Enriched environment ameliorates fear memory impairments induced by sleep deprivation via inhibiting PIEZO1/calpain/autophagy signaling pathway in the basal forebrain.

AIMS: To verify the hypothesis that an enriched environment (EE) alleviates sleep deprivation-induced fear memory impairment by modulating the basal forebrain (BF) PIEZO1/calpain/autophagy pathway. METHODS: Eight-week-old male mice were housed in a closed, isolated environment (CE) or an EE, before 6-h total sleep deprivation. Changes in fear memory after sleep deprivation were observed using an inhibitory avoidance test. Alterations in BF PIEZO1/calpain/autophagy signaling were detected. The PIEZO1 agonist Yoda1 or inhibitor GsMTx4, the calpain inhibitor PD151746, and the autophagy inducer rapamycin or inhibitor 3-MA were injected into the bilateral BF to investigate the pathways involved in the memory-maintaining role of EE in sleep-deprived mice. RESULTS: Mice housed in EE performed better than CE mice in short- and long-term fear memory tests after sleep deprivation. Sleep deprivation resulted in increased PIEZO1 expression, full-length tropomyosin receptor kinase B (TrkB-FL) degradation, and autophagy, as reflected by increased LC3 II/I ratio, enhanced p62 degradation, increased TFEB expression and nuclear translocation, and decreased TFEB phosphorylation. These molecular changes were partially reversed by EE treatment. Microinjection of Yoda1 or rapamycin into the bilateral basal forebrain induced excessive autophagy and eliminated the cognition-protective effects of EE. Bilateral basal forebrain microinjection of GsMTx4, PD151746, or 3-MA mimicked the cognitive protective and autophagy inhibitory effects of EE in sleep-deprived mice. CONCLUSIONS: EE combats sleep deprivation-induced fear memory impairments by inhibiting the BF PIEZO1/calpain/autophagy pathway.

Postpartum stress urinary incontinence: a clinical study of 6,302 cases in Jiangsu Province.

OBJECTIVES: To investigate the prevalence and influencing factors of stress urinary incontinence (SUI) within 6-8 weeks postpartum in Jiangsu Province. MATERIAL AND METHODS: We designed a multi-center cross-sectional study involving seven hospitals in Jiangsu province, and enrolled women who underwent postpartum examination at 6-8 weeks in these hospitals between July 2019 and June 2021. According to the presence or absence of SUI, the enrolled patients were divided into two groups: the SUI group and the non-SUI group, respectively. We assessed the general health status, noted the details of delivery, and checked the pelvic floor electromyographic parameters of the postpartum women in both groups. RESULTS: Among 6,302 cases of postpartum women in Jiangsu province, there were 1,579 cases of SUI, with a prevalence of 25.06%. The prevalence of SUI increased significantly with age, BMI, increasing parity, coexisting constipation, organ prolapse, and diastasis recti abdominis. Compared to the non-SUI group, the SUI group had a lower mean value of the pre-baseline rest phase, shorter rise and fall times of fast muscle contractions, and a lower mean value of the endurance contraction phase. Multiple regression analysis revealed associations with weight (especially overweight and obesity), coexisting organ prolapse, constipation, parity, gestational week of delivery, mode of delivery, and mean value of endurance contraction phase. CONCLUSIONS: The prevalence of postpartum stress urinary incontinence in Jiangsu Province was 25.06%, and was linked to being overweight, parity > 2, coexisting organ prolapse, constipation, and a decrease in the mean value of the endurance contraction phase of the electromyograph. In this report, we offer a theoretical basis for the effective prevention of postpartum SUI clinically.

[Dryness-alleviating effect of processed Aurantii Fructus from Lingnan based on correlation of fecal metabolomics and dryness].

