Prognostic Utility of Left Atrial Strain From MRI Feature Tracking in Ischemic and Nonischemic Dilated Cardiomyopathy: A Multicenter Study.

BACKGROUND. MRI-based prognostic evaluation in patients with dilated cardiomyopathy (DCM) has historically used markers of late gadolinium enhancement (LGE) and feature tracking (FT)-derived left ventricular global longitudinal strain (LVGLS). Early data indicate that FT-derived left atrial strain (LAS) parameters, including reservoir, conduit, and booster, may also have prognostic roles in such patients. OBJECTIVE. The purpose of our study was to evaluate the prognostic utility of LAS parameters, derived from MRI FT, in patients with ischemic or nonischemic DCM, including in comparison with the traditional parameters of LGE and LVGLS. METHODS. This retrospective study included 811 patients with ischemic or nonischemic DCM (median age, 60 years; 640 men, 171 women) who underwent cardiac MRI at any of five centers. FT-derived LAS parameters and LVGLS were measured using two- and four-chamber cine images. LGE percentage was quantified. Patients were assessed for a composite outcome of all-cause mortality or heart failure hospitalization. Multivariable Cox regression analyses including demographic characteristics, cardiovascular risk factors, medications used, and a wide range of cardiac MRI parameters were performed. Kaplan-Meier analyses with log-rank tests were also performed. RESULTS. A total of 419 patients experienced the composite outcome. Patients who did, versus those who did not, experience the composite outcome had larger LVGLS (-6.7% vs -8.3%, respectively; p < .001) as well as a smaller LAS reservoir (13.3% vs 19.3%, p < .001), LAS conduit (4.7% vs 8.0%, p < .001), and LAS booster (8.1% vs 10.3%, p < .001) but no significant difference in LGE (10.1% vs 11.3%, p = .51). In multivariable Cox regression analyses, significant independent predictors of the composite outcome included LAS reservoir (HR = 0.96, p < .001) and LAS conduit (HR = 0.91, p < .001). LAS booster and LGE were not significant independent predictors in the models. LVGLS was a significant independent predictor only in a model that initially included LAS booster but not the other LAS parameters. In Kaplan-Meier analysis, all three LAS parameters were significantly associated with the composite outcome (p < .001). CONCLUSION. In this multicenter study, LAS reservoir and LAS conduit were significant independent prognostic markers in patients with ischemic or nonischemic DCM, showing greater prognostic utility than the currently applied markers of LVGLS and LGE. CLINICAL IMPACT. FT-derived LAS analysis provides incremental prognostic information in patients with DCM.

Transcutaneous electrical cranial-auricular acupoint stimulation versus escitalopram for mild-to-moderate depression: An assessor-blinded, randomized, non-inferiority trial.

AIM: Transcutaneous electrical cranial-auricular acupoint stimulation (TECAS) is a novel non-invasive therapy that stimulates acupoints innervated by the trigeminal and auricular vagus nerves. An assessor-blinded, randomized, non-inferiority trial was designed to compare the efficacy of TECAS and escitalopram in mild-to-moderate major depressive disorder. METHODS: 468 participants received two TECAS sessions per day at home (n = 233) or approximately 10-13 mg/day escitalopram (n = 235) for 8 weeks plus 4-week follow-up. The primary outcome was clinical response, defined as a baseline-to-endpoint ≥50% reduction in Montgomery-Åsberg Depression Rating Scale (MADRS) score. Secondary outcomes included remission rate, changes in the severity of depression, anxiety, sleep and life quality. RESULTS: The response rate was 66.4% on TECAS and 63.2% on escitalopram with a 3.2% difference (95% confidence interval [CI], -5.9% to 12.9%) in intention-to-treat analysis, and 68.5% versus 66.2% with a 2.3% difference (95% CI, -6.9% to 11.4%) in per-protocol analysis. The lower limit of 95% CI of the differences fell within the prespecified non-inferiority margin of -10% (P ≤ 0.004 for non-inferiority). Most secondary outcomes did not differ between the two groups. TECAS-treated participants who experienced psychological trauma displayed a markedly greater response than those without traumatic experience (81.3% vs 62.1%, P = 0.013). TECAS caused much fewer adverse events than escitalopram. CONCLUSIONS: TECAS was comparable to escitalopram in improving depression and related symptoms, with high acceptability, better safety profile, and particular efficacy in reducing trauma-associated depression. It could serve an effective portable therapy for mild-to-moderate depression.

