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Abstract Static magnetic field (SMF) promoting bone tissue remodeling is a potential non-invasive therapy technique to accelerate orthodontic tooth movement (OTM). The periodontal ligament stem cells (PDLSCs), which are mechanosensitive cells, are essential for force-induced bone remodeling and OTM. However, whether and how the PDLSCs influence the process of inflammatory bone remodeling under mechanical force stimuli in the presence of SMFs remains unclear. In this study, we found that local SMF stimulation significantly enhanced the OTM distance and induced osteoclastogenesis on the compression side of a rat model of OTM. Further experiments with macrophages cultured with supernatants from force-loaded PDLSCs exposed to an SMF showed enhanced osteoclast formation. RNA-seq analysis showed that interleukin-6 (IL-6) was elevated in force-loaded PDLSCs exposed to SMFs. IL-6 expression was also elevated on the pressure side of a rat OTM model with an SMF. The OTM distance induced by an SMF was significantly decreased after injection of the IL-6 inhibitor tocilizumab. These results imply that SMF promotes osteoclastogenesis by inducing force-loaded PDLSCs to secrete the inflammatory cytokine IL-6, which accelerates OTM. This will help to reveal the mechanism of SMF accelerates tooth movement and should be evaluated for application in periodontitis patients.

Abstract The use of titanium implants as fixed supports following fractures in patients with OP can often result in sterile loosening and poor osseointegration. Oxidative stress has been shown to play a particularly important role in this process. While TSA has been reported to facilitate in vivo osteogenesis, the underlying mechanisms remain to be clarified. It also remains unclear whether TSA can improve the osseointegration of titanium implants. This study investigated whether TSA could enhance the osseointegration of titanium rods by activating AKT/Nrf2 pathway signaling, thereby suppressing oxidative stress. MC3T3-E1 cells treated with CCCP to induce oxidative stress served as an in vitro model, while an OVX-induced OP rat model was employed for in vivo analysis of titanium rod implantation. In vitro, TSA treatment of CCCP-treated MC3T3-E1 cells resulted in the upregulation of osteogenic proteins together with increased AKT, total Nrf2, nuclear Nrf2, HO-1, and NQO1 expression, enhanced mitochondrial functionality, and decreased oxidative damage. Notably, the PI3K/AKT inhibitor LY294002 reversed these effects. In vivo, TSA effectively enhanced the microstructural characteristics of distal femur trabecular bone, increased BMSCs mineralization capacity, promoted bone formation, and improved the binding of titanium implants to the surrounding tissue. Finally, our results showed that TSA could reverse oxidative stress-induced cell damage while promoting bone healing and improving titanium rods' osseointegration through AKT/Nrf2 pathway activation.

BackgroundBoth alcohol consumption and HIV infection are associated with worse brain, cognitive, and clinical outcomes in older adults. However, the extent to which brain and cognitive dysfunction is reversible with reduction or cessation of drinking is unknown. ObjectiveThe 30-Day Challenge study was designed to determine whether reduction or cessation of drinking would be associated with improvements in cognition, reduction of systemic and brain inflammation, and improvement in HIV-related outcomes in adults with heavy drinking. MethodsThe study design was a mechanistic experimental trial, in which all participants received an alcohol reduction intervention followed by repeated assessments of behavioral and clinical outcomes. Persons were eligible if they were 45 years of age or older, had weekly alcohol consumption of 21 or more drinks (men) or 14 or more drinks (women), and were not at high risk of alcohol withdrawal. After a baseline assessment, participants received an intervention consisting of contingency management (money for nondrinking days) for at least 30 days followed by a brief motivational interview. After this, participants could either resume drinking or not. Study questionnaires, neurocognitive assessments, neuroimaging, and blood, urine, and stool samples were collected at baseline, 30 days, 90 days, and 1 year after enrollment. ResultsWe enrolled 57 persons with heavy drinking who initiated the contingency management protocol (mean age 56 years, SD 4.6 years; 63%, n=36 male, 77%, n=44 Black, and 58%, n=33 people with HIV) of whom 50 completed 30-day follow-up and 43 the 90-day follow-up. The planned study procedures were interrupted and modified due to the COVID-19 pandemic of 2020-2021. ConclusionsThis was the first study seeking to assess changes in brain (neuroimaging) and cognition after alcohol intervention in nontreatment-seeking people with HIV together with people without HIV as controls. Study design strengths, limitations, and lessons for future study design considerations are discussed. Planned analyses are in progress, after which deidentified study data will be available for sharing. Trial RegistrationClinicalTrials.gov NCT03353701; https://clinicaltrials.gov/study/NCT03353701 International Registered Report Identifier (IRRID)DERR1-10.2196/53684

