CRISPR/Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus.

Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/µL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.

Ultrasound-Based Deep Learning Radiomics Nomogram for Tumor and Axillary Lymph Node Status Prediction After Neoadjuvant Chemotherapy.

RATIONALE AND OBJECTIVES: This study aims to explore the feasibility of the deep learning radiomics nomogram (DLRN) for predicting tumor status and axillary lymph node metastasis (ALNM) after neoadjuvant chemotherapy (NAC) in patients with breast cancer. Additionally, we employ a Cox regression model for survival analysis to validate the effectiveness of the fusion algorithm. MATERIALS AND METHODS: A total of 243 patients who underwent NAC were retrospectively included between October 2014 and July 2022. The DLRN integrated clinical characteristics as well as radiomics and deep transfer learning features extracted from ultrasound (US) images. The diagnostic performance of DLRN was evaluated by constructing ROC curves, and the clinical usefulness of models was assessed using decision curve analysis (DCA). A survival model was developed to validate the effectiveness of the fusion algorithm. RESULTS: In the training cohort, the DLRN yielded area under the receiver operating characteristic curve values of 0.984 and 0.985 for the tumor and LNM, while 0.892 and 0.870, respectively, in the test cohort. The consistency indices (C-index) of the nomogram were 0.761 and 0.731, respectively, in the training and test cohorts. The Kaplan-Meier survival curves showed that patients in the high-risk group had significantly poorer overall survival than patients in the low-risk group (P < 0.05). CONCLUSION: The US-based DLRN model could hold promise as clinical guidance for predicting the status of tumors and LNM after NAC in patients with breast cancer. This fusion model can also predict the prognosis of patients, which could help clinicians make better clinical decisions.

Different Contributions of embB and ubiA Mutations to Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates.

Objective: To explore the association between the variant mutations within embB and ubiA, and the degree of ethambutol (EMB) resistance of Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods: A total of 146 M. tuberculosis isolates were used to determine the minimum inhibitory concentrations (MICs) of EMB with a 96-well microplate-based assay. The mutations within embB and ubiA among these isolates were identified with DNA sequencing. Moreover, a multivariate regression model and a computer model were established to assess the effects of mutations on EMB resistance. Results: Our data showed that overall 100 isolates exhibited 28 mutated patterns within the sequenced embB and ubiA. Statistical analysis indicated that embB mutations Met306Val, Met306Ile, Gly406Ala, and Gln497Arg, were strongly associated with EMB resistance. Of these mutations, Met306Val and Gln497Arg were significantly associated with high-level EMB resistance. Almost all multiple mutations occurred in high-level EMB-resistant isolates. Although the mutation within ubiA accompanied with embB mutation presented exclusively in EMB-resistant isolates, four single ubiA mutations (Ala39Glu, Ser173Ala, Trp175Cys, and Val283Leu) leading to protein instability were observed in EMB-susceptible isolates. Conclusion: This study highlighted the complexity of EMB resistance. Some individual mutations and multiple mutations within embB and ubiA contributed to the different levels of EMB resistance.

Diagnostic Value of Ultrasound Endoscopy-Guided Fine-Needle Aspiration for Peritoneal Lesions.

Background: Peritoneal lesions present diagnostic challenges, necessitating precise imaging techniques. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) offers a promising approach for accurate diagnosis, aiding in optimal patient management and treatment planning. Objective: This study aims to assess the diagnostic efficacy of EUS-FNA in peritoneal lesions to offer insight in guiding optimal patient management. Methods: A prospective observational study was conducted, and a total of 58 patients who underwent EUS-FNA of the peritoneum at our hospital between October 2021 and November 2021 were included. The ultrasound diagnostic instrument facilitated puncture guidance, with 2-5 punctures performed in various parts of the selected peritoneal lesion areas. The analysis encompassed evaluating the sensitivity, specificity, positive predictive value, and negative predictive value of biopsy for diagnosing peritoneal-associated lesions, alongside assessing the number of punctures, puncture satisfaction, and incidence of postoperative complications. Results: The included patients undergoing EUS-FNA revealed that 41 (70.69%) had malignant lesions, while 17 (29.31%) presented with benign lesions. The diagnostic accuracy of EUS-FNA for peritoneal lesions was determined to be 94.83%, with a diagnostic sensitivity of 97.30% for malignant tumors, specificity of 90.48%, positive predictive value of 94.74%, and negative predictive value of 95%. Lesions exhibited a size range of 2.5cm × 2.9cm to 15.2cm × 9.8cm. Each patient underwent 2-5 punctures (3.3 ± 1.4), with a puncture satisfaction rate of 96.55%. The incidence of postoperative complications following EUS-FNA was found to be 3.45%. Conclusion: EUS-FNA exhibits substantial diagnostic utility for peritoneal-related lesions, marked by exceptional accuracy, sensitivity, specificity, and favorable safety. Its clinical adoption is warranted, promising improved patient care and management.

