coastal estuarine ecology ,13,14,15The Duliujian River is the largest river in the southern region of the Haihe River plain with a significant salinity gradient. Thus, it was selected as the research object to study the response of the zooplankton community to environmental factors. In this study, 18 abiotic factors and zooplankton data from 15 stations were monitored and analyzed in the river estuary ecosystem of the Duliujian River. The objectives of this study were to (i) identify the main factors affecting the zooplankton community’s abundance and biomass; (ii) quantify the relative importance of salinity impacts on the zooplankton community; and (iii) find potential thresholds for the effects of salinity on zooplankton community abundance and biomass.2. The river basin is located in the warm temperate zone and has a semi-humid continental monsoon climate, which means a unique seasonal climate. The annual average temperature ranges from 12 °C to 15 °C within the region, while the precipitation is concentrated from June to September, with an average annual rainfall of approximately 571 mm. Additionally, the Duliujian River connects two important reservoirs, the Tuanbowa Reservoir and the Beida Harbor Reservoir. Despite sluice control, interactions can possibly occur through small tributaries. Frequent human activities have caused point and non-point source pollution in the Duliujian River, such as factory wastewater discharge, urban domestic sewage discharge, and fishery and agricultural pollution, which could also increase the salinity in the river to a certain extent. The river’s proximity to the ocean renders it susceptible to saltwater intrusion. Consequently, the entire river exhibits a salinity gradient that, in conjunction with other abiotic conditions, influences the survival, growth, and reproduction of aquatic creatures [The Duliujian River in Tianjin City, China  is the largest river in the southern Haihe River Basin’s lower portions. It flows from the confluence of the Daqing River and the Ziya River to Bohai Bay , with a 4+-N), nitrate–nitrogen (NO3−-N), nitrite–nitrogen (NO2−-N), and chemical oxygen demand (COD) were analyzed in the laboratory. The water samples were tested for TN, TP, COD, and TUR in the lab. The other samples, which had been filtered through 0.45 m filter membranes, were tested for the other nutrient factors. Every environmental factor was measured in accordance with the relevant standards , water depth (WD), dissolved oxygen (DO), salinity (SAL), pH, and oxidation–reduction potential (ORP) were some of the environmental factors that were measured in situ (YSI ProPlus), while the other factors, such as total phosphorus (TP), total nitrogen (TN), total dissolved phosphorus (TDP), total dissolved nitrogen (TDN), ammonia–nitrogen (NHtandards . Furtherv/v). After 24 h of standing, each supernatant fluid was slowly removed, and the final sample volume was 30 mL. At each station, three parallel samples were collected. For 3 parallel zooplankton samples of protozoa and rotifers, 1 L water samples were collected with polyethylene bottles at a 0.5 m surface water depth and immediately fixed with 10 mL of Lugol’s solution. In the laboratory, these samples were stored in glass containers for 24 h, and then the upper section of each solution was siphoned using a rubber hose. For each sample, the remaining 50 mL sample sediment was transferred into a polyethylene bottle and preserved by adding formaldehyde (4% v/v). After 24 h, it was condensed to 30 mL by sucking away the supernatant liquid with a pipette. Lastly, all the pretreated samples were well-packaged and transferred to the Center of Monitoring and Scientific Research of Ecology and Environment, the Administration of Ecology and Environment of Haihe Basin and Beihai Area, MEE, to identify zooplankton species and quantify zooplankton abundance and biomass.For copepods and Cladocera, 20 L water samples were repeatedly collected twice at a depth of 1 m and filtered with a 25# plankton collection net (with a mesh size of 0.064 mm), and the net was then washed once. Each filtrate was injected into a 50 mL polyethylene bottle and then fixed with a formaldehyde solution (4% Y) of each species was calculated with Equation (1) to determine the dominant species of zooplankton.The dominance value (10(x+1)-transformed to eliminate dimensional differences. In order to reduce sampling error, the data on environmental factors and zooplankton used for statistical analysis were the average values of three parallel samples of each sampling station in each quarter. Before statistical analysis, all species data and water environmental factors (except for pH) were logp < 0.05. In addition, Monte Carlo permutation tests (999 permutations) with Bonferroni adjustment were used to determine whether the first 2 constraint axes were relevant at a significance level of p < 0.05. Variance partitioning analysis (VPA) was employed to quantify the unique and shared fractions of variation in the zooplankton community, which were explained via SAL, NUT , and PC  . The zooplankton species names and corresponding codes can be checked in The responses of the zooplankton community abundance and biomass to environmental conditions were analyzed using redundancy analysis . Prior to the RDA, detrended correspondence analysis (DCA) was used to assess whether linear or unimodal ordination methods should be implemented. In this study, all DCA values were less than 4, and RDA was, therefore, the most reliable model for analyzing the interactions between the zooplankton community and environmental conditions. The forward or backward selection approach with Monte Carlo permutation tests (999 permutations) was used to find the driving environmental determinants of the variance in the zooplankton community . Then, the variance inflation factor (VIF) was used to check the collinearity between the selected elements. The significance of the association between the zooplankton community and the filtered environmental parameters was evaluated using the Mantel test. Last but not least, the validity and significance of the RDA results were assessed using Monte Carlo permutation tests (999 permutations), and the results were significant when p < 0.01). Moreover, the first 2 constraint axes were meaningful at p < 0.001, which was proved using Monte Carlo permutation tests (999 permutations) with Bonferroni adjustment. It explained 36.38% of the total variance in the zooplankton abundance, of which the eigenvalue of axis 1 was 1.12, accounting for 28.16%, while the eigenvalue of axis 2 was 0.33, accounting for 8.22%.A total of 17 zooplankton species were identified in 4 seasons with abundance values ranging from 21.00 ind./L to 1218.33 ind./L, with an average of 471.48 ind./L . RDA wasAs shown in p < 0.01). Moreover, the first 2 constraint axes were meaningful at p < 0.01, which was proved using Monte Carlo permutation tests (999 permutations) with Bonferroni adjustment. It explained 14.19% of the total variance in the zooplankton abundance, of which the eigenvalue of axis 1 was 1.48, accounting for 8.72%, and the eigenvalue of axis 2 was 0.93, accounting for 5.47%.For zooplankton abundance at the species level b, the RDStentor amethystinus presented notable positive correlations with TN, and TN could explain 11.7% (p < 0.01) of the variation in Stentor amethystinus  and 21.8% (p < 0.001) of the variation in Calanus sinicus and Paracyclopina nana, respectively  and 15% (p < 0.01) of the variance, respectively  of the variation in Calanus sinicus could be explained  and 10.1% (p < 0.05) of the variation, respectively . Moreover, the first 2 constraint axes were meaningful at p < 0.01 and proved using Monte Carlo permutation tests (999 permutations) with Bonferroni adjustment, which explained 23.94% of the total variance in the zooplankton biomass. TN, WD, pH, SAL, and WT significantly affected zooplankton biomass at the grouping level. It was found that the effects of the environmental factors on zooplankton biomass were similar to those of the environmental factors on zooplankton abundance. Specifically, TN and pH had positive and negative correlations with both protozoa and rotifera, respectively, while WD was negative for copepods and cladocera. SAL had positive and negative correlations with copepods and rotifera, respectively. According to the results of the variation partitioning, the variation could be explained by PC unique fraction > SAL unique fraction > NUT unique fraction . The first 2 constraint axes were also meaningful at p < 0.01 and proved using Monte Carlo permutation tests (999 permutations) with Bonferroni adjustment. They explained 13.38% of the total variance in the zooplankton abundance, with the eigenvalues of axis 1 and axis 2 being 1.34 and 0.94 and accounting for 7.88% and 5.5%, respectively. As shown in the figure, zooplankton biomass at the species level was mainly influenced by TN, WD, pH, SAL, and TDP, which was similar to zooplankton abundance. According to the results of the variation partitioning, the variation could be explained by PC unique fraction > NUT unique fraction > SAL unique fraction  of its variation  of the variation in Calanus sinicus and 19.7% (p < 0.001) of the variation in Paracyclopina nana  and 12.2% (p < 0.01) of their variations could be explained, respectively  of its dynamics  and 9.3% (p < 0.05) of their variations could be explained, respectively  (p < 0.01)  a, which  < 0.01) b. ChangeCalanus sinicus) with low abundance but high biomass. In an investigation in the Neva Estuary, an opposite trend of rotifera and copepods with increasing salinity was also observed [By analyzing the species distributions in freshwater and saltwater in the Baltic Sea, V. V. Khlebovich proposed the critical salinity concept, 5–8‰, in which the number of species reaches its minimum ,44. Howeobserved . Therefoobserved , salinitStentor amethystinus. The effects of WD on copepods might have been mainly induced by Calanus sinicus and Paracyclopina nana. pH affected protozoa primarily by inhibiting Stentor amethystinus and Vorticella lutea. Rotifera and copepods were affected by SAL, which may have been induced by influencing Brachionus calyciflorus, Calanus sinicus, and Ectocyclops phaleratus. In addition, salinity of approximately 4–5 was important for the responses of the zooplankton community composition. Finally, combined with the results of previous studies on the effects of salinity on phytoplankton, it was found that salinity may adjust the river ecosystem by influencing the abundance and biomass of zooplankton rather than those of phytoplankton.In this study, RDA and variation partitioning analysis were used to determine the important environmental factors influencing zooplankton abundance and biomass, and the relative importance of different environmental factors. The results show that TN, WD, pH, and SAL were all crucial abiotic factors shaping the zooplankton community in this studied area. TN affected protozoa by affecting