With the approach of untargeted metabolomics and correlation analysis, this study aimed to explore the mechanism of Aurantii Fructus from Lingnan region in alleviating dryness by analyzing the different effects of raw Aurantii Fructus(RAF) and processed Aurantii Fructus(PAF) on fecal endogenous metabolism in normal rats. Eighteen Sprague-Dawley(SD) rats were randomly divided into a control group(C), an RAF group(10 g·kg~(-1)), and a PAF group(10 g·kg~(-1)). After seven days of administration, the effects of RAF and PAF on dryness-related indexes were compared, including water intake, fecal water content, salivary secretion, the expression of AQP5, VIP, and 5-HT in the submandibular gland, as well as the expression of AQP3, VIP, and 5-HT in the colon. The fecal samples in each group were determined by LC-MS. Multivariate statistical analysis and Pearson correlation coefficient were used for screening the differential metabolites and metabolic pathways in alleviating dryness of RAF. The results indicated that both RAF and PAF showed certain dryness, and the dryness of RAF was more significant. Moreover, PAF could alleviate dryness of RAF to a certain extent by reducing the water intake, fecal water content, and the expression of AQP3, VIP, and 5-HT in the colon and increasing the salivary secretion and the levels of AQP5, VIP, and 5-HT in the submandibular gland. According to the analysis of fecal metabolomics, 99 and 58 metabolites related to dryness were found in RAF and PAF respectively, where 16 of them played an important role in alleviating dryness of RAF. Pathway analysis revealed that the mechanism of PAF in alleviating dryness of RAF was presumably related to the regulation of riboflavin metabolism, purine metabolism, arginine biosynthesis, pyrimidine metabolism, alanine metabolism, aspartate metabolism, glutamate metabolism, and retinol metabolism pathways. This study suggested that PAF might alleviate dryness of RAF by affecting the metabolic levels of the body, which provides a new basis for further clarifying the processing mechanism of PAF.

Microglial voltage-dependent anion channel 1 signaling modulates sleep deprivation-induced transition to chronic postsurgical pain.

STUDY OBJECTIVES: This study verified that sleep deprivation before and after skin/muscle incision and retraction (SMIR) surgery increased the risk of chronic pain and investigated the underlying roles of microglial voltage-dependent anion channel 1 (VDAC1) signaling. METHODS: Adult mice received 6 hours of total sleep deprivation from 1 day prior to SMIR until the third day after surgery. Mechanical and heat-evoked pain was assessed before and within 21 days after surgery. Microglial activation and changes in VDAC1 expression and oligomerization were measured. Minocycline was injected to observe the effects of inhibiting microglial activation on pain maintenance. The VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and oligomerization inhibitor VBIT-4 were used to determine the roles of VDAC1 signaling on microglial adenosine 5' triphosphate (ATP) release, inflammation (IL-1ß and CCL2), and chronicity of pain. RESULTS: Sleep deprivation significantly increased the pain duration after SMIR surgery, activated microglia, and enhanced VDAC1 signaling in the spinal cord. Minocycline inhibited microglial activation and alleviated sleep deprivation-induced pain maintenance. Lipopolysaccharide (LPS)-induced microglial activation was accompanied by increased VDAC1 expression and oligomerization, and more VDAC1 was observed on the cell membrane surface compared with control. DIDS and VBIT-4 rescued LPS-induced microglial ATP release and IL-1ß and CCL2 expression. DIDS and VBIT-4 reversed sleep loss-induced microglial activation and pain chronicity in mice, similar to the effects of minocycline. No synergistic effects were found for minocycline plus VBIT-4 or DIDS. CONCLUSIONS: Perioperative sleep deprivation activated spinal microglia and increases the risk of chronic postsurgical pain in mice. VDAC1 signaling regulates microglial activation-related ATP release, inflammation, and chronicity of pain.

Immunoglobulin G4-related disease: case report and literature review.

Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a rare and chronic progressive clinical entity, characterized by elevated serum IgG4 along with tissue infiltration by IgG4 + plasma cells. It is an immune-mediated fibro-inflammatory condition that can affect virtually any organ and tissue. IgG4-related lung disease (IgG4-RLD) occupies 14% of all IgG4-RD, with nonspecific symptoms and various abnormal radiographic patterns. Published data on IgG4-related hypertrophic pachymeningitis (IgG4-RHP), an increasingly recognized central nervous system manifestation of IgG4-RD, is also limited. Both lung and cranial dura involvement have not yet been reported until now. We further entail a review of the literature on the clinicopathologic features and differential diagnosis of this uncommon disease. We herein report an interesting case of a 70-year-old male patient admitted due to headache and fever. A magnetic resonance imaging (MRI) of the brain revealed extensive dural thickening with marked enhancement. Chest computed tomography (CT) scan showed nodular or mass-like consolidation and focal interstitial change. Thoracoscopic lung biopsy and lumbar puncture were conducted. After careful histopathological observation and consideration of alternative differential diagnoses, he was diagnosed with IgG4-related disease with lung and cranial dural involvement based upon significant elevation of serum and cerebrospinal fluid (CSF) IgG4 concentration. The patient was started on oral prednisolone 60 mg/day (1.0 mg/kg/day) for 14 days, and a tapering dose of 5 mg every 2 weeks followed by maintenance therapy at low dose for 3 months. His clinical manifestations, and serologic and imaging findings improved with steroid treatment. Currently, the patient remains well without disease progression. IgG4-RD should be considered as a differential when diagnosing other similar multisystemic lesions. Clinical examination, careful histological observation, and immunostaining for appropriate markers are essential in establishing the diagnosis. Clinicians should become familiar with this alternative differential diagnosis.