SARS-CoV-2 prevalence in wildlife 2020-2022: a worldwide systematic review and meta-analysis.

The widespread transmission of SARS-CoV-2 in humans poses a serious threat to public health security, and a growing number of studies have discovered that SARS-CoV-2 infection in wildlife and mutate over time. This article mainly reports the first systematic review and meta-analysis of the prevalence of SARS-CoV-2 in wildlife. The pooled prevalence of the 29 included articles was calculated by us using a random effects model (22.9%) with a high heterogeneity (I2 = 98.7%, p = 0.00). Subgroup analysis and univariate regression analysis found potential risk factors contributing to heterogeneity were country, wildlife species, sample type, longitude, and precipitation. In addition, the prevalence of SARS-CoV-2 in wildlife increased gradually over time. Consequently, it is necessary to comprehensively analyze the risk factors of SARS-CoV-2 infection in wildlife and develop effective control policies, as well as to monitor the mutation of SARS-CoV-2 in wildlife at all times to reduce the risk of SARS-CoV-2 transmission among different species.

The protective effect of intranasal immunization with influenza virus recombinant adenovirus vaccine on mucosal and systemic immune response.

Influenza virus is a kind of virus that poses several hazards of animal and human health. Therefore, it is important to develop an effective vaccine to prevent influenza. To this end we successfully packaged recombinant adenovirus rAd-NP-M2e-GFP expressing multiple copies of influenza virus conserved antigens NP and M2e and packaged empty vector adenovirus rAd-GFP. The effect of rAd-NP-M2e-GFP on the activation of dendritic cell (DC) in vitro and in vivo was detected by intranasal immunization. The results showed that rAd-NP-M2e-GFP promoted the activation of DC in vitro and in vivo. After the primary immunization and booster immunization of mice through the nasal immune way, the results showed that rAd-NP-M2e-GFP induced enhanced local mucosal-specific T cell responses, increased the content of SIgA in broncho alveolar lavage fluids (BALF) and triggered the differentiation of B cells in the germinal center. It is proved that rAd-NP-M2e-GFP can significantly elicit mucosal immunity and systemic immune response. In addition, rAd-NP-M2e-GFP could effectively protect mice after H1N1 influenza virus challenge. To lay the foundation and provide reference for further development of influenza virus mucosal vaccine in the future.

Gut microbiota-derived LCA mediates the protective effect of PEDV infection in piglets.

BACKGROUND: The gut microbiota is a critical factor in the regulation of host health, but the relationship between the differential resistance of hosts to pathogens and the interaction of gut microbes is not yet clear. Herein, we investigated the potential correlation between the gut microbiota of piglets and their disease resistance using single-cell transcriptomics, 16S amplicon sequencing, metagenomics, and untargeted metabolomics. RESULTS: Porcine epidemic diarrhea virus (PEDV) infection leads to significant changes in the gut microbiota of piglets. Notably, Landrace pigs lose their resistance quickly after being infected with PEDV, but transplanting the fecal microbiota of Min pigs to Landrace pigs alleviated the infection status. Macrogenomic and animal protection models identified Lactobacillus reuteri and Lactobacillus amylovorus in the gut microbiota as playing an anti-infective role. Moreover, metabolomic screening of the secondary bile acids' deoxycholic acid (DCA) and lithocholic acid (LCA) correlated significantly with Lactobacillus reuteri and Lactobacillus amylovorus, but only LCA exerted a protective function in the animal model. In addition, LCA supplementation altered the distribution of intestinal T-cell populations and resulted in significantly enriched CD8+ CTLs, and in vivo and in vitro experiments showed that LCA increased SLA-I expression in porcine intestinal epithelial cells via FXR receptors, thereby recruiting CD8+ CTLs to exert antiviral effects. CONCLUSIONS: Overall, our findings indicate that the diversity of gut microbiota influences the development of the disease, and manipulating Lactobacillus reuteri and Lactobacillus amylovorus, as well as LCA, represents a promising strategy to improve PEDV infection in piglets. Video Abstract.