Introduction: Post-traumatic stress disorder (PTSD) is a debilitating disorder experienced by a subgroup of individuals following a life-threatening trauma. Several US states have passed laws permitting the medical use of marijuana (MMJ) by individuals with PTSD, despite very little scientific indication on the appropriateness of marijuana as a therapy for PTSD. This prospective pilot study of adults with confirmed PTSD in Florida (FL) investigated whether PTSD symptoms, sleep quality, affect, and general physical and mental health/well-being improved post-initiation of MMJ treatment. Methods: Participants, N = 15, were recruited from two MMJ clinics in Gainesville and Jacksonville, FL. To be eligible, participants had to be 18 years of age or older, not currently on MMJ, and willing to abstain from recreational marijuana, if using any, until the State Medical Cannabis Card was obtained, screen positive for PTSD. Participants were assessed at baseline (pre-MMJ initiation) and 30 and 70 days post-MMJ initiation using the Pittsburgh Sleep Quality Index (PSQI), PTSD Checklist for DSM-5 (PCL-5), Positive and Negative Affect Schedule (PANAS), PROMIS Global Health V1.2, and semi-structured marijuana and other substance use assessment. Results: PTSD symptom severity as measured by total PCL-5 score improved significantly at 30- and 70-day follow-ups. Similarly, statistically significant reductions in nightmares were reported at 30- and 70-day follow-ups. Corresponding improvements in sleep were noticed with participants reporting increased duration of sleep hours, sleep quality, sleep efficiency, and total PSQI score. Likewise, negative affect and global mental health improved significantly at follow-up. According to the post hoc analyses, the most statistically significant changes occurred between baseline and 30-day follow-up. The exception to this pattern was nightmares, which did not show significant improvement until day 70. Conclusion: The findings of this study highlight the potential of MMJ in improving patient outcomes for those with PTSD, particularly concerning sleep disturbances, which often do not respond to currently available treatments.

Abstract Mage-D1 (MAGE family member D1) is involved in a variety of cell biological effects. Recent studies have shown that Mage-D1 is closely related to tooth development, but its specific regulatory mechanism is unclear. The purpose of this study was to investigate the expression pattern of Mage-D1 in rat dental germ development and its differential mineralization ability to ectomesenchymal stem cells (EMSCs), and to explore its potential mechanism. Results showed that the expression of Mage-D1 during rat dental germ development was temporally and spatially specific. Mage-D1 promotes the proliferation ability of EMSCs but inhibits their migration ability. Under induction by mineralized culture medium, Mage-D1 promotes osteogenesis and tooth-forming ability. Furthermore, the expression pattern of Mage-D1 at E19.5 d rat dental germ is similar to p75 neurotrophin receptor (p75NTR), distal-less homeobox 1 (Dlx1) and msh homeobox 1 (Msx1). In addition, Mage-D1 is binding to p75NTR, Dlx1, and Msx1 in vitro. These findings indicate that Mage-D1 is play an important regulatory role in normal mineralization of teeth. p75NTR, Dlx1, and Msx1 seem to be closely related to the underlying mechanism of Mage-D1 action.