Passivation Effect of the Chlorinated Paraffin Added in the Cutting Fluid on the Surface Corrosion Resistance of the Stainless Steel.

Cutting fluids are the most effective method to lower the cutting temperature and decrease the cutting tool wear. At the same time, the cutting fluids influence the corrosion resistance property of the machined surface. In this study, chlorinated paraffin (CP), which is a common additive in the cutting fluid, was selected as the research objective to study its corrosion resistance property. The passivation effect of CP with different concentrations on the machined surface of stainless steel was studied. Electrochemical measurements and surface morphology investigation were used to characterize the passivation effect of CP with different concentrations. The test results showed that the corrosion resistance of stainless steel in the cutting fluid was enhanced with the increase in CP additive. This reason is that the charge transfer resistance increases and the corrosion current density decreases with the increase in CP additive. The X-ray photoelectron spectroscopy (XPS) results show that the proportion of metal oxides on the processed surface of the stainless steel sample was increased from 20.4% to 22.0%, 32.9%, 26.6%, and 31.1% after adding 1 mL, 2 mL, 4 mL and 6 mL CP in the cutting fluid with a total volume of 500 mL, respectively. The oxidation reaction between CP and the stainless steel sample resulted in an increase in metal oxides proportion, which prevented the stainless steel sample from corrosion in cutting fluid.

[Role and mechanisms of CHI3L1 in coronary artery lesions in a mouse model of Kawasaki disease-like vasculitis]. / CHI3L1åœ¨å·å´Žç— æ ·è¡€ç®¡ç‚Žå°é¼ æ¨¡åž‹å† çŠ¶åŠ¨è„‰æŸä¼¤ä¸­çš„ä½œç”¨åŠæœºåˆ¶ç ”ç©¶.

OBJECTIVES: To explore the role and potential mechanisms of chitinase-3-like protein 1 (CHI3L1) in coronary artery lesions in a mouse model of Kawasaki disease (KD)-like vasculitis. METHODS: Four-week-old male SPF-grade C57BL/6 mice were randomly divided into a control group and a model group, with 10 mice in each group. The model group mice were intraperitoneally injected with 0.5 mL of lactobacillus casei cell wall extract (LCWE) to establish a mouse model of KD-like vasculitis, while the control group mice were injected with an equal volume of normal saline. The general conditions of the mice were observed on the 3rd, 7th, and 14th day after injection. Changes in coronary artery tissue pathology were observed using hematoxylin-eosin staining. The level of CHI3L1 in mouse serum was measured by enzyme-linked immunosorbent assay. Immunofluorescence staining was used to detect the expression and localization of CHI3L1, von Willebrand factor (vWF), and α-smooth muscle actin (α-SMA) in coronary artery tissue. Western blot analysis was used to detect the expression of CHI3L1, vWF, vascular endothelial cadherin (VE cadherin), Caspase-3, B cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), nuclear factor κB (NF-κB), and phosphorylated NF-κB (p-NF-κB) in coronary artery tissue. RESULTS: The serum level of CHI3L1 in the model group was significantly higher than that in the control group (P<0.05). Compared to the control group, the expression of CHI3L1 in the coronary artery tissue was higher, while the expression of vWF was lower in the model group. The relative expression levels of CHI3L1, Bax, Caspase-3, NF-κB, and p-NF-κB were significantly higher in the model group than in the control group (P<0.05). The relative expression levels of vWF, VE cadherin, and Bcl-2 were lower in the model group than in the control group (P<0.05). CONCLUSIONS: In the LCWE-induced mouse model of KD-like vasculitis, the expression levels of CHI3L1 in serum and coronary arteries increase, and it may play a role in coronary artery lesions through endothelial cell apoptosis mediated by inflammatory reactions.

Transcriptomic analysis of α-linolenic acid content and biosynthesis in Paeonia ostii fruits and seeds.

