ObjectiveTo investigate the effects and underlying mechanisms of Shenqi Wenfei prescription （SQWF） on chronic obstructive pulmonary disease （COPD）. MethodsA rat model of COPD with lung Qi deficiency was established using lipopolysaccharide （LPS） combined with cigarette smoke. Forty-eight SD rats were randomly divided into a blank group， a model group， low-， medium-， and high-dose SQWF groups （2.835， 5.67， 11.34 g·kg^-1）， and a Yupingfeng group （1.35 g·kg^-1）. Drug administration began on day 29 after modeling and continued for 2 weeks. The general condition of the rats was observed， and the lung function in each group was assessed. Hematoxylin-eosin （HE） staining was used to observe pathological changes in lung tissue. The proportion of inflammatory cells in bronchoalveolar lavage fluid （BALF） was measured. Apoptosis in lung tissue was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） staining. The release level of lactate dehydrogenase （LDH） in BALF was detected by a microplate assay. Reactive oxygen species （ROS） levels in lung tissue were detected using fluorescent probes. The levels of malondialdehyde （MDA）， total superoxide dismutase （SOD）， and reduced glutathione （GSH） in BALF were measured by biochemical methods. Ultrastructural changes in lung cells were observed via transmission electron microscopy. Double immunofluorescence staining was performed to detect the expression of thioredoxin-interacting protein （TXNIP） and nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） in lung tissue. Western blot analysis was used to detect the protein expression of TXNIP， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， cysteinyl aspartate-specific protease-1 （Caspase-1）， Caspase-1 p20， gasdermin D （GSDMD）， GSDMD N-terminal active fragment （GSDMD-N）， interleukin-1β （IL-1β）， and IL-18 in lung tissue. Serum IL-1β and IL-18 levels were measured by ELISA. ResultsCompared with the blank group， the model group showed lassitude， fatigue， tachypnea， and audible phlegm sounds， and lung function significantly declined （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were obvious. The level of inflammatory cells in BALF increased significantly （P0.05）. The number of TUNEL-positive cells increased （P0.01）. Levels of LDH， ROS， and MDA in BALF increased significantly （P0.01）， while GSH and SOD activities decreased significantly （P0.01）. Lung tissue cells showed irregular morphology， swollen mitochondria， disrupted cell membranes， and abundant vesicles， i.e.， pyroptotic bodies. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue were significantly elevated （P0.01）， and serum IL-1β and IL-18 levels also increased significantly （P0.01）. Compared with the model group， each medication group showed alleviation of qi deficiency symptoms and improved lung function （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were reduced. Inflammatory cell levels decreased （P0.05）. The number of TUNEL-positive cells decreased significantly （P0.01）. Levels of LDH， ROS， and MDA decreased significantly （P0.05）， while GSH and SOD activities significantly increased （P0.01）. Morphological and structural damage in lung tissue was improved to varying degrees. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue significantly decreased （P0.01）， and serum IL-1β and IL-18 levels also decreased significantly （P0.05）. ConclusionSQWF can improve lung function and alleviate inflammatory responses in COPD rats. Its mechanism may be related to regulating the ROS/TXNIP/NLRP3 pathway and inhibiting pyroptosis.