Background Obesity is a chronic disease with a high prevalence rate and is an established risk factor for human health. Body mass index (BMI) is a common and primary indicator used in assessing obesity. This work aims to investigate the putative causal relationship among BMI, sex hormone-binding globulin (SHBG), bioavailable testosterone (BioT), and estradiol levels. Materials and Methods We conducted a bidirectional Mendelian randomization study, using single-nucleotide polymorphisms (SNPs) strongly associated with BMI, SHBG, BioT, and estradiol as instrumental variables. All SNPs were identified from the genome-wide association study (GWAS) summary data of large sample studies recruiting more than 150,000 European adult male individuals. The inverse-variance-weighted (IVW) approach was used as a primary algorithm for putative causal estimation. Results Genetically predicted elevated BMI was associated with decreased SHBG (IVW, β = −0.103, 95% confidence interval [CI] [−0.113 to −0.092], P = 1.50 × 10−77) and BioT levels (IVW, β = −0.139, 95% CI [−0.165 to −0.113], P = 9.54 × 10−26) and high estradiol levels (IVW, β = 0.014, 95% CI [0.009–0.019], P = 2.19 × 10−7). Increased SHBG levels were causally associated with low BMI (IVW, β = −0.051, 95% CI [−0.098 to −0.005], P = 0.030) and BioT (IVW, β = −0.126, 95% CI [−0.175 to −0.077], P = 5.97 × 10−7) and high estradiol levels (IVW, β = 0.046, 95% CI [0.035–0.056], P = 6.51 × 10−17). Conversely, no evidence of an effect of estradiol imbalance on SHBG levels (IVW, β = 1.035, 95% CI [−0.854 to 2.926], P = 0.283) and BMI (IVW, β = 0.091, 95% CI [−0.094 to 0.276], P = 0.336) was obtained. However, increased BioT levels were causally associated with lower SHBG levels (IVW, β = −0.044, 95% CI [−0.061 to −0.026], P = 8.76 × 10−7), not BMI (IVW, β = −0.006, 95% CI [−0.035 to 0.023], P = 0.679). Conclusions The findings support a network putative causal relationship among BMI, SHBG, BioT, and estradiol. SHBG, BioT, and estradiol may partly mediate the effect of obesity on male health. Reasonably modulating BioT and estradiol, especially SHBG, facilitated the attenuation of the harmful effects of obesity on male health.

Objective(s): This study aimed to investigate the function of transient receptor potential vanilloid 1 (TRPV1) in regulating periodontal lesions. In addition, we explored the underlying mechanism of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway.Materials and Methods: Lipopolysaccharide (LPS) stimulation of human periodontal ligament cells (HPDLCs) was used to construct a periodontitis cell model, and experimental periodontitis (EP) rats were established by ligation. The mechanism by which TRPV1 regulates periodontitis was further verified by injecting the TRPV1 agonist capsaicin (CPS) and antagonist capsazepine (CPZ) into the gingiva of rats; the alveolar bone losses in each group were measured by stereomicroscopy. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting (WB) were used to research the expression of TRPV1 and proinflammatory cytokines, and WB was performed to test the phosphorylation of PI3K and AKT.Results: In vitro experiments showed that LPS induced the upregulation of TRPV1 and proinflammatory cytokines and promoted the phosphorylation of PI3K and AKT proteins in HPDLCs, which was consistent with their expression in the rat periodontitis model. Moreover, in vivo studies indicated that CPZ had anti-inflammatory effects through the PI3K/AKT pathway and inhibited bone loss induced by periodontal ligation in rats, while CPS had the opposite effect.Conclusion: TRPV1 was involved in the process of alveolar bone defects and the inflammatory response in rats with periodontitis induced by ligation. Its mechanism might be related to the phosphorylation of related proteins in the PI3K/AKT signaling pathway.