BACKGROUND: Paeonia ostii is a potentially important oilseed crop because its seed yield is high, and the seeds are rich in α-linolenic acid (ALA). However, the molecular mechanisms underlying ALA biosynthesis during seed kernel, seed testa, and fruit pericarp development in this plant are unclear. We used transcriptome data to address this knowledge gap. RESULTS: Gas chromatograph-mass spectrometry indicated that ALA content was highest in the kernel, moderate in the testa, and lowest in the pericarp. Therefore, we used RNA-sequencing to compare ALA synthesis among these three tissues. We identified 227,837 unigenes, with an average length of 755 bp. Of these, 1371 unigenes were associated with lipid metabolism. The fatty acid (FA) biosynthesis and metabolism pathways were significantly enriched during the early stages of oil accumulation in the kernel. ALA biosynthesis was significantly enriched in parallel with increasing ALA content in the testa, but these metabolic pathways were not significantly enriched during pericarp development. By comparing unigene transcription profiles with patterns of ALA accumulation, specific unigenes encoding crucial enzymes and transcription factors (TFs) involved in de novo FA biosynthesis and oil accumulation were identified. Specifically, the bell-shaped expression patterns of genes encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE, CLE, and SLE in the kernel were similar to the patterns of ALA accumulation in this tissue. Genes encoding BCCP, BC, KAS I- III, and FATA were also upregulated during the early stages of oil accumulation in the kernel. In the testa, the upregulation of the genes encoding SAD, FAD2, and FAD3 was followed by a sharp increase in the concentrations of ALA. In contrast, these genes were minimally expressed (and ALA content was low) throughout pericarp development. CONCLUSIONS: We used three tissues with high, moderate, and low ALA concentrations as an exemplar system in which to investigate tissue-specific ALA accumulation mechanisms in P. ostii. The genes and TFs identified herein might be useful targets for future studies of ALA accumulation in the tree peony. This study also provides a framework for future studies of FA biosynthesis in other oilseed plants.

CEBPG promotes acute myeloid leukemia progression by enhancing EIF4EBP1.

BACKGROUND: Acute myeloid leukemia (AML) is a myeloid neoplasm accounts for 7.6% of hematopoietic malignancies. AML is a complex disease, and understanding its pathophysiology is contributing to the improvement in the treatment and prognosis of AML. In this study, we assessed the expression profile and molecular functions of CCAAT enhancer binding protein gamma (CEBPG), a gene implicated in myeloid differentiation and AML progression. METHODS: shRNA mediated gene interference was used to down-regulate the expression of CEBPG in AML cell lines, and knockdown efficiency was detected by RT-qPCR and western blotting. The effect of knockdown on the growth of AML cell lines was evaluated by CCK-8. Western blotting was used to detect PARP cleavage, and flow cytometry were used to determine the effect of knockdown on apoptosis of AML cells. Genes and pathways affected by knockdown of CEBPG were identified by gene expression analysis using RNA-seq. One of the genes affected by knockdown of CEBPG was Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), a known repressor of translation. Knockdown of EIF4EBP1 was used to assess its potential role in AML progression downstream of CEBPG. RESULTS: We explored the ChIP-Seq data of AML cell lines and non-AML hematopoietic cells, and found CEBPG was activated through its distal enhancer in AML cell lines. Using the public transcriptomic dataset, the Cancer Cell Line Encyclopedia (CCLE) and western blotting, we also found CEBPG was overexpressed in AML. Moreover, we observed that CEBPG promotes AML cell proliferation by activating EIF4EBP1, thus contributing to the progression of AML. These findings indicate that CEBPG could act as a potential therapeutic target for AML patients. CONCLUSION: In summary, we systematically explored the molecular characteristics of CEBPG in AML and identified CEBPG as a potential therapeutic target for AML patients. Our findings provide novel insights into the pathophysiology of AML and indicate a key role for CEBPG in promoting AML progression.

X-Linked Hyper IgM Syndrome Manifesting as Recurrent Pneumocystis jirovecii Pneumonia: A Case Report.

We reported a Chinese boy with X-linked hyper IgM (XHIGM) syndrome, manifesting as recurrent and severe pneumonia caused by Pneumocystis jirovecii. His parents were healthy and unrelated. In August 2018, the 5-month-old boy manifested as cough and dyspnea, and then in July 2019, he was admitted because of the same symptoms. Immunological results of the two admissions both showed low IgG, low IgA, normal IgM and high levels of 1,3-ß-D-glucan (BDG). Using next-generation sequencing (NGS), great reading counts of P. jirovecii were identified from the deep sputum in both admissions. Caspofungin combined with trimethoprim-sulfamethoxazole were used to anti-infection, and he recovered quickly. Whole-exome sequencing was performed for this family because of immune suppression, the disease-causing gene (exon 10-22 of CD40L) deletion for XHIGM syndrome was identified. NGS is beneficial for etiology diagnosis. Pneumocystis jirovecii pneumonia as an opportunistic infection could be recurrent in patients with XHIGM syndrome.