ObjectiveTo investigate the effects and underlying mechanisms of Shenqi Wenfei prescription （SQWF） on chronic obstructive pulmonary disease （COPD）. MethodsA rat model of COPD with lung Qi deficiency was established using lipopolysaccharide （LPS） combined with cigarette smoke. Forty-eight SD rats were randomly divided into a blank group， a model group， low-， medium-， and high-dose SQWF groups （2.835， 5.67， 11.34 g·kg^-1）， and a Yupingfeng group （1.35 g·kg^-1）. Drug administration began on day 29 after modeling and continued for 2 weeks. The general condition of the rats was observed， and the lung function in each group was assessed. Hematoxylin-eosin （HE） staining was used to observe pathological changes in lung tissue. The proportion of inflammatory cells in bronchoalveolar lavage fluid （BALF） was measured. Apoptosis in lung tissue was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） staining. The release level of lactate dehydrogenase （LDH） in BALF was detected by a microplate assay. Reactive oxygen species （ROS） levels in lung tissue were detected using fluorescent probes. The levels of malondialdehyde （MDA）， total superoxide dismutase （SOD）， and reduced glutathione （GSH） in BALF were measured by biochemical methods. Ultrastructural changes in lung cells were observed via transmission electron microscopy. Double immunofluorescence staining was performed to detect the expression of thioredoxin-interacting protein （TXNIP） and nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） in lung tissue. Western blot analysis was used to detect the protein expression of TXNIP， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， cysteinyl aspartate-specific protease-1 （Caspase-1）， Caspase-1 p20， gasdermin D （GSDMD）， GSDMD N-terminal active fragment （GSDMD-N）， interleukin-1β （IL-1β）， and IL-18 in lung tissue. Serum IL-1β and IL-18 levels were measured by ELISA. ResultsCompared with the blank group， the model group showed lassitude， fatigue， tachypnea， and audible phlegm sounds， and lung function significantly declined （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were obvious. The level of inflammatory cells in BALF increased significantly （P0.05）. The number of TUNEL-positive cells increased （P0.01）. Levels of LDH， ROS， and MDA in BALF increased significantly （P0.01）， while GSH and SOD activities decreased significantly （P0.01）. Lung tissue cells showed irregular morphology， swollen mitochondria， disrupted cell membranes， and abundant vesicles， i.e.， pyroptotic bodies. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue were significantly elevated （P0.01）， and serum IL-1β and IL-18 levels also increased significantly （P0.01）. Compared with the model group， each medication group showed alleviation of qi deficiency symptoms and improved lung function （P0.01）. Pulmonary emphysema and inflammatory cell infiltration were reduced. Inflammatory cell levels decreased （P0.05）. The number of TUNEL-positive cells decreased significantly （P0.01）. Levels of LDH， ROS， and MDA decreased significantly （P0.05）， while GSH and SOD activities significantly increased （P0.01）. Morphological and structural damage in lung tissue was improved to varying degrees. Protein levels of TXNIP， NLRP3， ASC， Caspase-1， Caspase-1 p20， GSDMD， GSDMD-N， IL-1β， and IL-18 in lung tissue significantly decreased （P0.01）， and serum IL-1β and IL-18 levels also decreased significantly （P0.05）. ConclusionSQWF can improve lung function and alleviate inflammatory responses in COPD rats. Its mechanism may be related to regulating the ROS/TXNIP/NLRP3 pathway and inhibiting pyroptosis.

ObjectiveTo investigate the effect of lower extremity exoskeleton robots on balance and walking function of patients with post-stroke cerebellar ataxia. MethodsA total of 60 patients with post-stroke cerebellar ataxia in Beijing Bo'ai Hospital from October, 2022 to October, 2024 were selected, and randomly divided into control group (n = 30) and exoskeleton group (n = 30) randomly. Both groups were given conventional exercise training, including trunk control training, rotation axis training and Frenkel training; the exoskeleton group received additional training with lower limb exoskeleton robots, for four weeks. Before and after treatment, the Gait Watch three-dimensional gait analyzer and the Holden Functional Ambulation Classification (HFAC) were used to evaluate the walking spatiotemporal parameters such as walking speed, walking frequency and step length deviation, as well as the walking ability. Berg Balance Scale (BBS) and the International Cooperative Ataxia Rating Scale (ICARS) were used to access the balance and ataxia functions, respectively. ResultsAfter treatment, the walking speed, walking frequency and step length deviation of both groups improved (|t| > 19.676, P < 0.001), the BBS score improved (|t| > 29.032, P < 0.001), and the ICARS scores decreased (t > 33.192, P < 0.001) in both groups, and they were better in the exoskeleton group than in the control group (|t| > 2.284, P < 0.05). There was no significant difference in the improvement rate of HFAC between two groups (P > 0.05). ConclusionLower extremity exoskeleton robots can effectively improve the balance and walking function of patients with post-stroke cerebellar ataxia.