In contrast to their well-known physiological properties, phytochemicals, such as flavonoids, have been less frequently examined for their physiochemical properties . A natural quercetin self-stabilizing Pickering emulsion was fabricated and characterized in the present study. The antisolvent precipitation method was used to modify quercetin (in dihydrate form), and the obtained particles were characterized by light microscope, atom force microscope, XRD, and contact angle. The antisolvent treatment was found to reduce the particle size, crystallinity, and surface hydrophobicity of quercetin. We then examined the effects of the antisolvent ratio, particle concentration, and oil fraction on the properties of the quercetin particle-stabilized emulsions. In addition, increasing the antisolvent ratio (1:1~1:10) effectively improved the emulsification performance of the quercetin particles. The emulsion showed good storage stability, and the particle size of the emulsion decreased with the rising particle concentration and increased with the rising oil phase ratio. The findings indicate that natural quercetin treated with antisolvent method has a good ability to stabilize Pickering emulsion, and this emulsion may have good prospective application potential for the development of novel and functional emulsion foods. Due to their surfactant-free nature, high stability, and good delivery capacity, Pickering emulsions, or particle-stabilized emulsions, have good prospects for application in food and pharmaceutical fields ,10,11,12In addition to their well-known physiological properties, natural phytochemicals, such as flavonoid, have also been found to stabilize Pickering emulsions ,15. MurrQuercetin is a flavonoid compound widely found in vegetables and fruits, which has antioxidant, anti-inflammatory, antiviral, and cardiovascular protective effects ,34,35. HIn this paper, quercetin was treated by an antisolvent precipitation method with low energy consumption and the properties of its emulsion were investigated. Firstly, the quercetin particles obtained by antisolvent precipitation were characterized in terms of their morphology, size, crystallinity, and contact angle. Secondly, the basic properties of the above quercetin particle-stabilized emulsions, including the type of emulsion, particle size, and microstructure, were investigated.The quercetin  was purchased from Shanghai Aladdin Reagent Co., Ltd. . The soybean oil was purchased from a local supermarket in Guangzhou (China). All of the other reagents were analytically pure.w/v) and kept at 45 °C for 5 min. The organic phase containing quercetin was added to the aqueous phase  within 1 min using a high-speed homogenizer  under vigorous stirring at 8000 rpm, and homogenized for 2.5 min after complete addition. The antisolvent ratios (defined as “the volume ratio of organic phase to aqueous phase”) were 1:1, 1:2.5, 1:5, 1:10, respectively. After rotary evaporation , the ethanol and part of the water were removed until the solids content of the quercetin aqueous dispersion was 3% (w/v). In order to obtain dried quercetin pellets with different antisolvent ratios, the aqueous dispersion was freeze-dried for 72 h in a freeze-dryer .Quercetin was dissolved in anhydrous ethanol  with different antisolvent ratios was taken and left in a transparent serum bottle to observe its appearance. The visual photos were taken by a cell phone . The quercetin without antisolvent treatment at the same concentration was used as a control.A certain amount of the aqueous dispersion of quercetin pellets at 1% (The microstructure of quercetin in the aqueous dispersions was studied using light microscopy (LM) with 40× objective . w/v), dropped onto freshly exfoliated mica sheets and dried naturally, and observed using AFM  with a tapping mode.The microstructure of the quercetin particles was also studied using atomic force microscopy (AFM). The aqueous dispersions of the native quercetin particles and the quercetin particles obtained by the antisolvent precipitation method were diluted to 0.01%  ,52,53. AThe crystallinity of the quercetin pellets was analyzed using X-ray diffraction (XRD) . The dried quercetin particles were placed on a glass sample table, flattened, and then placed on the bench for measurement. The scanning range was between 3° and 80° with a step size of 0.02°, and the subsequent analysis was performed using X-ray diffraction analysis software.For a better comparison of the difference in crystallinity between the different samples, the relative crystallinity (%) was calculated as follows: In surfactant stabilized emulsions, the HLB is often used to characterize the hydrophilicity/hydrophobicity of the surfactant, whereas in the case of Pickering emulsions, the contact angle is used to characterize the wettability of particles, which is important for emulsion stabilization. In general, solid particles with contact angles less than 90° are considered hydrophilic, while those with contact angles greater than 90° are considered hydrophobic. The contact angle can be used to indicate changes in the surface properties of quercetin particles after antisolvent precipitation treatment and to determine whether the particles are stable as O/W emulsions or stable W/O emulsions . The conc) of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3% (w/v). Subsequently, the quercetin dispersions were mixed with soy oil at different ratios. The mixture was homogenized for 2 min using a T25 high-speed homogenizer  at 10,000 rpm to obtain the emulsions. All of the emulsions were then immediately sealed and stored at room temperature.The aqueous dispersions of the quercetin particles were diluted using 10 mM PBS (pH 7.0) to obtain aqueous dispersions with quercetin concentrations  using a digital microscope  at 0 and 7 days, respectively. For the LM observations, the emulsion was gently dropped onto a slide and observed without a coverslip using a 10× objective. If necessary, dilute the emulsion with deionized water for clearer observation.The droplet size of emulsions at 0 and 7 days was measured using a laser diffraction particle size analyzer .The data statistics are expressed as mean ± standard deviation and combined with graphical analysis using SPSS and origin 2018 software.The quercetin pellet dispersions were obtained using the antisolvent treatment method, followed by rotary evaporation to remove solvents and part of the antisolvent see . As wateThe antisolvent treatment significantly changed the particle morphology and size of the quercetin. The optical microscopy results of the particle dispersions  showed tAtomic force microscopy (AFM) was used to observe the detail of the morphological changes of the quercetin particles after the antisolvent treatment see A–E. The The effect of the antisolvent treatment on the crystalline nature of quercetin was evaluated using the XRD technique. As shown in uercetin . Compareuercetin ,32. The The hydrophilic/hydrophobic characteristics (wettability) of particles can be reflected by the contact angle  is shown in φ) increased (from 0.2 to 0.5), the emulsion layer (upper layer) gradually increased, while the height of the lower layer (aqueous layer) gradually decreased. At a fixed particle concentration in the aqueous phase, the total volume of the emulsion droplets formed increases as the oil fraction increases, and thus the emulsion layer also increases. The changes in the droplets’ size at various oil fractions on days zero and seven are shown in The effect of the oil fraction on the particle size and stability of the quercetin emulsions was evaluated, and the results are shown in The antisolvent treatment can transform quercetin into a good oil-in-water Pickering emulsion stabilizer. The antisolvent treatment could significantly change the characteristics of quercetin particles, including a reduced size, crystallinity, and contact angle, which is strongly influenced by the antisolvent ratio. Compared with native quercetin, the ability of antisolvent-treated quercetin to stabilize emulsions and its emulsion stability against coalescence were significantly improved. The properties of quercetin-stabilized emulsions can be modulated by changing the antisolvent ratio, quercetin particle concentration, and oil fraction. The results of this paper are instructive for the development of highly stable emulsions, as well as novel quercetin functional foods.