Abstract Background Lipid abnormality and obesity have been proposed to be associated with lumbar disc degeneration, but little is known about the effect of ‘lipid healthy but obese’ (LH-O) and ‘lipid abnormal but not obese’ (LA-NO) phenotypes on lumbar disc degeneration in Chinese. The study aims to determine the impact and distinction of LH-O and LA-NO phenotypes on lumbar disc degeneration in Chinese, and to identify the association of related factors with risk of lumbar disc degeneration. Methods A total of 678 individuals were included with lumbar magnetic resonance imaging, serum lipid levels and anthropometric measurements. Obesity was defined on the basis of body mass index or waist to hip ratio (WHR). Pfirrmann score and Weishaupt’s scale were utilized to assess the degree of disc degeneration and facet joint degeneration. Results The incidence of the LH-O and LA-NO phenotypes were 11.4% and 18.1%, respectively. LA-NO phenotype demonstrates a high incidence for disc degeneration (P 0.05). Elevated triglycerides and greater WHR may be the risk factors for lumbar disc degeneration in Chinese. Conclusion LH-O and LA-NO phenotypes are common with different status of disc degeneration in Chinese. Elevated triglycerides and abdominal obesity appear to play crucial roles in the development of lumbar disc degeneration.

Purpose This study examined factors associated with TB among persons living with HIV (PLWH) in Florida and the agreement between self-reported and medically documented history of tuberculosis (TB) in assessing the risk factors. Methods Self-reported and medically documented data of 655 PLWH in Florida were analyzed. Data on sociodemographic factors such as age, race/ethnicity, place of birth, current marital status, education, employment, homelessness in the past year and ‘ever been jailed’ and behavioural factors such as excessive alcohol use, marijuana, injection drug use (IDU), substance and current cigarette use were obtained. Health status information such as health insurance status, adherence to HIV antiretroviral therapy (ART), most recent CD4 count, HIV viral load and comorbid conditions were also obtained. The associations between these selected factors with self-reported TB and medically documented TB diagnosis were compared using Chi-square and logistic regression analyses. Additionally, the agreement between self-reports and medical records was assessed. Results TB prevalence according to self-reports and medical records was 16.6% and 7.5% respectively. Being age ≥55 years, African American and homeless in the past 12 months were statistically significantly associated with self-reported TB, while being African American homeless in the past 12 months and not on antiretroviral therapy (ART) were statistically significantly associated with medically documented TB. African Americans compared to Whites had odds ratios of 3.04 and 4.89 for self-reported and medically documented TB, respectively. There was moderate agreement between self-reported and medically documented TB (Kappa = 0.41). Conclusions TB prevalence was higher based on self-reports than medical records. There was moderate agreement between the two data sources, showing the importance of self-reports. Establishing the true prevalence of TB and associated risk factors in PLWH for developing policies may therefore require the use of self-reports and confirmation by screening tests, clinical signs and/or microbiologic data.

Breast cancer is the most common malignancy among women worldwide, accordingly, numerous chemotherapeutic drugs have been discovered thus far. However, the development and application of these drugs is severely constrained because of their unclear mechanism. To address this issue, our previous work has defined 3-acyl isoquinolin-1(2H)-one derivatives as potent anti-tumor agents, among which the compound 4f possessed relatively higher activity in vitro. In this study, we aim to further explore the anti-cancer effect and the underlying molecular mechanism of 4f in breast cancer cells. Therefore, CCK8 assay was used to detect cell viability and flow cytometry was used to analyze cell cycle and apoptosis. Meanwhile, related proteins that regulate cell cycle and apoptosis were detected. The results showed that 4f induced cell apoptosis and inhibited cell proliferation in breast cancer cells in a dose-depended manner without significant toxicity to human normal mammary epithelial cell. The cell cycle was arrested at G2 phase with the suppressed expression of the CDK1 protein. Additionally, 4f was confirmed to induce the cell apoptosis with the up-regulation of bax, down-regulation of bcl-2, activation of cleaved-caspase3/7/9 and cleaved-PARP, together with the inhibition of MEK/ERK and p38 MAPK pathway. Moreover, the GSDME-mediated pyroptosis was also induced by 4f in breast cancer cells. Together, these results demonstrated that 4f could serve as a new and promising candidate for the treatment of breast cancer.