Oral squamous cell carcinoma (OSCC) is a common head and neck malignancy. Approximately 50% to 60% of patients with OSCC are diagnosed at a locally advanced stage (clinical staging III-IVa). Even with comprehensive and sequential treatment primarily based on surgery, the 5-year overall survival rate remains below 50%, and patients often suffer from postoperative functional impairments such as difficulties with speaking and swallowing. Programmed death receptor-1 (PD-1) inhibitors are increasingly used in the neoadjuvant treatment of locally advanced OSCC and have shown encouraging efficacy. However, clinical practice still faces key challenges, including the definition of indications, optimization of combination regimens, and standards for efficacy evaluation. Based on the latest research advances worldwide and the clinical experience of the expert group, this expert consensus systematically evaluates the application of PD-1 inhibitors in the neoadjuvant treatment of locally advanced OSCC, covering combination strategies, treatment cycles and surgical timing, efficacy assessment, use of biomarkers, management of special populations and immune related adverse events, principles for immunotherapy rechallenge, and function preservation strategies. After multiple rounds of panel discussion and through anonymous voting using the Delphi method, the following consensus statements have been formulated: 1) Neoadjuvant therapy with PD-1 inhibitors can be used preoperatively in patients with locally advanced OSCC. The preferred regimen is a PD-1 inhibitor combined with platinum based chemotherapy, administered for 2-3 cycles. 2) During the efficacy evaluation of neoadjuvant therapy, radiographic assessment should follow the dual criteria of Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 and immune RECIST (iRECIST). After surgery, systematic pathological evaluation of both the primary lesion and regional lymph nodes is required. For combination chemotherapy regimens, PD-L1 expression and combined positive score need not be used as mandatory inclusion or exclusion criteria. 3) For special populations such as the elderly (≥ 70 years), individuals with stable HIV viral load, and carriers of chronic HBV/HCV, PD-1 inhibitors may be used cautiously under the guidance of a multidisciplinary team (MDT), with close monitoring for adverse events. 4) For patients with a poor response to neoadjuvant therapy, continuation of the original treatment regimen is not recommended; the subsequent treatment plan should be adjusted promptly after MDT assessment. Organ transplant recipients and patients with active autoimmune diseases are not recommended to receive neoadjuvant PD-1 inhibitor therapy due to the high risk of immune related activation. Rechallenge is generally not advised for patients who have experienced high risk immune related adverse events such as immune mediated myocarditis, neurotoxicity, or pneumonitis. 5) For patients with a good pathological response, individualized de escalation surgery and function preservation strategies can be explored. This consensus aims to promote the standardized, safe, and precise application of neoadjuvant PD-1 inhibitor strategies in the management of locally advanced OSCC patients.

Objective: To evaluate the performance of 5T magnetic resonance imaging (MRI) in visualizing dental pulp and periodontal ligament (PDL) tissues in vivo in the young adult population, thereby providing a basis for the application of high-field MRI technology in clinical oral examinations. Methods: The study was approved by the Ethics Committee of the hospital. A total of 15 healthy volunteers (413 permanent teeth altogether) were recruited and underwent full-mouth 5T MRI scans. Among them, six volunteers (168 permanent teeth) also received both 3T MRI and cone-beam computed tomography (CBCT) scans. Two dental specialists independently evaluated the imaging quality of the dental pulp and PDL on the images