Dual mutations in the whitefly nicotinic acetylcholine receptor ß1 subunit confer target-site resistance to multiple neonicotinoid insecticides.

Neonicotinoid insecticides, which target insect nicotinic acetylcholine receptors (nAChRs), have been widely and intensively used to control the whitefly, Bemisia tabaci, a highly damaging, globally distributed, crop pest. This has inevitably led to the emergence of populations with resistance to neonicotinoids. However, to date, there have been no reports of target-site resistance involving mutation of B. tabaci nAChR genes. Here we characterize the nAChR subunit gene family of B. tabaci and identify dual mutations (A58T&R79E) in one of these genes (BTß1) that confer resistance to multiple neonicotinoids. Transgenic D. melanogaster, where the native nAChR Dß1 was replaced with BTß1A58T&R79E, were significantly more resistant to neonicotinoids than flies where Dß1 were replaced with the wildtype BTß1 sequence, demonstrating the causal role of the mutations in resistance. The two mutations identified in this study replace two amino acids that are highly conserved in >200 insect species. Three-dimensional modelling suggests a molecular mechanism for this resistance, whereby A58T forms a hydrogen bond with the R79E side chain, which positions its negatively-charged carboxylate group to electrostatically repulse a neonicotinoid at the orthosteric site. Together these findings describe the first case of target-site resistance to neonicotinoids in B. tabaci and provide insight into the molecular determinants of neonicotinoid binding and selectivity.

Preliminary Application of a Continuous Functional Contrast Visual Acuity System in the Assessment of Visual Function in Dry Eye Patients.

Purpose: To investigate the feasibility and efficacy of a continuous functional contrast visual acuity (CFCVA) system in the assessment of visual function in dry eye disease (DED). Methods: Twenty patients with DED and 15 normal controls were recruited. Subjective symptoms were evaluated using the Ocular Surface Disease Index (OSDI) questionnaire, and tear film stability was assessed by a noninvasive corneal topographer. Under natural blinking conditions, the custom-built CFCVA system was used to take serial visual acuity measurements at 100%, 25%, 10%, and 5% contrast for 60 seconds. A 5-minute measurement at a 100% contrast level was defined as the stress test (ST). Mean CFCVA was defined, and visual maintenance ratio (VMR) was the ratio of mean CFCVA divided by baseline visual acuity. Results: In both groups, VMR decreased and mean CFCVA (logarithm of the minimum angle of resolution) increased with decreasing optotype contrast (from 100% to 5%). In ST, the ST VMR at the fourth and fifth minutes (VMR54 and VMR55) showed the strongest correlations with OSDI total, ocular symptoms, and vision-related function (-0.646 and -0.598, -0.688 and -0.693, and -0.599 and -0.555, respectively, P < 0.05). VMR54 and VMR55 also demonstrated the best discriminating ability for detecting DED, with areas under the curve of 0.903 and 0.867, respectively. Conclusions: Extending the continuous measuring time was more effective for detecting vision-related functional abnormalities in patients with DED than simply decreasing the optotype contrast level. Translational Relevance: The proposed CFCVA system and associated parameters offer a potential method for quantifying and interpreting the visual symptoms of DED in clinical care.

Over-expression of UDP-glycosyltransferase UGT353G2 confers resistance to neonicotinoids in whitefly (Bemisia tabaci).

The whitefly, Bemisia tabaci, comes up high metabolic resistance to most neonicotinoids in long-term evolution, which is the key problem of pest control. UGT glycosyltransferase, as a secondary detoxification enzyme, plays an indispensable role in detoxification metabolism. In this study, UGT inhibitors, 5-nitrouracil and sulfinpyrazone, dramatically augmented the toxic damage of neonicotinoids to B. tabaci. A UGT named UGT353G2 was identified in whitefly, which was notably up-regulated in resistant strain (3.92 folds), and could be induced by most neonicotinoids. Additionally, the using of RNA interference (RNAi) suppresses UGT353G2 substantially increased sensitivity to neonicotinoids in resistant strain. Our results support that UGT353G2 may be involved in the neonicotinoids resistance of whitefly. These findings will help further verify the functional role of UGTs in neonicotinoid resistance.