In this study, we identified multidimensional profiles in students’ math anxiety, math self-concept, and math interest using data from a large generalizable sample of 16,547 9th grade students in the United States who participated in the National Study of Learning Mindsets. We also analyzed the extent that students’ profile memberships are associated with related measures such as prior mathematics achievement, academic stress, and challenge-seeking behavior. Five multidimensional profiles were identified: two profiles which demonstrated relatively high levels of interest and self-concept, along with low math anxiety, in line with the tenets of the control-value theory of academic emotions (C-VTAE); two profiles which demonstrated relatively low levels of interest and self-concept, and high levels of math anxiety (again in accordance with C-VTAE); and one profile, comprising more than 37% of the total sample, which demonstrated medium levels of interest, high levels of self-concept, and medium levels of anxiety. All five profiles varied significantly from one another in their association with the distal variables of challenge seeking behavior, prior mathematics achievement, and academic stress. This study contributes to the literature on math anxiety, self-concept, and interest by identifying and validating student profiles that mainly align with the control-value theory of academic emotions in a large, generalizable sample. In a brTo extend this multi-dimensional perspective, the current project extends on previous research related to student STEM learning by applying constructs related to the control-value theory of academic emotions  and emplIn the sections that follow, we first situate this work in Control-Value Theory of Emotions . We then2.2.1.The control-value theory of achievement emotions (C-VTAE) provides a framework explaining how emotions are tied to achievement activities and outcomes. Through this framework, math anxiety is hypothesized to develop when a student experiences low control of a math activity, especially when also experiencing a higher value appraisal . Hence, When both control and value appraisal are studied together, the negative association between math self-concept and anxiety is stronger when the student’s math value is perceived as higher . The recRelatedly, 2.2.According to Ramirez and colleagues , math anNumerous studies have found that math anxiety is negatively related to math achievement, both concurrently and longitudinally , with poPerforming poorly on math tasks can diminish learners’ math self-efficacy beliefs, or their beliefs about their ability to succeed on math tasks . In otheAlthough much of this research attests that math anxiety may disrupt learning and tends to have an overall negative effect on math performance, there may be some cases in which some math anxiety may be beneficial . Famousl2.3.Interest refers to “the psychological state of engaging or the predisposition to re-engage with particular classes of objects, events, or ideas over time” , p. 112.Across content areas, interest in a specific subject is positively related to achievement, engagement with tasks in that subject, attention, goal-setting, competence beliefs, and expertise development . HoweverInterest may facilitate deeper processing and more sustained engagement with tasks in that area . Interes2.4.Self-concept refers to the perception one has of one’s self through experiences and how they interact and interpret their environment . Self-co2.5.Person-centered approaches such as latent profile analysis LPA;  and clusFor example, in a study investigating Taiwanese junior high school students’ conceptualizations of what comprises learning in math, Wang and colleagues  used LPAThis study will also utilize LPA to better understand the relationship between math anxiety, self-concept, and interest in a nationally representative sample of 9th grade students in the United States. This study will also determine the extent to which membership in each profile is associated with academic stress, challenge seeking behaviors, prior mathematics GPA. Given the demonstrated salience of math motivation and the complex interplay of control, value, and emotions demonstrated by C-VTAE, and the importance of measuring it with person-centered approaches, our work sought to answer the following research questions:2.6.Are there distinct profiles, or classes, of students who show similar patterns of performance across measures of math anxiety, math self-concept, and math interest?Hypothesis 1: Given prior literature using related constructs and the theoretical relationship between anxiety, math self-concept, and math interest articulated in C-VTAE, we hypothesize that students in this sample can be classified into a number of distinct latent profiles. Given the exploratory focus of this paper, we do not offer a hypothesis as to the number of profiles which may exist.To what extent are students’ profile memberships associated with related measures, including prior mathematics achievement, perceived academic stress and challenge-seeking behavior?Hypothesis 2: Our hypothesis for this question is more general in nature, given the exploratory purpose of the paper. We would hypothesize, based on the theoretical relationships between our variables of interest  and prior math achievement, academic stress, and challenge-seeking behavior, that latent profiles defined by our variables of interest would also exhibit significant differences on these related constructs.3.3.1.Data for this paper come from a nationally generalizable dataset collected as part of the National Study of Learning Mindsets . These dvia application to the Inter-university Consortium for Political and Social Research (ICPSR).Data used in the current study were all collected online via a restricted web survey portal in Fall 2015. Students were asked to complete a 25 min survey using computers provided by their school. 3.2.Student measures used in this study included: (1) prior math achievement, (2) math anxiety, (3) math interest, (4) math self-concept, (5) math challenge-seeking behavior, and (6) perceived academic stress. Math prior achievement was operationalized as each students’ 8th grade math grade point average (GPA). This measure ranged from 0 (F) to 4.30 (A).Math anxiety (“Math makes me anxious”), interest (“Math is interesting to me”), and academic stress (“Schoolwork is stressful for me”) were all measured single items from the time-1 student survey, with a 5-point Likert-type response scale that ranged from 1 (“Strongly disagree”) to 5 (“Strongly agree”). Math self-concept (“I am a math person”) was a single item ranging from 1 (“Strongly disagree”) to 7 (“Strongly agree”). Mathematics challenge seeking behavior was also measured using a single item, which described a challenging math problem and then asked the student how likely they were to try and solve the problem. Responses here were also measured on a 0 (“Highly unlikely”) to 4 (“Highly likely”) Likert-type scale. Given that items used somewhat different scale lengths, for purposes of interpreting profile differences we used a percent of maximum possible  transfor3.3.Latent profile analysis is a method that attempts to identify hidden subgroups within a sample or population  and is aThe main variables of interest used in our LPA were mathematics self-concept, mathematics anxiety, and mathematics interest, all measured by self-report items and described above. These three indicators are the basis for determining the optimal number of student subgroups to answer Research Question (RQ) 1. As described in RQ 2, several additional measures were used as distal outcomes to test the validity of the LPA solution, including academic stress and challenge-seeking behavior, both measured by self-report items, and prior math achievement, as measured by students’ 8th grade mathematics grade point average (GPA).via a global Chi-square test, which is significant if any pairwise comparisons are significant, as well as via individual pairwise comparisons.The LPA modeling approach used was the parametric procedure outlined by MLR in Mplus) was used. To assess model fit, the scaled Satorra-Bentler Chi-Square statistic  enumerating the mathematics learning profile groups (latent profiles) and (b) assessing mathematics learning profile differences with respect to distal measures.4.1.4.2.Having chosen the 5-profile solution based on the available fit evidence, we next created a conditional means plot to visualize the relative shapes and levels of each profile. Conditional means, adjusting for the classification error for each student, were generated for math self-concept, anxiety, and interest for each of the 5 profiles see . The POMProfiles 1 (3% of sample) and 3 (16% of sample) include students that tended to have higher levels of anxiety and lower levels of self-concept and interest. Profile 1 demonstrated the highest average levels of anxiety , and profile 3 had the second highest at 3.1 out of 5 points. Compared to Profile 3, Profile 1 had lower levels of both self-concept and interest. Profiles 2 (10% of sample) and 5 (34%) are characterized by lower levels of anxiety and high levels of self-concept and interest. Profile 2 had the lowest level of anxiety (1.70 out of 5) but differing slightly levels of interest (3.70 out of 5) and self-concept (7 out of 7) with profile members having the highest self-concept possible. Students in profile 5 had slightly lower interest (3.10 out of 5) and self-concept (6 out of 7), and higher anxiety than profile 2 (2.20 out of 5). This aligns with prior literature that higher value and control appraisal have negative associations with math anxiety. Profile 4 (37% of sample) represents the largest group in our sample and is characterized by students that had average math interest (2.40 out of 5) with slightly higher anxiety (2.70 out of 5), and higher self-concept (5 out of 7).4.3.We next analyzed the extent to which profile membership might be associated with differential responses on distal variables, including challenge seeking behavior, prior math achievement, and academic stress. Results of this analysis are available in 4.3.1.Overall, the percentage of students who attempted a challenging math problem ranged from a minimum of 25% in Profile 1 to a maximum of 59% in Profile 2. In a variable-centered analysis, we might hypothesize that challenge-seeking is positively associated with self-concept and interest, and negatively associated with math anxiety. If this holds within-person, then we might expect that students in profiles with high self-concept and low anxiety might demonstrate higher levels of challenge seeking behavior. Our analysis confirms this. For example, Profiles 2 and 5 both have high self-concept and high interest. Although they exhibit slightly different levels of challenge seeking, 59% in Profile 2 and 49% in Profile 5, they have the highest levels of challenge seeking amongst all the profiles. We also found that Profile 4, a profile with medium anxiety, had the next highest levels of challenge seeking, at 40%.4.3.2.Overall, 8th grade math GPA ranged from a minimum conditional mean of 2.19 (out of 4.30) in Profile 1 to a maximum conditional mean of 3.39 in Profile 2. We would expect that math prior achievement would likely relate positively to self-concept and interest, and negatively to anxiety. Thus, we expected that Profiles 2 and 5 might have the highest math prior achievement. Our results confirm this, as Profile 2 exhibited the highest mean prior achievement . Profile 5 had the second-highest prior achievement.4.3.3.Overall, academic stress ranged from a minimum conditional mean of 2.76 (out of 5) in Profile 2 to a maximum conditional mean of 3.66 in Profile 1. This is less variation than was observed for math anxiety, which ranged from a minimum of 1.70 in Profile 1 to a maximum of 3.70 in Profile 2. In theory, academic stress should map most closely onto math anxiety, so we hypothesized that students in Profiles 1 and 3 should also have the highest academic stress. Our results confirm this. We found that the profile with the highest academic stress, Profile 1, had high levels of anxiety. Profile 3, the next highest in academic stress, was high for anxiety as well.5.Using an exploratory approach to latent profile analysis and including math anxiety, self-concept, and math interest as indicators, we found significant evidence that extends on previous research on students’ math learning, emotions, and motivation by investigating control-value profiles of students’ math motivation and emotions in a nationally representative sample of more than 16,000 ninth grade students in the United States. We also examined the extent to which student profile membership predicts academic stress and challenge-seeking behaviors, and the relationship between student profile membership and prior math achievement.Results call for the need to look beyond linear relations among these complex, multifaceted constructs. Several prior studies that analyze both control and value appraisal have found results that do not strictly align with the framework that value appraisal has negative associations with math anxiety . Pekrun’Math interest is consistently found to have a positive association with achievement . InterveHowever, the largest profile in our sample did not exhibit a clear alignment with the framework. Although in prior research, when control and value appraisal are studied together, the negative association between math self-concept and anxiety is stronger when the student’s math value is perceived as higher. This group showed lower math interest than self-concept, which may explain the higher math anxiety. Profiles aligned with theory in predicting whether students would attempt a challenging problem. The differences noted here were the anxiety levels between the two groups. We expected self-concept to relate positively to interest and negatively to anxiety, which aligns with the profiles with highest math self-concept. The research literature demonstrates that math anxiety can have conflicting, and at times unexpected, relationships with students’ math interest and self-concept. For example, as This work furthers the research related to STEM disciplines in that achievement is not the only factor contributing to STEM learning, but psychological and socio-emotional factors have an impact as well. This theme was a key component of a recent report by the 5.1.This work has several limitations that are important to emphasize. First, the profile indicators we used here for student math anxiety and self-concept were responses to a single item on the NSLM survey. We acknowledge that single items are typically not ideal for measuring psychological constructs. However, in this case, given that the NSLM was not primarily designed to assess the constructs we use here (and rather to focus on learning mindsets), we believe that the advantage of having a large, generalizable sample of responses outweighs the drawbacks of only having single item responses. In some cases, single-item measures can be appropriate to measure psychological constructs provided that the item is clearly written, with a clear object and attribute . We belivia latent transition analysis or a similar approach. These profiles are likely not static, and as high school students continue to develop there could be both changes in profile membership as well as potential changes in the pattern and structure of the profiles overall. This work also suggests a number of future directions in this area that might be beneficial. For example, it would be important to explore potential differences in profile composition related to gender, minoritized racial and ethnic groups, and socioeconomic status, especially given the longstanding inequities and underrepresentation in STEM . It is a5.2.This work builds on and extends prior research on students’ math motivation and emotions in the transition to high school. We found a variety of complex student profiles which conform to many of the tenets of C-VTAE. However, we also found that our largest profile, comprising nearly 40% of the total sample, displayed a pattern of anxiety, interest, and self-concept that did not clearly separate as might be expected. It is possible that this group is made up of students who are relatively average or typical in their levels of these indicators, and as such do not display the separation seen in the other profiles. Nonetheless, this finding is interesting, given the size of the profile and suggests that for many students, the hypothesized relationship between these three constructs may not be as clear. We hope that this work can further elucidate the tenets of C-VTAE in a within-person context while also contributing to a more nuanced and complex understanding of the interplay between student motivation, emotion, and achievement in mathematics.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by Institutional Review Board, University of Texas at Austin. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.MB led all aspects of the study, including conceptualization, data analysis and interpretation, manuscript drafting, revising, and editing. ER assisted with results interpretation, manuscript drafting, revising, and editing. NS assisted with manuscript revising and editing. EE assisted with the initial draft of the manuscript. All authors contributed to the article and approved the submitted version.Research reported in this summary was supported by the National Study of Learning Mindsets Early Career Fellowship with funding provided by the Bezos Family Foundation. The University of Texas at Austin receives core support from the National Institute of Child Health and Human Development under the award number 5R24 HD042849. This publication uses data from the National Study of Learning Mindsets , which was made possible through methods and data systems created by the Project for Education Research That Scales (PERTS), data collection carried out by ICF International, meetings hosted by the Mindset Scholars Network at the Center for Advanced Study in the Behavioral Sciences at Stanford University, assistance from C. Hulleman, R. Ferguson, M. Shankar, T. Brock, C. Romero, D. Paunesku, C. Macrander, T. Wilson, E. Konar, M. Weiss, E. Tipton, and A. Duckworth, and funding from the Raikes Foundation, the William T. Grant Foundation, the Spencer Foundation, the Bezos Family Foundation, the Character Lab, the Houston Endowment, the National Institutes of Health under award number R01HD084772-01, the National Science Foundation under grant number 1761179, Angela Duckworth , and the President and Dean of Humanities and Social Sciences at Stanford University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Bezos Family Foundation, the Mindset Scholars Network, The University of Texas at Austin Population Research Center, the National Institutes of Health, the National Science Foundation, or other funders.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