using a 5-point Likert scale and recorded the number of detectable root canals for each tooth. Inter-rater agreement was assessed using weighted kappa statistics and intraclass correlation coefficient (ICC). Non-parametric tests were employed to compare differences in imaging performance among different tissue structures, tooth positions, and imaging modalities. Results: 5T MRI can achieve in vivo imaging for most dental pulp tissues and partial periodontal membrane structures. There was a high level of agreement between the two raters in their imaging scores for the dental pulp and PDL (dental pulp κ = 0.934, PDL κ = 0.737). The imaging scores for dental pulp were significantly higher than those for PDL (P < 0.001), and the scores for molar dental pulp were lower than those for premolars and anterior teeth. In the multimodal comparison involving six volunteers, the raters showed good consistency in scoring dental pulp and PDL imaging across 5T MRI, 3T MRI, and CBCT, as well as in root canal counts (5T MRI for dental pulp κ = 0.971, 3T MRI for dental pulp κ = 0.933, CBCT for dental pulp κ = 0.964; 5T MRI for PDL κ = 0.625, 3T MRI for PDL κ = 0.667, CBCT for PDL κ = 0.571; ICC for root canal counts all ≥ 0.990). The imaging scores for dental pulp and PDL using 5T MRI were significantly higher than those using 3T MRI (dental pulp: P < 0.001; PDL: P = 0.022), but there was no statistically significant difference in the detection rate of the number of root canals between the two (P > 0.05). Although the imaging scores for dental pulp and PDL as well as the detection rate of the number of root canals with 5T MRI were inferior to those with CBCT (dental pulp: P < 0.001; PDL: P = 0.02; number of root canals: P < 0.05), 5T MRI can truly achieve "direct imaging" of these two soft tissues. Conclusion 5T MRI enables effective in vivo direct imaging of dental pulp and PDL tissues in the young adult population, indicating its potential clinical application value in the diagnosis and treatment of pulp and periodontal diseases.

Objective: To analyze the changes and related factors of influenza vaccine hesitancy among college students in Shanghai from 2022 to 2025, so as to provide evidence for influenza prevention and control in universities. Methods: A stratified random cluster sampling method was used to select 1 393 college students from Shanghai universities for electronic questionnaire surveys in June 2022 and January to February 2025. The survey mainly included influenza vaccination status, sources of vaccine related information, and potential related factors. Data were analyzed using the Chi-square test, Mann-Whitney U test, and binary Logistic regression. Results: The influenza vaccine hesitancy rate among college students in Shanghai was higher in 2025 ( 40.63 %) than in 2022 (31.95%) ( χ 2=22.73), and the hesitancy score [2022:0 (0,5);2025:0 (0,5)] also increased ( Z = -4.16 ) (both P <0.01). The proportion of students exposed to reports of vaccine adverse events rose from 36.90% in 2022 to 59.22 % in 2025 ( χ 2=139.08, P <0.01). Binary Logistic regression analysis showed that basic influenza knowledge (2022: OR = 0.79 , 95% CI =0.68-0.92; 2025: OR =0.79, 95% CI =0.69-0.90) and previous self paid vaccination with other vaccines (2022: OR =0.52, 95% CI =0.37-0.75; 2025: OR =0.63, 95% CI =0.48-0.83) were associated with influenza vaccine hesitancy among college students in both years; medical professional background ( OR =0.34, 95% CI =0.17-0.67) and trust in healthcare providers recommendations ( OR =0.31, 95% CI =0.11-0.86) were associated with influenza vaccine hesitancy among college students in 2025 (all P <0.05). Conclusions The phenomenon of influenza vaccine hesitancy among college students in Shanghai has intensified. Increased knowledge has failed to offset concerns arising from exposure to negative information. Medical background and recommendations from healthcare providers have become new related factors.