Carfilzomib inhibits the growth of lung adenocarcinoma via upregulation of Gadd45a expression.

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.

Infiltrating regulatory T cells promote invasiveness of liver cancer cells via inducing epithelial-mesenchymal transition.

BACKGROUND: Regulatory T (Treg) cells are a major component of the microenvironment of hepatocellular carcinoma (HCC) contributing to immunosuppression. The present study aimed to evaluate the effects of Treg cells on the invasion potential of HCC. METHODS: Infiltrating Treg cells were isolated from fresh HCC tissues by immunomagnetic bead separation and detected by flow cytometry. Circulating tumor cells (CTCs) were detected using the CellSearch platform. The cell migration and invasion potentials were evaluated by Transwell assays. The cell viability was tested by the cell counting kit-8 (CCK8) approach, and the apoptosis rates were determined by flow cytometry. The concentrations of active transforming growth factor-ß1 (TGFß1) were measured by enzyme-linked immunosorbent assay. RESULTS: Infiltrating Treg cells significantly correlated with the number of CTCs and vascular invasion (both P<0.05). Moreover, these cells could greatly promote HCC migration, invasion, and proliferation, and inhibit HCC apoptosis. Polymerase chain reaction and Western blot assays revealed that Treg cells significantly decreased the expression levels of epithelium-related molecules and increased the expression levels of mesenchyme-related molecules. Treg cells could activate Smad2/3 via secreting TGFß1, and these effects could be impaired by knocking down the expression of TGFß1 in Treg cells. CONCLUSIONS: The involvement of infiltrating Treg cells in triggering the TGFß1 signaling pathway and promoting the epithelial-mesenchymal transition (EMT) of cancer cells during tumor hematogenous dissemination is presumably responsible for increasing the invasiveness potential of HCC cells. Targeting Treg cells in microenvironments can be a promising therapeutic strategy to improve the prognosis for patients with HCC undergoing resection.

Comparsion between Intravenous Delivered Human Fetal Bone Marrow Mesenchymal Stromal Cells and Mononuclear Cells in the Treatment of Rat Cerebral Infarct.