Peripheral CD4+ /CD8+ T cell composition distinct from healthy individuals is shared by ankylosing spondylitis and rheumatoid arthritis.

OBJECTIVE: Ankylosing spondylitis (AS) and rheumatoid arthritis (RA) are chronic inflammatory joint diseases, linking to the alterations of immune cells. We attempted to assess whether the alterations in the composition of CD4+ /CD8+ T cells are different between AS and RA and identify the characteristic cells between male and female patients. METHODS: The proportions of CD3+ or double positive T cells, 6 CD4+ T subsets and 9 CD8+ T cell subsets were detected by flow cytometry and compared in 30 healthy individuals, 42 AS patients and 45 RA patients. The differentially altered cells were individually analyzed for associations with disease activity parameters. In addition, their proportions were compared between different genders in the 3 groups. RESULTS: The proportions of CD4+ T cells, naive CD4+ T cells and central memory CD4+ T cells were lower in AS patients (P = 0.001, P = 0.002 and P = 0.007, respectively) and RA patients (P = 0.032, P < 0.001 and P = 0.016, respectively), but the proportion of effector memory ones was higher when compared with healthy populations (both P < 0.001), as were the decrease of naive/central memory CD8+ T cells in AS (P = 0.003 and P = 0.016, respectively) and RA (P < 0.001 and P = 0.006, respectively), and the increased tendency of terminally differentiated CD8+ T cells. However, these above-mentioned cells, regulatory T (Treg) cells and CD8+ T cells with different CD127 expressions between AS and RA were similar in proportion. Furthermore, naive CD4+ T cells were positively associated with C-reactive protein (CRP) in AS, whereas CD4+ T cells and terminally differentiated CD8+ T of RA patients were associated with CRP in RA. The gender-related alterations predominantly displayed the overexpressions of Treg cells and naive CD8+ T cells in female patients with AS and RA, respectively. CONCLUSIONS: AS patients and RA patients have some similar peripheral CD4+ /CD8+ T cell subsets but are distinct from healthy individuals, which may contribute to disease severity. Females are respectively characterized by the up-regulation of Treg cells and naive CD8+ T cells in AS patients and RA patients. The study offers an in-depth understanding of the role of T cell subsets in the similarities of the disorders and helps us to monitor disease changes and may offer a theoretical basis of developing novel therapies against common targets.

The study of the effect of HLA-B27 on THP-1 monocytic cells survival and its mechanism.

OBJECTIVE: Previous studies have shown that human leukocyte antigen (HLA)-B27 induces the accumulation of unfolded proteins in the endoplasmic reticulum (ER) to cause ER stress, resulting in the unfold protein response (UPR), apoptosis and autophagy. However, it is still unknown whether it affects the survival of monocytes. In this study, we attempted to examine the effect of HLA-B27 gene knockout on the proliferation and apoptosis of THP-1 monocytic cell line and its potential mechanism. METHODS: HLA-B27 gene knockout THP-1 cell line was constructed by lentivirus infection, and knockout efficiency was detected by immunofluorescence, quantitative reverse transcription - polymerase chain reaction (qRT-PCR) and western blot. Cell Counting Kit-8 (CCK-8) method and Annexin-V/PI double staining were used to detect the proliferation and apoptosis of the constructed THP-1 cell line, respectively. qRT-PCR was used to detect the effect of HLA-B27 inhibition on the expressions of ER molecular chaperone binding immunoglobulin protein (BiP) and genes about the UPR pathway. The proliferation rate of human BiP protein-stimulated THP-1 cells was detected by CCK-8 method. RESULTS: HLA-B27 gene knockout THP-1 cells were successfully constructed by lentivirus infection. Knockout of HLA-B27 effectively promoted the proliferation of THP-1 cells and inhibited the apoptosis induced by cisplatin. qRT-PCR showed that BiP was synchronously increased, while activation of UPR pathway was inhibited. Stimulation with human BiP promoted the proliferation of THP-1 cells in a concentration-dependent manner. CONCLUSIONS: HLA-B27 inhibition can promote the proliferation and inhibit the apoptosis of THP-1 cells. The inhibition function may be achieved through promotion of BiP and inhibition of UPR pathway activation.

Lactobacillus plantarum surface-displayed Eimeria tenella profilin antigens with FliC flagellin elicit protection against coccidiosis in chickens.