ObjectiveTo investigate the protective effect of α-ketoglutarate （α-KG） on hepatic lipid deposition in offspring caused by arsenic exposure during pregnancy. Methods8-week-old institute of cancer research （ICR） mice were mated in a ratio of 2∶1 between females and males， and the detection of vaginal plugs confirmed pregnant. A total of 32 pregnant mice were randomly divided into four groups： control group， arsenic group， α-KG group， arsenic+α-KG group. On gestational day 0-16 （GD0-GD16）， the arsenic and arsenic+α-KG groups were exposed to sodium arsenite （NaAsO₂ ，15 mg/L） in drinking water everyday， and the α-KG and arsenic+α-KG groups were gavaged with α-KG （2 g/kg） everyday. On GD16， pregnant mice were euthanized to collect fetal liver， and fetal body weight and crown-rump length were measured. Gene expression differences between the control group and the arsenic group were analyzed by transcriptome. The total triglycerides （TGs） and subtypes in fetal liver were detected by liquid chromatography tandem mass spectrometry （LC-MS/MS）. Oil red O staining was used to observe the histopathological changes in the liver. Quantitative polymerase chain reaction （qPCR） was used to detect the expression level of genes related to lipid synthesis， transport， and degradation， and phosphatidylinositol 3' -kinase/ protein kinase B （PI3K/AKT） in the liver of fetus. ResultsTranscriptomics analysis showed that 2 144 genes were downregulated and 1 675 genes were upregulated in the arsenic exposed fetal liver； body weight and crown-rump length were reduced （P_TuKey<0.05）； the level of hepatic TGs was elevated in arsenic group （P_TuKey<0.05）； oil-red O staining showed a significant increase in lipid droplets in arsenic group （P_TuKey<0.01）； the expression of lipid synthesis-related genes were significantly upregulated （P_TuKey<0.05）； the expression of β-oxidation-related genes and lipid degradation-related genes were downregulated （P_TuKey<0.05）； the expression of PI3K， AKT decreased（P_TuKey<0.05）. Compared with the arsenic group， the body weight and crown-rump length of fetus increased in the arsenic+α-KG group （P_TuKey<0.05）； the level of hepatic TGs decreased in the arsenic+α-KG group （P_TuKey<0.05）； oil red O staining showed lipid droplets significantly decreased （P_TuKey<0.01）； the expression of lipid synthesis-related genes were downregulated （P_TuKey<0.05）， the expression of β-oxidation-related genes and lipid degradation-related genes were upregulated （P_TuKey<0.05）； the expression levels of PI3K and AKT increased （P_TuKey<0.05）. Conclusionα-KG alleviated hepatic lipid deposition in offspring exposed to arsenic during pregnancy through activating PI3K/AKT signaling pathway.

Objective To identify factors affecting and key environmental factors of the Oncomelania hupensis snail density in the Yangtze River Delta region using machine learning methods. Methods Administrative village-level O. hupensis snail survey data in the Yangtze River Delta (including Shanghai Municipality, Jiangsu Province, Zhejiang Province and Anhui Province) from 2011 to 2021 were retrieved from the Information Management System for Parasitic Disease Control of Chinese Center for Disease Control and Prevention. Environmental factor data were captured from the Google Earth Engine platform, including elevation, slope, terrain, normalized difference vegetation index (NDVI), vegetation type, soil type, total petroleum hydrocarbon (TPH), ammonium nitrogen, inorganic nitrogen, dissolved oxygen, pH of water, chemical oxygen demand (COD) and inorganic phosphorus, and climatic factor data in the study region were retrieved from the Copernicus Climate Data Store, including annual precipitation, aridity index and annual mean temperature (AMT). O. hupensis snail survey data in the Yangtze River Delta region from 2011 to 2021 were randomly divided into a training set (70%) and a test set (30%), and five machine learning models were selected for machine learning model construction and comparative analysis of the O. hupensis snail density using the software R 4.3.0, including random forest (RF), eXtreme gradient boosting (XGBoost), support vector machine (SVM), gradient boosting machine (GBM) and neural network (NN). The XGBoost model was employed to construct a predictive model for the O. hupensis snail density, and the impact of each environmental factor on O. hupensis snail distribution was quantified. The SHapley Additive exPlanations (SHAPs) values were calculated to estimate the average contribution of each variable to the model prediction, and the core environmental factors affecting the O. hupensis snail population density were screened. Results Among the five machine learning models, the XGBoost model exhibited the optimal comprehensive performance, with the coefficient of determination (R²) of 0.855, mean squared error (MSE) of 0.188, root mean squared error (RMSE) of 0.434 and mean absolute error (MAE) of 0.155, respectively. Analysis of factors affecting the O. hupensis snail density with the XGBoost model showed that among the 16 environmental factors, the top four high-impact factors ranked by SHAPs values included annual precipitation, elevation, aridity index and NDVI, with cumulative SHAPs contributions of 75%, which was higher than that of other environmental factors. If NDVI was higher than 0.6, the O. hupensis snail density increased with NDVI and peaked if NDVI was 0.8 (1.60 snails/0.1 m²). The O. hupensis snail density increased with elevation if the elevation ranged from 14 to 40 m, and slowly rose if the annual precipitation ranged from 900 to 1 300 mm, and then increased rapidly to the peak (1.52 snails/0.1 m²) if the annual precipitation ranged from 1 300 to 1 500 mm. In addition, the O. hupensis snail density increased rapidly to the maximum (1.60 snails/0.1 m²) if the aridity index ranged from 0.8 to 1.1, and decreased gradually if the aridity index exceeded 1.1. Conclusions The XGBoost model shows excellent performance in prediction of the O. hupensis snail density and identification of key environmental factors in the Yangtze River Delta region. Annual precipitation, elevation, aridity index and NDVI are key environmental factors affecting the distribution and density of O. hupensis snails in the Yangtze River Delta region.