After 4.6 ± 4.0 years of follow-up, 72 patients (50.3%) remained hypercapnic. In bivariable analysis, clinical data showed no difference in follow-up duration, number of comorbidities, comorbidities, or circumstance of discovery. Patients with persistent hypercapnia on NIV were generally older, with lower BMI and more comorbidities. , female sex (87.5% versus 71.8%), was treated by NIV , had lower FVC , lower TLC , lower RV , higher pCO2 at diagnosis  and lower pH , higher pressure support  and lower EPAP . There was no difference in non-intentional leaks and daily use between patients between both groups. By multivariable analysis, sex, BMI, pCO2 at diagnosis, and TLC were independent risk factors for persistent hypercapnia on home NIV. In individuals with OHS, persistent hypercapnia on home NIV therapy is frequent. Sex, BMI, pCO2 at diagnosis, and TLC were all associated with an increased risk of persistent hypercapnia in persons treated with home NIV.Obesity-hypoventilation syndrome (OHS) is a respiratory complication of obesity characterized by chronic hypercapnic respiratory failure. It is often associated with several comorbidities and is treated by positive airway pressure (PAP) therapy. This study aimed to identify factors associated with persistent hypercapnia in patients receiving home non-invasive ventilation (NIV). We performed a retrospective study including patients with documented OHS. In total, 143 patients were included (79.7% women, age 67 ± 15.5 years, body mass index 41.6 ± 8.3 kg/m Obesity hypoventilation syndrome is associated with many comorbidities, including cardiovascular and metabolic. Persistent hypercapnia is frequent and deleterious for patients. There is a paucity of data on the factors associated with persistent hypercapnia on home NIV after optimizing PAP settings. Sex, BMI, pCO2 at diagnosis, and TLC are independently associated with persistent hypercapnia on home NIV.Obesity is a major public health problem worldwide and its prevalence has increased significantly in recent years . ObesityMartinique is a French department of the West Indies. The Caribbean population is predominantly composed of French citizens of Afro-Caribbean origin. The prevalence of obesity, diabetes, and arterial hypertension is higher in this population than in metropolitan France ,7. ObesiTo the best of our knowledge, no study has evaluated the impact of comorbidities on the course of OHS and the factors that may affect outcomes. The objective of this study was therefore to describe the profiles of OHS patients in Martinique and to assess the factors associated with persistent hypercapnia in patients receiving nocturnal positive airways pressure (PAP) therapy at home.We performed an observational, single-centre, retrospective study in the Department of Respiratory Medicine of the University Hospital (CHU) of Martinique, which is the referral centre for patients with respiratory diseases for the whole island, and where all patients with chronic respiratory failure, including OHS, are treated.Patients were included from 1 January 2019 to 31 December 2022. All patients followed in our Department for OHS and hospitalized in the department during the study period were eligible. The inclusion criteria were:Age 18 years or older2, daytime partial pressure of carbon dioxide (PCO2) ≥ 45 mmHg, and absence of COPD, neuromuscular disease, or hypothyroidism.Diagnosis of OHS, including body mass index (BMI) ≥ 30 Kg/mWe excluded patients with a smoking history of more than 10 pack-years in women or 15 pack-years in men, as this may be associated with obstructive syndrome where associated COPD cannot be ruled out.Patients had regular follow-ups every 6 to 12 months in our Department to optimize NIV settings and monitor comorbidities. In follow-up, patients were admitted for 3 to 5 days, to optimize the device settings for the home NIV device, and to monitor comorbidities. The objective is to correct hypercapnia by reaching an optimal balance between the increase in pressure support leaks, apnea-hypopnea index (AHI), and monitoring ventilator asynchrony.20. Inspiratory assistance was then programmed to achieve the optimal balance between efficiency and leaks. For patients who were not receiving CPAP, parameters were set empirically, by adjusting according to residual apnea-hypopnea index (AHI), asynchrony, leaks, and patient complaints.BIPAP was initiated during hospitalisation. Most patients being followed-up were diagnosed in the context of acute decompensation, and BIPAP was initiated at that time. For patients already receiving CPAP, expiratory positive airway pressure (EPAP) was set at a level equal to CPAP minus 2 cm HComorbidities were assessed based on the biological work-up and the patient’s ongoing treatment. If there were any doubts, a relevant specialist was consulted to confirm or rule out the diagnosis. Cardiovascular comorbidities were diagnosed based on electrocardiographic and/or echocardiographic findings, and a cardiologist’s opinion. Respiratory comorbidities, notably asthma, were assessed based on clinical symptoms, obstructive ventilatory disorders, and absence of heavy smoking or professional exposures. Patients all underwent polygraphy or polysomnography, either before diagnosis and were already receiving CPAP for severe obstructive sleep apnea, or at the time of diagnosis. Severe obstructive sleep apnea was defined as an AHI > 30/hour.We recorded sociodemographic data , comorbidities at diagnosis, comorbidities at inclusion in the cohort, treatment initiated , and follow-up data on persistent hypercapnia for all patients.Persistent hypercapnia was defined as a diurnal PaCO2 > 45 mmHg (6 KPa) in room air without acute on chronic respiratory failure. We considered the value of arterial gas, as well as treatment data  during the last routine visit without exacerbation.t-test. Categorical variables are described as numbers and percentages and were compared using Fisher’s exact test. Bivariable and multivariable analyses for the associations between covariates and the persistence of hypercapnia were performed using binary logistic regression modelling. Statistical analyses were performed using SAS version 9.4 . Tests were considered significant for p-values < 0.05.A descriptive analysis was performed. Quantitative variables are described as mean ± standard deviation (SD), and we compared them using the Student The study was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of the University Hospitals of Martinique under the number 2020/073. At each follow-up consultation or hospitalization, patients received an information leaflet explaining that their routine medical data could be used for research purposes, and indicating how they could explicitly oppose this use, if they so desired, in accordance with French legislation governing retrospective studies using routine medical data. No patient explicitly opposed the use of their medical data for the purposes of the present study.2.One hundred and forty-three patients were included, of whom 114 (79.7%) were women. The patients included were diagnosed between 5 October 2001 and 30 September 2022. The mean age at diagnosis was 67.3 ± 15.5 years, and the mean age at inclusion was 71.7 ± 15.2 years. The mean BMI was 41.6 ± 8.3 kg/mAfter a mean follow-up period of 4.58 ± 4.04 years, 72 patients (50.3%) had persistent hypercapnia, despite optimal settings on their home NIV device and management of comorbidities. The characteristics of the study population are displayed in All patients with BIPAP had persistent hypercapnia, while none of the patients receiving CPAP had persistent hypercapnia. There was no difference in the length of daily use, or unintentional leaks, between patients with and without persistent hypercapnia, but in ventilator settings, the