Objective To compare the effecacy of human mesenchymal stromal cell (hMSC) with human mononuclear cell (hMNC) in treating rat cerebral infarct.Methods The SD rat models of cerebral infarct were established by distal middle cerebral artery occlusion (dMCAO). Rats were divided into four groups: sham,ischemia vehicle,MSC,and MNC transplantation groups. For the transplantation group,1×106 hMSCs or hMNCs were intravascularly transplanted into the tail vein 1 hour after the ischemia onset. The ischemia vehicle group received dMCAO surgery and intravascular saline injection 1,3,5,and 7 days after the ischemia onset,and then behavioral tests were performed. At 48 h after the ischemia onset,the abundance of Iba- 1,the symbol of activated microglia,was evaluated in the peri-ischemia striatum area; meanwhile,the neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in ipsilateral peri-ischemia striatum area were also measured. Results The relative infarct volume in ischemia vehicle group,hMSC group,and hMNC transplantation group were (37.85±4.40)%,(33.41±3.82)%,and (30.23±3.63)%,respectively. The infarct volumes of MSC group (t=2.100,P=0.034) and MNC group (t=2.109,P=0.0009) were significantly smaller than that of ischemia vehicle group,and that of MNC group was significantly smaller than that of MSC group (t=1.743,P=0.043). One day after transplantation,the score of ischemia vehicle group in limb placing test was (4.32±0.71)%,which was significantly lower than that in sham group (9.73±0.36)% (t=2.178,P=8.61×10-11). The scores of MSC and MNC group,which were (5.09±0.62)% (t=2.1009,P=0.024) and (5.90±0.68)% (t=2.1008,P=0.0001),respectively,were significantly higher than that of ischemia vehicle group; also,the score of MNC group was significantly higher than that of MSC group(t=2.1009,P=0.0165). The contralateral forelimb scores of MSC and MNC groups in beam walking test were (5.56±0.86)% (t=2.120,P=0.020) and (5.13±0.95)% (t=2.131,P=0.003),were both significantly lower than that of ischemia vehicle group [(6.47±0.61)%]. Three days after the transplantation,the limb placing test score of MNC group [(6.91±1.10)%] was significantly higher than that of ischemia vehicle group (5.80±0.82)% (t=2.110,P=0.027). The score of MSC group [(6.30±0.77)%] showed no statistic difference with that of ischemia vehicle group(t=2.101,P=0.199).The contralateral forelimb scores of MNC group in beam walking test [(4.34±0.58)%] was significantly lower than that of ischemia vehicle group [(5.31±0.65)%] (t=2.100,P=0.006) and MSC group [(4.92±0.53)%] (t=2.100,P=0.041); there was no statistic difference between MSC group and ischemia vehicle group (t=2.109,P=0.139). The relative abundance of Iba- 1 in sham,ischemia vehicle,MSC,and MNC groups was 1.00+0.00,1.72±0.21,1.23±0.08,and 1.48±0.06,respectively. The Iba-1 relative abundance of ischemia vehicle group was significantly higher than that of sham group (t=2.262,P=2.9×10-6). The Iba-1 relative abundances of both MSC (t=2.178,P=3.91×10-5)and MNC (t=2.200,P=0.007)groups were significantly lower than that of ischemia vehicle group. It was also significantly lower in MNC group than in MSC group also (t=2.120,P=7.09×10-6). Three days after transplantation,the BDNF and GDNF levels of MSC group,which were (531.127±73.176)pg/mg (t=2.109,P=0.003)and(127.780±16.733)pg/mg(t=2.100,P=2.76×10-5),respectively,were significantly higher than those of ischemia vehicle group,which were (401.988±89.006)pg/mg and (86.278±14.832) pg/mg,respectively. The BDNF and GDNF levels of MNC group,which were (627.429±65.646)pg/mg (t=2.144,P=0.017) and (153.117±20.443)pg/mg (t=2.109,P=0.010),respectively,were all significantly higher than that of MSC group. At day 7,the BDNF and GDNF levels of MSC group,which were (504.776±83.282)pg/mg (t=2.101,P=0.005) and (81.641±11.019)pg/mg (t=2.100,P=0.002),respectively,were significantly higher than those of ischemia vehicle group,which were (389.257±70.440)pg/mg and (64.322±9.855) pg/mg,respectively. The BDNF and GDNF levels of MNC group,which were (589.068±63.323)pg/mg (t=2.100,P=0.027) and (102.161±19.932)pg/mg (t=2.144,P=0.017),respectively,were all significantly higher than that of MSC group. Conclusions Both hMSC and hMNC are beneficial to the ischemia-damaged brain when they are intravascularly transplanted within 1 h after the onset of ischemia. The anti-inflammation ability and secretion of neurotrophic factors are the underlying mechanisms of the therapeutic effects. MNC is more effective than MSC in reducing infarct area and improving behaviors,which might be explained by the fact that MNC induces more GDNF and BDNF in brain than MSC.

Malignant glomus tumor of the lung with multiorgan metastases: case report and literature review.

Glomus tumor is an exceedingly rare neoplasm that is derived from cells of the neuromyoarterial glomus or glomus body. It rarely occurs in the visceral organs where glomus body may be sparse or even absent, such as the stomach, intestines, mediastinum, and respiratory tract. It is unusual for a glomus tumor to demonstrate atypical or malignant histopathological characteristics. It is also rare for such a tumor to express clinically aggressive behavior. However, when metastasis does occur, this disease is often fatal. We herein report an interesting case of a middle-age woman admitted due to progressive cough and hemoptysis. A polypoid mass was found to occlude the left lingular lobar bronchus. Final histopathologic examination showed the presence of malignant glomus tumor, confirmed by immunoreactivity for smooth muscle actin and vimentin. Two months later, the patient developed abdominal distension and gastrointestinal bleeding. Further evaluation lead to the discovery of widespread metastatic disease to the gastrointestinal tract, spleen, and the left adrenal gland. We further entail a review of the literature on the clinicopathologic features and diagnosis of this uncommon tumor.

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses.

Enteroviruses are found in most environments and cause several diseases in humans. Loop-mediated isothermal amplification (LAMP) was adapted and evaluated for the rapid detection of enteroviruses. Based on the highly conserved 5' untranslated region (5'-UTR) of the human enteroviruses (HEVs), particularly human enterovirus A (HEV-A) and HEV-B, a set of universal primers was designed. The LAMP amplification was carried out under isothermal conditions at 61 °C, depending on the template concentration results were obtained within 45-90 min. The detection limits were found to be 10(1) copies of cloned enterovirus 71 fragments, more sensitive than conventional PCR. Nine water samples collected from drinking water sources during three seasons and 19 stool specimens collected from HFMD patients were analyzed. By using the LAMP assay, the majority of samples was tested positive, 9/9 (100 %) and 18/19 (94.7 %), respectively. LAMP is a practical method for the rapid detection of enteroviruses in environmental and clinical samples.

Artesunate exerts an anti-immunosuppressive effect on cervical cancer by inhibiting PGE2 production and Foxp3 expression.