Objective To investigate the effect of resin infiltrant on resistance of sound permanent enamel surfaces to demineralization. Method Eighty healthy premolars were sectioned to obtain enamel blocks from the buccal surface. Specimens with baseline surface microhardness values of 320–370 were selected. The experimental group were treated with resin infiltrant, while the control group was not. Specimens from each group were artificially demineralized and the surface microhardness values were measured again. Confocal laser scanning microscopy was used to measure the depth of demineralization and detect the penetration ability of the resin infiltrant. The specimens were subjected to a simulated toothbrushing abrasion test. Scanning electron microscopy was used to observe changes in the surface morphology of specimens after each of these procedures. Results No significant differences between the experimental and control groups were observed in the baseline microhardness values or in the experimental group after resin infiltration compared with the baseline conditions. After artificial demineralization, the microhardness value in the control group was significantly lower than that in the experimental group (266.0 (±34.5) compared with 304.0 (±13.0), P = 0.017). Confocal laser scanning microscopy results showed that the demineralization depth in the control group was significantly deeper than that in the experimental group (97.9 (±22.8) µm vs. 50.4 (±14.3) µm, P < 0.001), and that resin infiltrant completely penetrated the acid-etched demineralized area of the tooth enamel with a mean penetration depth of 31.6 (±9.0) µm. Scanning electron microscopy showed that the surface morphology was more uniform and smoother after simulated toothbrushing. The enamel surface structure was more severely destroyed in the control group after artificial demineralization compared with that of the experimental group. Conclusion Resin infiltrant can completely penetrate an acid-etched demineralized enamel area and improve resistance of sound enamel surfaces to demineralization. Our findings provide an experimental basis for preventive application of resin infiltrant to sound enamel surfaces to protect tooth enamel against demineralization.

A lack of replicable test systems that realistically simulate hot water premise plumbing conditions at the laboratory-scale is an obstacle to identifying key factors that support growth of opportunistic pathogens (OPs) and opportunities to stem disease transmission. Here we developed the convectively-mixed pipe reactor (CMPR) as a simple reproducible system, consisting of off-the-shelf plumbing materials, that self-mixes through natural convective currents and enables testing of multiple, replicated, and realistic premise plumbing conditions in parallel. A 10-week validation study was conducted, comparing three pipe materials (PVC, PVC-copper, and PVC-iron; n = 18 each) to stagnant control pipes without convective mixing (n = 3 each). Replicate CMPRs were found to yield consistent water chemistry as a function of pipe material, with differences becoming less discernable by week 9. Temperature, an overarching factor known to control OP growth, was consistently maintained across all 54 CMPRs, with a coefficient of variation

Despite campaigns to increase safer sex practices, there are people living with HIV/AIDS (PLWH) who do not disclose their HIV status to sexual partners and engage in condomless sex. The purpose of this research was to: 1) describe factors associated with disclosure of HIV status to sexual partners; and 2) determine if disclosure and/or receipt of prevention counseling are independently associated with condomless sex. We used the Florida Medical Monitoring Project to analyze data from 376 HIV positive men with more than one sexual partner. Results indicated that 55% consistently disclosed their HIV status to sexual partners, 30% inconsistently disclosed, 15% did not disclose, and 48% reported any condomless sex. The odds of having condomless sex was 3.3 (CI = 1.5, 7.3) times greater in men who disclosed to all partners. Results suggest that men who disclose are also those who are more likely to have condomless sex. More research is needed to better understand the complex nature of disclosure and sexual risk behaviors and how disclosure impacts sexual risk.

Methane production by methanogens in mangrove sediments is known to contribute significantly to global warming, but studies on the shift of methanogenic community in response to anthropogenic contaminations were still limited. In this study, the effect of anthropogenic activities in the mangrove sediments along the north and south coastlines of Singapore were investigated by pyrosequencing of the mcrA gene. Our results showed that hydrogenotrophic, acetoclastic and methylotrophic methanogens coexist in the sediments. The predominance of the methylotrophic Methanosarcinales reflects the potential for high methane production as well as the possible availability of low acetate and high methylated C-1 compounds as substrates. A decline in the number of acetoclastic/methylotrophic methanogens in favor of hydrogenotrophic methanogens was observed along a vertical profile in Sungei Changi, which was contaminated by heavy metals. The diversity of methanogens in the various contaminated stations was significantly different from that in a pristine St. John's Island. The spatial variation in the methanogenic communities among the different stations was more distinct than those along the vertical profiles at each station. We suggest that the overall heterogeneity of the methanogenic communities residing in the tropical mangrove sediments might be due to the accumulated effects of temperature and concentrations of nitrate, cobalt, and nickel.