pressure support was higher in the group with persistent hypercapnia .By multivariate analysis, sex, BMI, pCO2 at diagnosis, and total lung capacity were found to be significantly associated with the risk of persistent hypercapnia .2 of BMI decreased the risk of persistent hypercapnia by 8%, a 1% increase in TLC decreased the risk by 4%, an increase in pCO2 at diagnosis of 1 mmHg increased the risk by 5%, and being a man decreased the risk by 75%. Daily use of NIV, non-intentional leaks, and settings were similar in both groups, but patients with persistent hypercapnia had higher pressure support. Surprisingly, BMI seems to have a protective effect against persistent hypercapnia. The restrictive syndrome, measured by total lung capacity, increases the risk of persistent hypercapnia, as does pCO2 at the time of diagnosis.The most frequent comorbidities in our population were arterial hypertension, diabetes mellitus, hypercholesterolemia, chronic cardiac failure, and asthma. After a follow-up period of 4.8 ± 4 years, 50.3% of patients still had PaCO2 > 45 mmHg (6 kPa). Sex, BMI, pCO2 at diagnosis, and TLC were found to be independently associated with the risk of persistent hypercapnia. An increase of 1 kg/mThe mechanisms of diurnal hypercapnia in OHS are complex, and include hypoventilation during nighttime sleep and especially, persisting during the daytime ,16. TherIn our study, patients who had persistent hypercapnia were less obese but had great restrictive syndrome. This restrictive syndrome seems to have been the determinant feature driving the persisting hypercapnia, but this result is likely influenced by several specificities unique to our population.Firstly, the subjects in our study were, in general, older, with an average age at diagnosis was 67.3 ± 15.5 years, and 71.7 ± 15.2 at the time of inclusion. This is higher than reported elsewhere; for example, BaHammam et al. reported a mean age of 61.5 ± 11.9 years in women, and 49.1 ± 12.5 in men . Maza etSecond, regarding comorbidities, there were high rates of certain comorbidities in our study, including asthma and chronic heart failure. Asthma is a chronic pulmonary disease with a higher prevalence in the French West Indies compared to mainland France ,22. BoreThere is also the question of the possible role of pulmonary resistance and compliance in response to NIV. Further prospective studies with adequately powered sample sizes are warranted to elucidate the particularity of OHS in black people, the exact role between obesity, restrictive syndrome, and persisting hypercapniaRegarding the role of sex, a few studies found a female predominance, as in our study ,29,30, bRegarding age, it was not found to be an independent factor associated with persistent hypercapnia in our analysis.Normalization of PaCO2 after initiation of nocturnal home ventilation varies between 2% and 74% . SoghierThere is a paucity of data in the literature regarding the factors potentially associated with persistent hypercapnia over the long-term. Mokhlesi et al. identified variables that predicted changes in PaCO2 after PAP therapy and found that average daily hours of positive pressure therapy, FEV1 percentage of predicted, and baseline PaCO2 were all significant modifiable predictors of improvement in pCO2 . Howard 2, vs a mean of 44 kg/m2 among 757 OHS patients from 10 studies involving mostly non-Asian subjects [2 in the two groups, which is in line with the average values reported for non-Asian subjects. Nevertheless, there may be other factors at play. Although the differences did not reach statistical significance, the subjects with persistent hypercapnia were older and also had numerically higher rates of arterial hypertension, diabetes, and a lower rate of pulmonary hypertension. There may therefore be macro- and microvascular mechanisms involved that cannot be elucidated by the present data, and warrant further investigation, in addition to the effect of ethnic origin.We expected that the severity of obesity would be associated with the restriction, but paradoxically, very obese patients with less restriction are probably easier to ventilate than patients who are less obese but with greater restriction. The mechanisms of OHS are extremely complex and do not solely depend on obesity, but also on its impact on respiratory function . The spesubjects . Of noteTo the best of our knowledge, few previous studies have evaluated the factors associated with persistent alveolar hypoventilation in patients on nocturnal home NIV for OHS after optimization of the device settings. Similarly, no study has been performed on this subject in the Martinican population, a population comprising predominantly persons of African descent, with their inherent specificities, and indeed, who are often excluded from studies on the subject. This population has a higher prevalence of obesity, diabetes, and arterial hypertension than the population of metropolitan France.The strengths of this study include the follow-up of patients in the same center, which ensures consistent follow-up methods. However, limitations include the fact that this was a retrospective, single-centre study with all the related shortcomings, including reduced statistical power. Data are missing for certain variables, particularly those regarding the BMI of some obese and bedridden patients that our center is not able to weigh. Further studies are warranted to elucidate the relationship between factors such as age, sex or BMI, and persistent hypercapnia.OHS is a complication of obesity that affects mainly women and the elderly in the Martinican population. It is associated with many comorbidities such as cardiovascular diseases, metabolic diseases, and asthma. Persistent hypercapnia under NIV is present in 50.3% of patients. Female sex, lower BMI, total lung capacity, and high baseline pCO2 were all independently associated with persistent hypercapnia under home NIV. Knowing the independent factors that may positively impact blood gas results could help to guide measures to optimize the management of these factors for better follow-up. Special attention should be paid to these factors in the follow-up of these patients. Further studies are warranted to further elucidate the relationship between these factors and persistent hypercapnia.


Leptospira have been described in ill dogs, the most commonly detected being Icterohaemorragiae (ICT) ST 17, Australis (AUS) ST 24 and ST 198, Pomona (POM) ST 117 and ST 289, and Sejroe (SEJ) ST 155. However, there is little information available on the environmental exposure to Leptospira of wild and synanthropic animals. The aim of this study was to identify the circulating genotypes in potential reservoirs to fill this gap of knowledge. Between 2015 and 2022, 681 animal carcasses collected by the Public Veterinary Service were analyzed for Leptospira with a real-time PCR-based screening test, while positive samples were genotyped by multi-locus sequence typing analysis. To carry out our study, we tested 330 hedgehogs, 105 red foxes, 108 Norway rats, 79 mice, 22 coypus, 10 bank voles, 13 grey wolves, 5 common shrews and 9 greater mouse-eared bats. Five sequence types (STs) common in dogs were also found in wild animals: ST 24, ST 198, ST 17 and ST 155 in hedgehogs, ST 17 and ST 24 in foxes, ST 17 in rats, ST 17 and ST 155 in mice, and ST 117 in a wolf. In addition, to the best of the authors’ knowledge, this is the first Italian report of SEJ ST 197 in a bank vole. Furthermore, this study described a previous survey conducted in 2009 on coypus , referring to a serological positivity (L. Bratislava) without any molecular detection of Leptospira. This study on Leptospira in synanthropic and wild animals highlighted the importance of increasing our epidemiological knowledge of leptospirosis and its zoonotic risks.Leptospirosis is an infectious disease widely reported in veterinary practice and a worldwide zoonosis. In Northeastern Italy, different serogroups and genotypes of  Leptospira, which consists of 22 known species , including more than 300 serovars Leptospira isolation was attempted from urine and kidney samples, as previously described [Leptospira spp. through an end-point PCR assay targeting a 423 bp fragment within the region of the lipL32 gene, conserved among pathogenic Leptospira serovars [escribed . Kidney serovars .Leptospira. Epidemiological data  were recorded for each sample; however, due to the study design, no additional information was available to be used for a complete spatial and modelling analysis.Passive surveillance of synanthropic and wild animals was carried out on animal carcasses collected by the Public Veterinary Service during the routine surveillance program, therefore no deliberate killing of animals was contemplated for this sampling. Between 2015 and April 2022, 681 synanthropic and wild animal carcasses were submitted for analyses . Kidney tissue or urine were collected and further investigated for the presence of pathogenic Kidney tissue samples were homogenized at a 1:10 dilution in 600 µL of sterile PBS, with TissueLyser II . Two mL of urine samples were centrifuged at 12,000 g for 20 min at 4 °C and the pellet was re-suspended in 0.2 mL of sterile PBS, in order to possibly concentrate the leptospires and increase the yield of DNA. Given the poor cellular matrices, 20 µg of a poly-A carrier  was added to each urine sample to increase the recovery efficiency of nucleic acids.