Exploring potential proteins associated with cyclin B 3'UTR in Procambarus clarkii oocytes.

Cyclin B is a ubiquitous regulatory molecule and has been implicated in mitosis and meiosis in oocytes. Phenomenon that differ in the length of cyclin B 3'UTR in crustacean has attracted much attention, although molecular details are poorly understood. The study of 3'UTR-interacting proteins could yield much information in translational regulation and the mRNA localization process. Previous studies on crayfish suggested that the 3'UTR (1300 bp) probably contains the potential regulatory sequence/motifs such as CPEs and K-box et al. In present study, using pull-down assay coupled with mass spectrometry approach allowing us to explore the potential proteins associated with the 3'UTR. We finally identified four candidate proteins including Hspg 2, Vtg, eef1a and Tuba1a, which annotated as significant roles involved in cell differentiation, lipid transporter activity, and meiotic cell cycle process. The preliminary results will contribute to the advance in understanding the translational activation of cyclin B in oocyte maturation regulation in crustacean.

[Value of three scoring systems in evaluating the prognosis of children with severe sepsis].

OBJECTIVE: To study the predictive value of Pediatric Age-adapted Sequential Organ Failure Assessment Score (pSOFA), Pediatric Risk of Mortality Score III (PRISM III), and Pediatric Critical Illness Score (PCIS) in children with severe sepsis. METHODS: A retrospective analysis was performed for the clinical data of 193 hospitalized children with severe sepsis. According to the final outcome, these children were divided into a survival group with 151 children and a death group with 42 children. The scores of pSOFA, PRISM III, and PCIS were determined according to the worst values of each index within 24 hours after admission. The receiver operating characteristic (ROC) curve was used to analyze the efficiency of each scoring system in predicting the risk of death due to sepsis. Smooth curve fitting was used to analyze the correlation between the three scoring systems and the threshold effect of each scoring system. Decision curve analysis (DCA) was used to evaluate the application value of each scoring system. RESULTS: The ROC analysis showed that PCIS and pSOFA had a similar predictive value (P=0.182) and that PRISM III and pSOFA had a similar predictive value (P=0.210), while PRISM III had a better predictive value than PCIS (P=0.045). PRISM III had the highest degree of fitting with prognosis, followed by pSOFA and PCIS. The DCA analysis showed that when the risk of death was 0.4 and 0.6 in children with severe sepsis and the three scoring systems were used as the basis for emergency intervention decision-making, pSOFA achieved the highest standardized net benefit, followed by PRISM III and PCIS. CONCLUSIONS: All three scoring systems have a certain value in predicting the prognosis of children with severe sepsis, and pSOFA has a better value than PRISM III and PCIS.

Policy uncertainty and corporate performance in government-sponsored voluntary environmental programs.

This study combines insights from the policy uncertainty literature and neo-institutional theory to examine corporate performance in implementing a government-sponsored voluntary environmental program (VEP) during 2004-2012 in Guangzhou, China. In this regulatory context, characterized by rapid policy changes, corporate performance in VEPs is affected by government surveillance, policy uncertainty, and peer pressures. Specifically, if VEP participants have experienced more government surveillance, they tend to perform better in program implementation. Such positive influence of government surveillance is particularly evident among those joining under high and low, rather than moderate uncertainty. Participants also perform better if they belong to an industry with more certified VEP firms, but worse if they are located in a regulatory jurisdiction with more certified VEP firms. At a moderate level of policy uncertainty, within-industry imitation is most likely to occur but within-jurisdiction imitation is least likely to occur.

Establishment of a markerless multiple-gene deletion method based on Cre/loxP mutant system for Bacillus pumilus.