T follicular helper cells (Tfh) provide help to B cells to support their activation, expansion and differentiation. However, the role of Tfh cells in chronic HBV infection is poorly defined. The aim of this research was to examine the function of Tfh cells and whether they are involved in HBV related disease. Blood CXCR5+CD4+T cells and B cells in 85 patients with chronic HBV infection (HBV patients) and health controls (HC) were examined by flow cytometry. The molecule expression in blood CXCR5+CD4+ T cells was detected by real-time PCR. Blood CXCR5+CD4+ T cells and B cells were co-cultured and the production of Ig and cytokines was detected by ELISA. Autoantibodies were detected by indirect immunofluorescence and immunospot assay. We found that blood CXCR5+CD4+ T cells in patients with chronic HBV infection (HBV patients) expressed higher level of activation related molecules and cytokines than that from health controls (HC).In HBV patients, the frequency of blood CXCR5+CD4+ T cells was significantly correlated with serum ALT and AST. We also found that blood CXCR5+CD4+ T cells from HBV patients could induce B cells to secret higher level of immunoglobulin than that from HC. Several autoantibodies, including ANA, ss-A, ss-B, Scl-70, Jo-1, ect, were indeed positive in 65% HBV patients. Among HBV patients, expression of function related molecules was significantly higher in blood CXCR5+CD4+ T cells from patients with autoantibodies than that without autoantibodies. Our research indicated that blood CXCR5+CD4+ T cells from HBV patients were over activated and show augmented capacity to help B cells for antibody secreting, which might correlated with liver inflammation and the production of autoantibodies in extrahepatic manifestations.

In 2012, an unprecedented large-scale outbreak of disease in pigs in China caused great economic losses to the swine industry. Isolates from pseudorabies virus epidemics in swine herds were characterized. Evidence confirmed that the pathogenic pseudorabies virus was the etiologic agent of this epidemic.

BACKGROUND: MicroRNAs (miRNAs) represent a class of small ncRNAs that repress gene expression on the post-transcriptional level by the degradation or translation inhibition of target mRNA. METHODOLOGY: Three small RNA libraries from oyster haemocytes were sequenced on the Illumina platform to investigate the latent immunomodulation of miRNAs after bacteria challenge and heat stress. Totally, 10,498,663, 8,588,606 and 9,679,663 high-quality reads were obtained in the control, bacteria and bacteria+heat library, respectively, from which 199 oyster miRNAs including 71 known and 128 novel ones were identified. Among these miRNAs, 6 known and 23 novel ones were predicted to possess more than one precursor-coding region, and cgi-miR-10a, cgi-miR-184b, cgi-miR-100, cgi-miR-1984 and cgi-miR-67a were observed to be the most abundant miRNAs in the control library. The expression levels of 22 miRNAs in the bacteria library were significantly higher than those in the control library, while there were another 33 miRNAs whose expression levels were significantly lower than that in the control library. Meanwhile, the expression levels of 65 miRNAs in the bacteria+heat library changed significantly compared to those in the bacteria library. The target genes of these differentially expressed miRNAs were annotated, and they fell in immune and stress-related GO terms including antioxidant, cell killing, death, immune system process, and response to stimulus. Furthermore, there were 42 differentially expressed miRNAs detected in both control/bacteria and bacteria/bacteria+heat comparisons, among which 9 miRNAs displayed the identical pattern in the two comparisons, and the expression alterations of 8 miRNAs were confirmed using quantitative RT-PCR. CONCLUSIONS: These results indicated collectively that immune challenge could induce the expression of immune-related miRNAs, which might modulate the immune response such as redox reaction, phagocytosis and apoptosis, and the expression of some immune-related miRNAs could be also regulated by heat stress to improve the environmental adaption of oyster.