Coccidiosis is a parasitic disease in the intestine caused by the genus Eimeria that poses a substantial economic threat to the broiler breeding industry. The misuse of chemoprophylaxis and live oocyst vaccines has a negative impact on chicken reproductivity. Therefore, there is a pressing need to develop safe, convenient, and effective vaccines. Lactic acid bacteria can be used as a means to deliver mucosal vaccines against intestinal pathogens, which is a promising strategy. In this study, a recombinant Lactobacillus plantarum (L. plantarum) with surface-expressed antigens constructed from the fusion of Eimeria tenella (E. tenella) antigen profilin and the Salmonella enterica serovar Typhimurium flagellin protein FliC was created. After oral immunization with the recombinant L. plantarum, T-cell differentiation was analyzed by flow cytometry, and specific antibody levels were determined via indirect ELISA. Oocyst shedding, body weight, and cecum lesions were assessed as measures of protective immunity after challenge with E. tenella. The results of this study demonstrate the effectiveness of recombinant L. plantarum as an immunization agent for chickens. Specific IgA titers in the intestine and specific IgG antibody titers in the serum were significantly higher in chickens immunized with recombinant L. plantarum (P < 0.001). Additionally, the levels of IL-2 (P < 0.05) and IFN-γ (P < 0.01) in the serum were markedly increased. Recombinant L. plantarum induced T-cell differentiation, resulting in a higher proportion of CD4+ and CD8+ T cells in splenocytes (P < 0.001). Fecal oocyst shedding in the immunized group was significantly reduced (P < 0.001). Additionally, recombinant L. plantarum significantly relieved pathological damage in the cecum, as evidenced by lesion scores (P < 0.01) and histopathological cecum sections. In conclusion, the present study provides evidence to support the possibility of using L. plantarum as a promising carrier for the delivery of protective antigens to effectively protect chickens against coccidiosis.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

PKM2 promotes proinflammatory macrophage activation in ankylosing spondylitis.

Macrophages play a critical role in ankylosing spondylitis by promoting autoimmune tissue inflammation through various effector functions. The inflammatory potential of macrophages is highly influenced by their metabolic environment. Here, we demonstrate that glycolysis is linked to the proinflammatory activation of human blood monocyte-derived macrophages in ankylosing spondylitis. Specifically, ankylosing spondylitis macrophages produced excessive inflammation, including TNFα, IL1ß, and IL23, and displayed an overactive status by exhibiting stronger costimulatory signals, such as CD80, CD86, and HLA-DR. Moreover, we found that patient-derived monocyte-derived M1-type macrophages (M1 macrophages) exhibited intensified glycolysis, as evidenced by a higher extracellular acidification rate. Upregulation of PKM2 and GLUT1 was observed in ankylosing spondylitis-derived monocytes and monocyte-derived macrophages, especially in M1 macrophages, indicating glucose metabolic alteration in ankylosing spondylitis macrophages. To investigate the impact of glycolysis on macrophage inflammatory ability, we treated ankylosing spondylitis M1 macrophages with 2 inhibitors: 2-deoxy-D-glucose, a glycolysis inhibitor, and shikonin, a PKM2 inhibitor. Both inhibitors reduced proinflammatory function and reversed the overactive status of ankylosing spondylitis macrophages, suggesting their potential utility in treating the disease. These data place PKM2 at the crosstalk between glucose metabolic changes and the activation of inflammatory macrophages in patients with ankylosing spondylitis.

[Main Infection Control Measures for Respiratory Infectious Diseases in Medical Institutions and Public Places in China].

Respiratory infectious diseases (RID) are the major public health problems threatening the people's lives and health.Infection control (IC) is one of the effective tools to contain the occurrence and spread of RID.We collected the articles and data on IC published since January 1,2018 and summarized the achievements,problems,and challenges of IC from administrative control,management control,environment and engineering control,and personal protection in the medical institutions and public places in China.The efforts for IC vary in different regions and medical institutions of different levels.There are still links to be improved for IC from administrative control,management control,environment and engineering control,and personal protection,especially in community-level medical institutions and public areas.It is urgent to strengthen the implementation of IC policies and conduct IC precisely according to local situations.We proposed the following suggestions.First,the existing IC products and tools should be applied to precisely implement the IC measures;second,modern high technology should be employed to develop efficient and convenient IC products and tools;finally,a digital or intelligent IC platform should be built for monitoring infections,so as to contain the occurrence and spread of RID.