In this study, we established a Cre/loxP mutant recombination system (Cre/lox71-66 system) for markerless gene deletion to facilitate our follow-up rational genetic engineering to the strain Bacillus pumilus W3. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. Two selected genes, cotA and sigF, were continuously knocked out and verified at different levels using this method. This method is simple and efficient and can be easily implemented for multiple gene deletions in B. pumilus.

Stakeholder demands and corporate environmental coping strategies in China.

This paper examines how stakeholder demand and compliance capacity jointly shape corporate environmental coping strategies and subsequently environmental protection practices. A four-dimensional classification of coping strategies-formalism, accommodation, referencing, and self-determination-is conceptualized. Drawing on survey and interview data collected from manufacturing enterprises in China between 2010 and 2012, the paper shows that compared with formalism and accommodation, coping strategies of referencing and self-determination are associated with stronger environmental protection practices. Enterprises adjust their coping strategies by taking into account the constraints defined by both their internal and external environments. The results also demonstrate the potential synergetic effects of state and non-state stakeholders working together in promoting better corporate environmental coping strategies and environmental practices in China.

[Chrysin inhibits adipogenic differentiation of mouse embryonic fibroblasts].

Chrysin is an active flavonoid wildly presented in many herbs. It has the effect to reduce serum lipid. To investigate the effect of chrysin on the adipogenic differentiation of mouse embryonic fibroblasts, methyl thiazolyl tetrazolium (MTT) and crystal violet were used to detect the cytotoxic effect of chrysin on Immortalized mouse embryonic fibroblasts (iMEFs). Propidium iodide (PI) staining combined with flow cytometry (FCM) was employed to detect the effects of different concentrations of chrysin on iMEFs cell cycle. The effect of chrysin on adipogenic differentiation ability of iMEFs was determined by oil red O staining. Semi-quantitative PCR was employed to detect the effect of chrysin on mRNA transcriptional levels of adipogenic differentiation markers, including perilipin 2, adiponectin (adipoq), Fabp4, LPL, MCP-1 and adipogenic differentiation key transcription factor peroxisome proliferators-actiated receptor-gamma 2(PPAR-γ2). Results indicated that chrysin had certain cytotoxic effect for iMEFs in a dose-dependent manner, and the IC50 was identified nearly to 30 µmol•L⁻¹. FCM analysis showed that chrysin could affect the cell-cycle distribution of iMEFs, increasing the ratio of cells in G1 phase. Adipogenic differentiation inducing experiment showed that 30 µmol•L⁻¹ chrysin significantly reduced lipid drops accumulation induced by insulin and dexamethasone. In addition, the mRNA transcriptional levels of PPAR-γ2 and LPL were significantly decreased and mRNA levels of fabp 4, MCP-1, adipoq were also affected after chrysin treatment. The experiment results suggest that chrysin attenuates the adipogenic differentiation capacity of mesenchymal stem cells.

Using a laser source to measure the refractive index of glass beads and Debye theory analysis.

Using a monochromatic laser beam to illuminate a homogeneous glass bead, some rainbows will appear around it. This paper concentrates on the study of the scattering intensity distribution and the method of measuring the refractive index for glass beads based on the Debye theory. It is found that the first rainbow due to the scattering superposition of backward light of the low-refractive-index glass beads can be explained approximately with the diffraction, the external reflection plus the one internal reflection, while the second rainbow of high-refractive-index glass beads is due to the contribution from the diffraction, the external reflection, the direct transmission, and the two internal reflections. The scattering intensity distribution is affected by the refractive index, the radius of the glass bead, and the incident beam width. The effects of the refractive index and the glass bead size on the first and second minimum deviation angle position are analyzed in this paper. The results of the measurements agree very well with the specifications.

Enterprise-Level Motivations, Regulatory Pressures, and Corporate Environmental Management in Guangzhou, China.