® Mag Universal Extraction Kit , in accordance with the manufacturer’s instructions. Each DNA extraction session included a negative process control (water).DNA isolation from 100 µL of tissue homogenate or urine pellet, was performed after a pre-lysis treatment with 2.5 μL of lysozyme  for 15 min at +37 °C. The DNA extraction was performed on the KingFisher™ Flex Purification System  platform using the ID GeneLeptospira was searched by means of a screening real time PCR (qPCR) targeting a 87 bp fragment that corresponded to a portion of the gene encoding the 16S rDNA [Leptospira biflexa serovar Patoc), and a positive control (DNA of L. interrogans serovar Icterohaemorrhagiae). The assay was performed under the following thermal conditions: a holding step at 95 °C for 10 min and 40 cycles at 95 °C for 15 s and 60 °C for 60 s. Samples with cycle threshold (Ct) < 38 were considered positive. Samples having Ct values within the 38–40 range were considered doubtful, whereas samples having no FAM fluorescence signal or with Ct ≥ 40 were considered negative.The presence of pathogenic species of 16S rDNA . The qPCDNA of positive samples were referred to the IZSLER National Reference Centre for Leptospirosis for the genomic characterization with Multi-locus Sequence Typing (MLST) technique. glmU, pntA, sucA, tpiA, pfkB, mreA and caiB, as previously described [Leptospira multilocus sequence typing (MLST) website (https://pubmlst.org/leptospira/) sited at the University of Oxford [Leptospira being tested.The genotyping was attempted in all qPCR positive samples by using the 7-loci scheme proposed by Boonsilp in 2013 , which iescribed . The seqf Oxford . CompariLeptospira spp. positivity, Bayesian generalized linear mixed models (GLMM) were used. These models were adapted by considering the following fixed effects: the origin of the samples (province), the species and the season of collection. To assess any correlation between Leptospira positivity in synanthropic and wild animals and seasonality, each sample was placed in a seasonal time slot, as follows: in spring, if sampled from March to May; in summer, if collected from June to August; in autumn, if collected from September to November; and in winter, if collected from December to February. Analyses were performed using the R software package “blme″ (R Development Core Team 2013) with binomial error (logit-link function) using the year of collection as a random effect [To identify the factors that may be significantly correlated with m effect .Data about the geographic origin of the samples (province) were depicted in a map which displayed the distribution of positive samples and genotypes, in order to make the spatial distribution easy to understand. The map was created using the open-source software QGIS 3.16.L. Australis serovar Bratislava (titres of 1:200 and 1:1600). From the province of Padua, 24/41 animals  resulted seropositive to different serovars: 21/41  reported serological positivity for L. Australis serovar Bratislava (antibodies titres ranging from 1:100 a 1:3200), 2/41 coypus  had low antibodies titres against L. Icterohaemorragiae serovar Icterohaemorragiae (titres 1:100 and 1:200), 1/41 subject  reported a low antibody positivity against L. Icterohaemorragiae serovar Copenhageni (1:100)  as seropositive, both with antibodies for  (1:100)  The moleBetween January 2015 and April 2022, the Public Veterinary services of Northeastern Italy collected 681 animal carcasses, belonging to different species of synanthropic and wild animals: 9 greater mouse-eared bats, 10 bank vole, 105 red foxes, 5 common shrew, 330 hedgehogs, 79 house mice, 108 Norway rats, 22 coypus, 13 grey wolves. For each animal, a molecular investigation through qPCR was performed on the kidney tissue or urine samples.Leptospira spp. in each enrolled animal is reported in n = 9/22), bank voles , Norway rats , red foxes , hedgehogs . Samples from house mice , grey wolves , greater-mouse eared bats  and shrews  show a lower prevalence.The overall prevalence of pathogenic Samples of grey wolves, bank voles, and common shrews were consistently distributed between the different collection sites, while red foxes were mainly concentrated in the provinces of Belluno, Trento and Bolzano, in the alpine and pre-alpine region. In contrast, hedgehogs were mostly obtained from the provinces of Venice, Padua and Rovigo, and the Norway rat from the provinces of Padua and Vicenza. These provinces are located in the geographical areas of the Po Valley. Most of the positive coypus samples were collected from a single geographical site and within a short period of time (7–21 days). The nine mouse-eared bats were collected from a single colony and only one tested weakly positive.Leptospira tested. Both the complete and the partial MLST profiles were reported and placed in a corresponding ST. For allelic number and ST identification, assembled and trimmed sequences were queried against the Bacterial Isolate Genome Sequence Database (BIGSdb) available on the Leptospira MLST website  sited at the University of Oxford [Leptospira being tested. To perform comparisons among historical serological studies (where serovars and serogroups were defined) and genotyping data (where species and genomic profiles were defined), we chose to assign to each identified ST a classification at the serogroup and serovar levels obtained from BIGSdb, knowing that this information was deduced and did not result from active serological typing.DNA of positive samples was submitted to the genomic characterization with MLST technique to deduce the species, the serotype and/or the serovar of the f Oxford . CompariLeptospira serogroups such as Australis and Icterohaemorrhagiae have been reported, as well as fewer other different serogroups .Leptospira spp. with qPCR, As described in Leptospira positivity was found between the different sampled species. The most represented serogroup among the positive samples was Australis , reporting positivity both for ST 24  and ST 198 . Among the species, ST 24 was reported in hedgehogs , house mouse  and red foxes . Furthermore, ST 198 was detected exclusively in hedgehogs .The overall prevalence in the sampled animals was 18.9% (CI95% 16.1–22.1), no statistically significant difference in L. interrogans Icterohaemorragiae ST 17 demonstrated an overall prevalence of 37.5%  among the positive samples: ST 17 was reported in Norway rats , in hedgehogs , house mice  and red foxes . L. borgpetersenii Javanica ST 146 was reported in 4.8% of positive animals , specifically in one hedgehog , one house mouse  and two red foxes . L. borgpetersenii Sejroe reported positivity for ST 155  in one hedgehog  and in one house mouse , respectively. Moreover, a positivity for L. borgpetersenii Sejroe ST 197 was reported in a bank vole . L. kirschneri Pomona ST 117 was reported in one grey wolf .Leptospira at molecular analysis, variability in STs is evident. ST 24 is more represented in hedgehogs from Padua and Venice, whereas ST 198 is more frequent in hedgehogs from Rovigo.Leptospira spp. than the other provinces , but this figure is difficult to relate in a standard model, as the sampling was carried out following the reporting of an outbreak of Leptospirosis .The province of Udine was found to have a fourfold higher prevalence of positivity to Leptospira spp. in the four seasons were calculated for the overall population of the study, and individually for the species with the highest number of collected samples . A GLMM was used to determine the seasonal differences in Leptospira positivity with the year and province of collection as random effects, the p values of the Wald chi-square tests for the GLMMs for each species is shown in The differences in prevalence of the number of animals positive to Leptospira spp. prevalence among the wild and synanthropic animal population revealed no significant correlation with the season, except in the model represented a statistically significant fluctuation in the seasonal prevalence of infection with pathogenic Leptospira (p < 0.05), with a higher prevalence in summer.The analysis of Leptospira in wild rodents in Europe report a high seroprevalence (75–86%), and the most represented serogroups are Australis, Autumnalis, Icterohaemorragiae, Grippotyphosa, Panama, and Sejroe. [Leptospira in European small mammals reported a different prevalence of Leptospira DNA detection [Leptospira after infection, hence their