Jiyan-1 puffer, the F1 hybrid of Takifugu rubripes and Takifugu flavidus, displays obvious heterosis in the growth performance, flavor and stress tolerance. In the present study, comparative analysis for the transcriptomes of T. rubripes, T. flavidus and Jiyan-1 was performed aiming to reveal the possible mechanisms of heterosis in pufferfish. Whole transcriptomes were sequenced using the SOLiD4 platform, and a total of 44,305 transcripts corresponding to 18,164 genes were identified collectively. A total of 14,148 transcripts were differentially expressed. By comparing the gene expression patterns of the three samples, the coexistence of overdominance, dominance, underdominance and additivity was observed in the gene action modes of Jiyan-1. There were 2,237 transcripts in the intersection of the differentially expressed transcripts from Jiyan-1 versus T. rubripes and Jiyan-1 versus T. flavidus, among which 213 transcripts were also in the T. rubripes versus T. flavidus. The potential functions of the remaining 2,024 transcripts were mainly associated with metabolic process, nucleotide binding and catalytic activity. The enrichment results indicated metabolism was the most activated biological function in the heterosis. In addition, 35 KEGG pathways were retrieved as affiliated with more than three differentially expressed transcripts and 8,579 potentially novel transcript isoforms were identified for Jiyan-1. The present study revealed the coexistence of multiple gene actions in the hybrid puffer, indicated the importance of metabolism, ion binding function and kinase activity, as well as provided a list of candidate genes and pathways for heterosis. It could be helpful for the better understanding of the determination and regulation mechanisms of heterosis.

Folliculogenesis is tightly controlled by a series of hormones, growth factors and cytokines, many of which are secreted as proproteins and require processing by proteases before becoming functional. Furin is a member of the subtilisin-like proteases that activate large numbers of proprotein substrates and is ubiquitously expressed and implicated in many physiological and pathological processes. However, the precise role of furin during folliculogenesis has not been thoroughly investigated. The goal of the present work is to identify the role of furin in the development of granulosa cells during folliculogenesis, using immunohistochemistry, RT-PCR, Western blot and functional studies in primary cultured rat granulosa cells. Our results demonstrate that furin is highly expressed in granulosa cells and oocytes of the ovary with very limited expression in other ovarian cells such as the epithelial, stromal or theca cells. Furin siRNA significantly increases apoptosis of the granulosa cells from large antral/preovulatory follicles, in part via downregulation of the anti-apoptotic proteins, XIAP and p-AKT. On the contrary, furin siRNA markedly decreases proliferation of granulosa cells based on the downregulation of proliferation cell nuclear antigen (PCNA). Taken together, these data suggest that furin may play an important role in regulating apoptosis and proliferation of granulosa cells.