This study examines the effects of internal motivations and external pressures on the integration of environmental management (EM) practices within manufacturing operations in China. The moderating role of perceptions toward the regulatory process is also considered along with comparisons between wholly Chinese-owned and foreign-owned enterprises. From a sample of 131 manufacturing companies in the Guangzhou area, it was found that the salience of fees and fines has a strong positive influence on perceptions toward the regulator (the local Environmental Protection Bureau, EPB). This also has a positive effect on perceptions toward regulations themselves for foreign-owned enterprises. Business-case motivations for EM positively shape enterprise perceptions toward regulations, whereas risk-reduction motivations have a negative effect on perceptions toward regulations in foreign-owned enterprises. Enterprise perceptions toward the regulatory process have direct effects on the integration of EM practices in wholly Chinese-owned enterprises, but in opposite directions. While positive perceptions toward regulations have positive influence, positive perceptions toward regulators (i.e., the EPB) negatively affect it. Overall, these results indicated that promoting the adoption of EM practices depends on convincing business leaders that EM practices contribute to profit making. The regulatory process can potentially promote these practices, but measures need to be taken to ensure that the regulator is not co-opted by the regulated, especially in wholly Chinese-owned enterprises.

Effect of Dietary Selenium on the Growth and Immune Systems of Fish.

Dietary selenium (Se) is an essential component that supports fish growth and the immune system. This review attempts to provide insight into the biological impacts of dietary Se, including immunological responses, infection defense, and fish species growth, and it also identifies the routes via which it enters the aquatic environment. Dietary Se is important in fish feed due to its additive, antioxidant, and enzyme properties, which aid in various biological processes. However, excessive intake of it may harm aquatic ecosystems and potentially disrupt the food chain. This review explores the diverse natures of dietary Se, their impact on fish species, and the biological methods for eliminating excesses in aquatic environments. Soil has a potential role in the distribution of Se through erosion from agricultural, industrial, and mine sites. The research on dietary Se's effects on fish immune system and growth can provide knowledge regarding fish health, fish farming strategies, and the health of aquatic ecosystems, promoting the feed industry and sustainable aquaculture. This review provides data and references from various research studies on managing Se levels in aquatic ecosystems, promoting fish conservation, and utilizing Se in farmed fish diets.

[Expression of IGLL1 Gene and Its Clinical Significance in Pediatric T-ALL].

OBJECTIVE: To detect the relative expression of IGLL1 (immunoglobulin lambda-like polypeptide 1) mRNA in bone marrow of children with T-cell acute lymphoblastic leukemia (T-ALL), and analyze its correlation with the clinical characteristics and prognosis of the patients, so as to clarify the clinical significance of IGLL1 in pediatric T-ALL patients. METHODS: A total of 56 pediatric T-ALL patients hospitalized in Children's Hospital of Soochow University from June 2012 to December 2017 and treated with CCLG-ALL 2008 regimen were selected. Transcriptome sequencing technology was used to detect the transcription level of IGLL1 gene in children with T-ALL. According to 25% of the IGLL1 transcription level (cutoff value:448), the enrolled children were divided into IGLL1 low expression group (17 cases) and IGLL1 high expression group (39 cases). Combined with clinical data, the correlation between the expression level of IGLL1 and prognosis of the patients was analyzed. RESULTS: The comparative analysis showed that the transcription level of IGLL1 was not correlated with the clinical characteristics of the patients, such as sex, age, bone marrow blast, white blood cell (WBC) count at initial diagnosis. The 5-year OS rate of patients with high IGLL1 expression was significantly higher than that of patients with low IGLL1 expression (76.9%±6.7% vs 47.1%±12.1%, P =0.018). Further comparison of relapse-free survival (RFS) rate between the two groups showed that the 5-year RFS rate of patients with high IGLL1 expression was higher than that of patients with low IGLL1 expression, but the difference between the two groups was not statistically significant (P =0.095). Multivariate COX analysis was conducted on common clinical prognostic factors (age, sex, WBC count at diagnosis, prednisone response on the 7th day, bone marrow response on the 15th day after treatment) and IGLL1 expression level, and the results showed that IGLL1 expression (P =0.012) and prednisone response (P =0.017) were independent risk factors for overall survival in pediatric T-ALL patients. CONCLUSION: In pediatric T-ALL, the OS rate of children with high expression of IGLL1 gene was significantly higher than that of children with low expression of IGLL1 gene, and the expression level of IGLL1 gene was an independent factor affecting the survival of children with T-ALL, which suggests that IGLL1 is a marker of good clinical prognosis of children with T-ALL.

Development of an in vitro culture system for immature oocytes of red swamp crayfish (Procambarus clarkii).