Artesunate (ART), derived from a common traditional Chinese medicine, has beeen used an antimalarial for several years. In this study, the effect and mechanism of ART on anti-human cervical cancer cells was examined. The level of prostaglandin E2 (PGE2 ) and the population of CD4+CD25+Foxp3 regulatory T cells (Treg) in peripheral blood were detected by flow cytometry. In vivo antitumor activity was investigated in mice with cervical cancer by the subcutaneous injection of various concentrations of ART. The concentrations of PGE2 in the supernatants of CaSki cells were measured using an ELISA kit. Cyclooxygenase-2 (COX-2) and Foxp3 expression were determined using quantitative polymerase chain reaction (qPCR) and western blot analysis. The effect of ART on the viability of CaSki and Hela cells was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. It was identified that the level of PGE2 and the population of CD4+CD25+Foxp3 Treg cells in the peripheral blood were significantly higher in cervical cancer patients and mice with cervical cancer. ART was capable of inhibiting orthotopic tumor growth, which correlated with a decrease in the level of PGE2 and the percentage of Treg cells in mice with cervical cancer. Furthermore, ART decreased COX-2 expression and the production of PGE2 in CaSki and Hela cells. Notably, the supernatants of CaSki cells treated with ART lowered the expression of Foxp3 in Jurkat T cells, which was capable of being reversed by exogenous PGE2 . Our data revealed that ART may elicit an anti-tumor effect against cervical cancer by inhibition of PGE2 production in CaSki and Hela cells, which resulted in the decrease of Foxp3 expression in T cells. Therefore, ART may be an effective drug for immunotherapy of cervical cancer.

The 40-80 nt region in the 5'-NCR of genome is a critical target for inactivating poliovirus by chlorine dioxide.

Chemical disinfection is the most common method used to inactivate viruses from drinking water throughout the world. In this study, cell culture, ELISA, RT-PCR, and spot hybridization were employed to investigate the mechanism underlying chlorine dioxide (ClO(2) )-induced inactivation of Poliovirus type 1 (PV1), which was also confirmed by recombinant viral genome RNA infection models. The results suggested that ClO(2) inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ClO(2) degraded specifically the 40-80 nucleotides (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 RNA lacking this 40-80 nt region was not infectious. This study not only elucidated the mechanism of PV1 inactivation by ClO(2), but also defined the critical genetic target for the disinfectant to inactivate Poliovirus. This study also provides a strategy by which rapid, accurate, and molecular methods based on sensitive genetic targets may be established for evaluating the effects of disinfectants on viruses.

[Culture and characterization of and lentiviral vectors mediated glial cell derived neurotrophic factor expression in mesenchymal stem cells from human umbilical cord blood].

OBJECTIVE: To isolate and culture mesenchymal stem cells from umbilical cord blood (UCB-MSCs), study its biological characterization in vitro, transfect UCB-MSCs using lentiviral vectors encoding glial cell derived neurotrophic factor (GDNF) gene, evaluate the biological function change of UCB-MSCs, and detect GDNF expression level in vitro. METHODS: We isolated monocyte by Ficoll density gradient, separated two kinds of adherent cells through different trypsin digestion time, and detected the cells surface markers by fluorescence activated cell sorting when it was proliferated for P7 passages. At the same time, we sub-cloned GDNF gene into lentiviral vectors and packaged lentiviral supernatant through three plasmids co-transfection method, then transfected the UCB-MSCs using lentiviral vectors encoding GDNF at different multiplicity of infection, and evaluated the change of biological function by observing the ability of proliferation and differentiation, morphology, and the cells surface markers. We detected the GDNF mRNA and protein expression level by using real-time polymerase chain reaction (real-time PCR) and enzyme-link immunosorbent assay (ELISA). RESULTS: The UCB-MSCs were successfully isolated and cultured in vitro, and induced it to differentiate into fat cells. FACS results showed that the UCB-MSCs expressed CD90, CD73, and CD105 positively, and CD14, CD34, CD45, CD19, HLA-DR, Stro-1, and CD106 negatively. Real-time PCR and ELISA showed that the expressions of GDNF protein and mRNA were correlated with the copy number of transfected cells: high copy number of transfected cells were associated with high GDNF expression. The biological characterization of UCB-MSCs did not obviously change after sub-cloning with GDNF. CONCLUSIONS: UCB-MSCs was successfully isolated and cultured in vitro. By transfecting UCB-MSCs with GDNF gene-containing lentiviral vectors, the secretion of GDNF protein and mRNA expression level can be controlled by the copy number of transfected cells, and thus make it constantly express GDNF at high level.