Objective To investigate the species of sandflies and the prevalence of Leishmania infections in sandflies from selected areas of northern and northwestern China, so as to provide insights into identification of leishmaniasis vectors and assessment of epidemiological trends of leishmaniasis in China. Methods Sandfly samples were collected from Mentougou District of Beijing Municipality, Xiangning County in Linfen City of Shanxi Province, Ejin Banner in Alxa League of Inner Mongolia Autonomous Region, and Payzawat County of Karamay District of Karamay City, Gaochang District of Turpan City in Xinjiang Uygur Autonomous Region from July 2023 to July 2024. Approximately 100 intact female sandfly samples were randomly selected from each site and the species of sandflies was identified according to morphological characteristics and molecular assays. Female sandflies originating from the same habitat were grouped into pools of 10 individuals. Leishmania infection was detected using polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 1 (ITS-1) gene, and the prevalence of Leishmania infection was calculated in sandflies from different sampling sites using the minimum infection rate (MIR) method. In addition, positive amplicons were sequenced and subjected to phylogenetic analysis. Results A total of 6 155 sandflies were collected from different environments at sampling sites across the six aforementioned regions from July 2023 to July 2024. Phlebotomus chinensis (96.00%) was the dominant sandfly species in Mentougou District, Beijing Municipality, with a small proportion of Ph. sergenti (4.00%), and only Ph. chinensis was found in Xiangning County, Linfen City, Shanxi Province. Ph. wui was the only sandfly species detected in Ejin Banner, Alxa League, Inner Mongolia Autonomous Region, and Payzawat County, Kashgar City, Xinjiang Uygur Autonomous Region, and Ph. caucasicus (97.70%) was the dominant sandfly species in Karamay District, Karamay City, Xinjiang Uygur Autonomous Region, with a small proportion of Ph. wui (2.30%), while Ph. alexandri was the only species in Gaochang District, Turpan City, Xinjiang Uygur Autonomous Region. A total of 40, 60, 34, 18, 18, and 22 pools of sandfly samples were tested from Mentougou District in Beijing Municipality, Xiangning County in Linfen City of Shanxi Province, Ejin Banner in Alxa League of Inner Mongolia Autonomous Region, Payzawat County in Kashgar City, Karamay District in Karamay City, and Gaochang District in Turpan City of Xinjiang Uygur Autonomous Region, respectively. L. infantum was detected in Ph. chinensis samples from Mentougou District in Beijing Municipality, and Xiangning County of Linfen City in Shanxi Province, with MIR of 0.25% to 1.00%, and L. donovani was detected in Ph. wui from Ejin Banner in Alxa League of Inner Mongolia Autonomous Region, and Payzawat County in Kashgar City of Xinjiang Uygur Autonomous Region, with MIR of 0.56% to 0.88%; however, no Leishmania infection was detected in Ph. caucasicus from Karamay District in Karamay City or Ph. alexandri from Gaochang District in Turpan City of Xinjiang Uygur Autonomous Region. Phylogenetic analysis showed that the Leishmania ITS-1 gene sequences obtained from Mentougou District in Beijing Municipality and Xiangning County in Linfen City of Shanxi Province were clustered into the same clade with the reference sequences of L. infantum ITS-1 gene, while the Leishmania ITS-1 gene sequences obtained from Ejin Banner in Alxa League of Inner Mongolia Autonomous Region and Payzawat County in Kashgar City of Xinjiang Uygur Autonomous Region were clustered into the same clade with the reference sequences of L. donovani ITS-1 gene. Conclusions There are variations in sandfly species in selected areas of northern and northwestern China, and variations in the species of Leishmania infecting sandflies. Improved surveillance of sandfly vectors and targeted control strategies with adaptations to geographical features and leishmaniasis vectors are recommended.