The present study assessed general strategies for the isolation and in vitro culture of immature ovary-derived cells (oocytes) in red swamp crayfish (Procambarus clarkii). In phase 1 of this study, the effects of different digestive enzymes on cell retrieval from developing ovarian tissues were examined to facilitate efficient oocyte isolation. Subsequently, the ovary-derived cells/oocytes were cultured under various conditions to evaluate the effects of basal media and supplement concentrations. Collagenase I treatment at 26 ℃ for 25 min was more effective (approx. 95 %) in oocytes isolation from ovarian tissues than other treatments. Furthermore, DMEM media with HEPES (DMEM-H) were more effective than L15 for oocyte culture (72.4 % survival rate), while oocyte survival improved to 90.6 % using a 10 % fetal bovine serum supplement. In phase 2, the top-performing media were used in immature oocyte culture to assess 17α-Hydroxyprogesterone (17-OHP) induction in vitro. An up-regulated expression of maturity-related genes, i.e., cyclin B, cdc2, ef1α, after 12 h of incubation was evident in qPCR results, demonstrating that the exogenous hormone 17-OHP could induce P. clarkii oocyte maturation in vitro. These results may provide a basis for developing an in vitro system for P. clarkii germline cell culture, which may ultimately lead to the genetic improvement of this species.

PcVDAC promotes WSSV infection by regulating the apoptotic activity of haemocytes in Procambarus clarkii.

Apoptosis is programmed cell death that is strictly regulated by a series of related genes and is of great importance in resisting pathogen invasion and maintaining cell environment homeostasis. Among apoptotic proteins, the voltage-dependent anion channel protein (VDAC) plays a key role in the mitochondrial apoptosis pathway because of its close connection with changes in mitochondrial membrane potential. However, the role of VDAC in apoptosis and immune regulation in Procambarus clarkii is poorly understood. In this study, the VDAC gene in P. clarkii (PcVDAC) was cloned by rapid amplification of cDNA ends (RACE) technology. The gene was found to have a total length of 2277 bp, including a 194-bp 5'-UTR, 1234-bp 3'-UTR and 849-bp open reading frame (ORF), and to encode 282 amino acids. PcVDAC was expressed in all tissues tested, and its expression was upregulated after white spot syndrome virus (WSSV) infection (P < 0.05). The RNA interference (RNAi) method was used to explore the role of PcVDAC in WSSV infection. The results showed that the number of WSSV copies in haemocytes was significantly reduced after RNAi (P < 0.05), and the survival rate was significantly increased. In addition, after RNAi, the apoptosis rate was significantly increased (P < 0.05), the mitochondrial membrane potential was reduced (P < 0.01), and the expression of caspase-3 and other genes was upregulated (P < 0.05). These results indicate that PcVDAC promotes the replication of WSSV in P. clarkii by inhibiting haemocytes apoptosis. Therefore, the results presented in this paper provide new insights into the immune response of P. clarkii infected with WSSV.

16S rRNA gene sequencing analysis reveals an imbalance in the intestinal flora of Eriocheir sinensis with hepatopancreatic necrosis disease.

Hepatopancreas necrosis disease (HPND) is a highly fatal disease that first appeared in Jiangsu Province, China, in 2015, and later spread to many other provinces, which had a severe impact on the culture of Chinese mitten crab (Eriocheir sinensis). Here, changes in the intestinal flora of healthy and HPND-affected Chinese mitten crabs were compared via 16S rRNA sequencing. Our findings indicated that Firmicutes, Bacteroidota, and Proteobacteria were the three dominant phyla in both healthy and HPND-affected crabs and exhibited no significant differences in α-diversity (richness p = 0.0892; evenness and diversity p = 0.0630). Furthermore, there were no significant changes in the abundance of Proteobacteria between the experimental groups. However, the abundance of Bacteroidota in the HPND group was significantly higher than that of the control group (HPND: 30.12%, Control: 16.60%), whereas the abundance of Firmicutes was significantly lower (HPND: 29.90%, Control: 50.55%). At the genus level, the abundance of Candidatus Bacilloplasma, Desulfovibrio, Bacteroides, and Aeromonas also differed significantly between groups (P < 0.05). Collectively, our study confirms an imbalance in the gut microbiota of Chinese mitten crabs with HPND and we speculate that this alteration may affect the metabolism and immune function of these organisms. Furthermore, we suspect that the structural changes in the intestinal flora of sick crabs observed in our study may be related to HPND.