Knockdown of the Nicotinic Acetylcholine Receptor ß1 Subunit Decreases the Susceptibility to Five Neonicotinoid Insecticides in Whitefly (Bemisia tabaci).

The sweet potato whitefly, Bemisia tabaci, (Gennadius) (Hemiptera:Aleyrodidae) is a global pest of crops. Neonicotinoids are efficient insecticides used for control of this pest. Insecticidal targets of neonicotinoids are insect nicotinic acetylcholine receptors (nAChRs). Here, we characterized and cloned the full length of the nAChR ß1 subunit (BTß1) in B. tabaci and confirmed the consistency of BTß1 in B. tabaci MEAM1 and MED. Expression levels of BTß1 in different developmental stages and body parts of adults were investigated and compared in B. tabaci MED. dsRNA was prepared to knock down BTß1 in adult B. tabaci and significantly decreases the susceptibility to five neonicotinoid insecticides, including imidacloprid, clothianidin, thiacloprid, nitenpyram, and dinotefuran. This study indicated BTß1 as a notable site influencing the susceptibility of B. tabaci to neonicotinoids.

Changes in the morphology traits, anatomical structure of the leaves and transcriptome in Lycium barbarum L. under salt stress.

Salt stress directly affects the growth of plants. The limitation of leaf grow is among the earliest visible effects of salt stress. However, the regulation mechanism of salt treatments on leaf shape has not been fully elucidated. We measured the morphological traits and anatomical structure. In combination with transcriptome analysis, we analyzed differentially expressed genes (DEGs) and verified the RNA-seq data by qRT-PCR. Finally, we analyzed correlation between leaf microstructure parameters and expansin genes. We show that the leaf thickness, the width, and the leaf length significantly increased at elevated salt concentrations after salt stress for 7 days. Low salt mainly promoted the increase in leaves length and width, but high salt concentration accelerated the leaf thickness. The anatomical structure results indicated that palisade mesophyll tissues contribute more to leaf thickness than spongy mesophyll tissues, which possibly contributed to the increase in leaf expansion and thickness. Moreover, a total of 3,572 DEGs were identified by RNA-seq. Notably, six of the DEGs among 92 identified genes concentrated on cell wall synthesis or modification were involved in cell wall loosening proteins. More importantly, we demonstrated that there was a strong positive correlation between the upregulated EXLA2 gene and the thickness of the palisade tissue in L. barbarum leaves. These results suggested that salt stress possibly induced the expression of EXLA2 gene, which in turn increased the thickness of L. barbarum leaves by promoting the longitudinal expansion of cells of the palisade tissue. This study lays a solid knowledge for revealing the underlying molecular mechanisms of leaf thickening in L. barbarum in response to salt stresses.

NLRP3 Plays a Key Role in Antihelminth Immunity in the Enteral and Parenteral Stages of Trichinella spiralis-Infected Mice.

Trichinellosis is an important foodborne zoonosis, and no effective treatments are yet available. Nod-like receptor (NLR) plays a critical role in the host response against nematodes. Therefore, we aimed to explore the role of the NLRP3 inflammasome (NLRP3) during the adult, migrating, and encysted stages of Trichinella spiralis infection. The mice were treated with the specific NLRP3 inhibitor MCC950 after inoculation with T. spiralis. Then, the role that NLRP3 plays during T. spiralis infection of mice was evaluated using enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, histopathological evaluation, bone marrow-derived macrophage (BMDM) stimulation, and immunofluorescence. The in vivo results showed that NLRP3 enhanced the Th1 immune response in the adult and migrating stages and weakened the Th2 immune response in the encysted stage. NLRP3 promoted the release of proinflammatory factors (interferon gamma [IFN-γ]) and suppressed the release of anti-inflammatory factors (interleukin 4 [IL-4]). Pathological changes were also improved in the absence of NLRP3 in mice during T. spiralis infection. Importantly, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days postinfection was observed in mice treated with the specific NLRP3 inhibitor MCC950. In vitro, we first demonstrated that NLRP3 in macrophages can be activated by T. spiralis proteins and promotes IL-1ß and IL-18 release. This study revealed that NLRP3 is involved in the host response to T. spiralis infection and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated T. spiralis expulsion. These findings may help in the development of protocols for controlling trichinellosis.