BACKGROUND: Nitric oxide synthase (NOS) is responsible for synthesizing nitric oxide (NO) from L-arginine, and involved in multiple physiological functions. However, its immunological role in mollusc was seldom reported. METHODOLOGY: In the present study, an NOS (CfNOS) gene was identified from the scallop Chlamys farreri encoding a polypeptide of 1486 amino acids. Its amino acid sequence shared 50.0~54.7, 40.7~47.0 and 42.5~44.5% similarities with vertebrate neuronal (n), endothelial (e) and inducible (i) NOSs, respectively. CfNOS contained PDZ, oxygenase and reductase domains, which resembled those in nNOS. The CfNOS mRNA transcripts expressed in all embryos and larvae after the 2-cell embryo stage, and were detectable in all tested tissues with the highest level in the gonad, and with the immune tissues hepatopancreas and haemocytes included. Moreover, the immunoreactive area of CfNOS distributed over the haemocyte cytoplasm and cell membrane. After LPS, β-glucan and PGN stimulation, the expression level of CfNOS mRNA in haemocytes increased significantly at 3 h (4.0-, 4.8- and 2.7-fold, respectively, P < 0.01), and reached the peak at 12 h (15.3- and 27.6-fold for LPS and β-glucan respectively, P < 0.01) and 24 h (17.3-fold for PGN, P < 0.01). In addition, TNF-α also induced the expression of CfNOS, which started to increase at 1 h (5.2-fold, P < 0.05) and peaked at 6 h (19.9-fold, P < 0.01). The catalytic activity of the native CfNOS protein was 30.3 ± 0.3 U mgprot(-1), and it decreased significantly after the addition of the selective inhibitors of nNOS and iNOS (26.9 ± 0.4 and 29.3 ± 0.1 U mgprot(-1), respectively, P < 0.01). CONCLUSIONS: These results suggested that CfNOS, with identical structure with nNOS and similar enzymatic characteristics to nNOS and iNOS, played the immunological role of iNOS to be involved in the scallop immune defense against PAMPs and TNF-α.

BACKGROUND: Acetycholinesterase (AChE; EC 3.1.1.7) is an essential hydrolytic enzyme in the cholinergic nervous system, which plays an important role during immunomodulation in vertebrates. Though AChEs have been identified in most invertebrates, the knowledge about immunomodulation function of AChE is still quite meagre in invertebrates. METHODOLOGY: A scallop AChE gene was identified from Chlamys farreri (designed as CfAChE), and its open reading frame encoded a polypeptide of 522 amino acids. A signal peptide, an active site triad, the choline binding site and the peripheral anionic sites (PAS) were identified in CfAChE. The recombinant mature polypeptide of CfAChE (rCfAChE) was expressed in Pichia pastoris GS115, and its activity was 71.3±1.3 U mg(-1) to catalyze the hydrolysis of acetylthiocholine iodide. The mRNA transcripts of CfAChE were detected in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in hepatopancreas. The relative expression level of CfAChE mRNA in haemocytes was both up-regulated after LPS (0.5 mg mL(-1)) and human TNF-α (50 ng mL(-1)) stimulations, and it reached the highest level at 12 h (10.4-fold, P

BACKGROUND: The C1q domain containing (C1qDC) proteins refer to a family of all proteins that contain the globular C1q (gC1q) domain, and participate in a series of immune responses depending on their gC1q domains to bind a variety of self and non-self binding ligands. METHODOLOGY: In the present study, the mRNA expression patterns, localization, and activities of a C1qDC protein from scallop Chlamys farreri (CfC1qDC) were investigated to understand its possible functions in innate immunity. The relative expression levels of CfC1qDC mRNA in hemocytes were all significantly up-regulated after four typical PAMPs (LPS, PGN, β-glucan and polyI:C) stimulation. During the embryonic development of scallop, the mRNA transcripts of CfC1qDC were detected in all the stages, and the expression level was up-regulated from D-hinged larva and reached the highest at eye-spot larva. The endogenous CfC1qDC was dominantly located in the hepatopancreas, gill, kidney and gonad of adult scallop through immunofluorescence. The recombinant protein of CfC1qDC (rCfC1qDC) could not only bind various PAMPs, such as LPS, PGN, β-glucan as well as polyI:C, but also enhance the phagocytic activity of scallop hemocytes towards Escherichia coli. Meanwhile, rCfC1qDC could interact with human heat-aggregated IgG, and this interaction could be inhibited by LPS. CONCLUSIONS: All these results indicated that CfC1qDC in C. farreri not only served as a PRR involved in the PAMPs recognition, but also an opsonin participating in the clearance of invaders in innate immunity. Moreover, the ability of CfC1qDC to interact with immunoglobulins provided a clue to understand the evolution of classical pathway in complement system.

BACKGROUND: Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps. METHODOLOGY: A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. CONCLUSIONS: LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp.

BACKGROUND: Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate. METHODOLOGY: The full-length cDNA encoding DDC (designated CfDDC) was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P