epidemiological role needs to be evaluated in comparison with multiple factors  [According to previous studies in the literature, serological studies of  Sejroe. . Conversetection ,31. It iization) ,32. In aization) .Leptospira, with a prevalence ranging from 50 to 90%, whereas coypus did not show a high prevalence of Leptospira in Europe [Australis as the most represented , but none of the animals tested positive after molecular screening. Similar results were reported in a study conducted in Northwestern Italy, in which L. Australis/Bratislava was the most represented serogroup among coypus [Leptospira with an increased antibodies reaction by cross- reaction [Many surveys worldwide ,34,35 ren Europe ,36. Thisg coypus , and in g coypus . DiffereLeptospira (40.9%), although the value obtained in the qPCR analysis indicates a low bacterial load. According with studies in the previous literature [We performed molecular analyses on the same species sampled from the coypu population between 2015 and 2022 during a passive surveillance program, reporting high prevalence of terature , this hiLeptospira. The present study highlights the need to further investigate the epidemiologic role of coypus as a non-autochthonous rodent in a Leptospira-contaminating environment (rate and duration of leptospires excretion) and transmission to other susceptible hosts.Previous studies reported that coypus seem to be a less efficient maintenance hosts rather than other species  , althougLeptospira, as well as some other infections given by multi-host pathogens, is not clearly defined, mainly due to the scarce epidemiological and ecological information. In this study, we reported a broad exposure and geographical distribution of Leptospira spp. in synanthropic species and wildlife in northeastern Italy. In agreement with studies in the previous literature [Leptospira spp. in Norway rats (23%), bank voles (30%), house mice (11.4%), a common shrew and in hedgehogs (17.6%). Among the carnivourous, one grey wolf reported positively for Leptospira and red foxes were also found to be positive with a prevalence of 21%. In addition, we detected a pathogenic Leptospira spp. positivity in a greater-mouse eared bat, but the low bacterial load did not allow the identification of the strain.The most shared and accepted definition of reservoir is “a population which is chronically infested with the causative agent of a disease and can infect other populations” . The conterature ,31, thisLeptospira infecting the animal host, providing a helpful and powerful tool in investigating the epidemiology of leptospirosis. In this study, the association of Leptospira ST with small mammals showed different patterns: serogroups Icterohaemorragiae, Sejroe, Javanica and Australis were detected in multiple host species  [L. Javanica ST 146 in this species suggests the broadening of the host spectrum of this serogroup among wild carnivores. Furthermore, we reported  (26.3%) , Croatia (26.3%) , Spain ( (26.3%)  and Norw (26.3%) . SignifiL. borgpetersenii Sejroe ST 197 in a bank vole. According to our results, ST 197 had already been reported in bank voles in Europe [Leptospira spp. and sources of infection for humans and livestock. As supposed for other Leptospira species, the detection of ST 197 in different geographical regions may suggest the development of new patterns of diffusion or transmission, questioning the ‘‘one pathogen-one carrier’’ hypothesis [To the best of the authors’ knowledge, this is the first Italian report of n Europe . The idepothesis .L. kirschneri, serogroup Pomona, serovar Mozdok (ST 117) in a grey wolf. This serogroup has also been reported among wild boars (Sus scrofa) with a seroprevalence of 4.18% in central Italy [L. kirschneri, serogroup Pomona, serovar Mozdok was the most frequently isolated Leptospira in small rodents [Leptospira through the analysis of specific fragments of bacterial loci. Through the assignment of sequence types (STs), MLST allows for objective comparisons to be made among Leptospira strains infecting the same host in different geographic regions or different host species within the same geographic area, thus providing a helpful and powerful tool to investigate the epidemiology of leptospirosis. As previously reported, these research teams described the circulation of Leptospira among domestic dogs: the most reported STs belong to L. interrogans Icterohaemorragiae , L. interrogans Australis . L. kirschneri serogroup Pomona serovar Mozdok (ST 117 and ST 289) and L. borgpetersenii serogroup Sejroe (ST 155) have also been identified in dogs with clinical leptospirosis [Leptospira strains distribution and the relationship at the wildlife/domestic animals/human interface.As previously reported in greater detail , in thisal Italy , 45% in al Italy . In east rodents , and seq rodents . In addi rodents . Therefo rodents  or to a  rodents  and mult rodents , can prospirosis . To dateLeptospira among wild and synanthropic animals is still not well defined. In our study, seasonality was not significantly related with the prevalence of Leptospira among the sampled animals, except in hedgehogs (p < 0.05), with a lower prevalence observed during winter, which could be associated with the winter hibernation period. By contrast, a recent study conducted in Spain [Leptospira prevalence in peri-urban micromammals ranged from 8% to 13%, and the probability of infection with Leptospira was three times higher in spring than in autumn. Other studies have reported a high probability of Leptospira detection among susceptible hosts in autumn and have demonstrated that seasonal patterns depend on the region and yearly rainfall in the period under investigation [To date, the role of seasonality in the prevalence of in Spain  reportedtigation ,23. In otigation .This study assessed the seasonality of Leptospira in red foxes caught predominantly in the alpine region, but no significant differences were found, even if previous studies had described that antibodies against specific serovars were more frequent during the winter period than during the summer .Leptospira; furthermore, this survey was conducted on a limited number of animals that certainly does not reflect the size of the animal population present in the studied area. In addition, the data reported in the present study refer to samples of animal carcasses, evaluated within the context of passive surveillance control programs; therefore, it was not possible to standardize sample collection, neither was it possible to collect specific epidemiological and time-related information.Generally, the present study assessed only the molecular detection of Leptospira in wild and synanthropic animals using standardized and repeatable protocols in order to assess prevalence trends among these species.In light of this, it would be useful to envisage monitoring programs for Leptospira strains in wild and synanthropic species, highlighting a possible connection between domestic-synanthropic-wild species and the shared environment. Therefore, the knowledge of regional epidemiology, which can be assessed only by the identification of locally prevalent strains either among domestic animals or wildlife, is necessary to understand infection patterns and transmission chains. Moreover, the lack of knowledge about the correlation between geographical, spatial, and meteorological information needs to be implemented in order to describe possible risk factors in the current climate conditions.The present study reported the presence of Leptospira across synanthropic species and wildlife in northeastern Italy and underlined the relevance of shared environmental exposure among the species. Particularly, L. Australis ST 24 and L. borgpetersenii Javanica ST 146 were reported in hedgehogs, mice, and foxes, suggesting the need to further investigate the possible prey–predator epidemiological scenario. Furthermore, L. Australis ST 198 was detected exclusively in hedgehogs. L. interrogans Icterohaemorragiae ST 17 was reported to be the most represented in rats, as expected, followed by hedgehogs, mice, and foxes. L. borgpetersenii Sejroe ST 155 was rarely reported (one hedgehog and one mouse), as well as L. borgpetersenii Sejroe ST 197 (one vole). L. kirschneri Pomona Mozdok ST 117 was described in one wolf, suggesting the importance of evaluating the wildlife/domestic interface. In the ecology of leptospirosis and of some other infections given by multi-host pathogens, the definition of reservoir is not totally clear. The detection of a particular ST in animal species belonging to specific ecological contexts , highlights the need to consider the challenge of characterizing the mechanisms and epidemiological pathways of Leptospira exposure or infection in a large number of host animals and the need to define their epidemiological role as environmental sentinel hosts or reservoirs.The study confirmed a broad exposure and geographical distribution of