Objective To examine the impact of extreme temperature and drought stress on the survival of Bulinus globosus, so as to provide the theoretical evidence for the genomic research of Bulinus in absence of reference genes. Methods B. globosus snail samples were collected from Kiwani Shehia in Pemba Island, Zanzibar, Tanzania, and offspring snails were obtained through laboratory breeding and reproduction. A total of 120 10-week-old B. globosus snails from the same generation were selected and randomly assigned into four groups, including the high-temperature drought (HD) group, normal temperature drought (D) group, low-temperature drought (LD) group, and the control (C) group, of 30 snails in each group. Snails in HD, D, and LD groups were placed in beakers containing dry soil at the bottom and subsequently housed in climate chambers at 35, 26 ℃, and 10 ℃, respectively, while snails in Group C were maintained in 500 mL petri dishes containing dechlorinated tap water at 26 ℃. Following 3 days of breeding, living snails in each group were collected, and soft tissues were dissected and isolated. Total RNA was extracted from snail soft tissues for library construction, followed by high-throughput sequencing on the Illumina HiSeq 4000 sequencing system. De novo transcriptome assembly was performed using the Trinity software, and the longest transcripts were selected as unigenes. Gene functional annotations of unigenes were conducted using the Diamond software against Gene Ontology (GO) knowledgebase, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, NCBI non-redundant (NR) protein sequences database, Protein Family (Pfam) database, and UniProtKB/Swiss-Prot (Swiss-Prot) knowledgebase. GO and KEGG enrichment analyses of differentially expressed genes (DEGs) were performed using the topGO and clusterProfiler software, respectively. In addition, four relevant genes were selected for validation using a real-time quantitative PCR (qRT-PCR) assay to verify the reliability of transcriptome sequencing results. Results Following 3 days of breeding, there were 7, 20, 28, and 30 survival B. globosus snails in HD, LD, D, and C groups, with corresponding survival rates of 23.33% (7/30), 66.67% (20/30), 93.33% (28/30), and 100.00% (30/30), respectively (χ² = 52.72, P < 0.001). De novo transcriptome assembly generated 176 942 unigenes, with annotation rates of 0.98%, 13.49%, 26.46%, 12.48%, and 14.39% against GO knowledgebase, KEGG pathway database, NR protein sequences database, Pfam database, and Swiss-Prot knowledgebase, respectively. There were 33 up-regulated and 72 down-regulated genes in Group D, 483 up-regulated and 815 down-regulated genes in Group HD, and 245 up-regulated and 172 down-regulated genes in Group LD relative to in Group C. Following removal of overlapping genes across groups and unmatched genes, 11 candidate genes were identified. GO and KEGG analyses revealed 3 heat shock protein (HSP)-related DEGs in these 11 candidate genes, which were annotated as HSP12.2, HSP70, and HSP20 genes and were all significantly up-regulated in each treatment group. Three immune and nervous system-related DEGs were identified, and were all significantly down-regulated in each treatment group, which were involved in the neural cell adhesion molecule L1-like protein pathway, fibrinogen binding protein pathway, and leukocyte elastase inhibitor-like protein pathway. qRT-PCR assay quantified that the expression trends of four genes related to temperature and drought stress across different treatment groups were highly consistent with transcriptome sequencing data. Conclusion The survival rate of B. globosus significantly reduces under combined stresses of extreme temperature and drought, possibly due to an imbalance in its cellular homeostasis regulatory system.