LSM14B controls oocyte mRNA storage and stability to ensure female fertility.

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b-deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability.

Glutamine exerts a protective effect on osteoarthritis development by inhibiting the Jun N-terminal kinase and nuclear factor kappa-B signaling pathways.

Strategies for treating osteoarthritis (OA) have become a research focus because an effective treatment for OA is unavailable. The objective of this study was to explore the effects and underlying mechanisms of glutamine (Gln) in OA. First, the chondrocytes were identified and a standard IL-1ß-induced OA model was established. After treatment with Gln or saline, the viability and apoptosis of chondrocytes were evaluated using a CCK-8 assay and flow cytometry analysis, which revealed that Gln can improve the IL-1ß-induced OA cells. Meanwhile, Gln can enhance the expression of aggrecan and collagen II, which are protective proteins for articular cartilage. Instead, Gln inhibited the expression of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13), which can degrade cartilage. To better understand the underlying mechanisms of Gln in IL-1ß-induced chondrocytes, the classical OA pathways of JNK and NF-κB were examined at the protein and mRNA levels using western blot and qRT-PCR analyses. We found that JNK and NF-κB were downregulated gradually depending on the Gln dose and protective and destructive factors changed based on changes of JNK and NF-κB. The effects of high-dose Gln were more effective than low-dose. Moreover, Gln was applied to the animal OA model to check the effects in vivo. The results showed that Gln attenuated cartilage degeneration and decreased OARSI scores, which demonstrated that Gln can improve OA. The experiments showed that Gln can benefit mice with OA by inhibiting the JNK and NF-κB signaling pathways.

Single-cell RNA sequencing of the Mongolia sheep testis reveals a conserved and divergent transcriptome landscape of mammalian spermatogenesis.

Spermatogenesis is a highly coordinated and complex process, and is pivotal for transmitting genetic information between mammalian generations. In this study, we investigated the conservation, differences, and biological functions of homologous genes during spermatogenesis in Mongolia sheep, humans, cynomolgus monkey, and mice using single-cell RNA sequencing technology. We compared X chromosome meiotic inactivation events in Mongolia sheep, humans, cynomolgus monkey, and mice to uncover the concerted activity of X chromosome genes. Subsequently, we focused on the dynamics of gene expression, key biological functions, and signaling pathways at various stages of spermatogenesis in Mongolia sheep and humans. Additionally, the ligand-receptor networks of Mongolia sheep and humans in testicular somatic and germ cells at different developmental stages were mapped to reveal conserved germ cell-soma communication using single-cell resolution. These datasets provided novel information and insights to unravel the molecular regulatory mechanisms of Mongolia sheep spermatogenesis and highlight conservation in gene expression during spermatogenesis between Mongolia sheep and humans, providing a foundation for the establishment of a large mammalian disease model of male infertility.

Dysbiosis of gut microbiome affecting small intestine morphology and immune balance: a rhesus macaque model.

There is a growing appreciation for the specific health benefits conferred by commensal microbiota on their hosts. Clinical microbiota analysis and animal studies in germ-free or antibiotic-treated mice have been crucial for improving our understanding of the role of the microbiome on the host mucosal surface; however, studies on the mechanisms involved in microbiome-host interactions remain limited to small animal models. Here, we demonstrated that rhesus monkeys under short-term broad-spectrum antibiotic treatment could be used as a model to study the gut mucosal host-microbiome niche and immune balance with steady health status. Results showed that the diversity and community structure of the gut commensal bacteria in rhesus monkeys were both disrupted after antibiotic treatment. Furthermore, the 16S rDNA amplicon sequencing results indicated that Escherichia-Shigella were predominant in stool samples 9 d of treatment, and the abundances of bacterial functional genes and predicted KEGG pathways were significantly changed. In addition to inducing aberrant morphology of small intestinal villi, the depletion of gut commensal bacteria led to increased proportions of CD3 + T, CD4 + T, and CD16 + NK cells in peripheral blood mononuclear cells (PBMCs), but decreased numbers of Treg and CD20 + B cells. The transcriptome of PBMCs from antibiotic-treated monkeys showed that the immune balance was affected by modulation of the expression of many functional genes, including IL-13, VCAM1, and LGR4.

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1 -/-mice infected with influenza virus (H1N1).

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI). The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo. IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation. We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice. Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6, TNF-α, G-CSF, KC, and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1 -/- mice in comparison with that of wild type infected mice. The adaptive immune response against the H1N1 virus in IL-1R1 -/- mice was impaired with downregulated anti-viral Th1 cell, CD8+ cell, and antibody functions, which contributes to attenuated viral clearance. Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1 -/- mice compared with that in WT infected mice. Moreover, the infected IL-1R1 -/- mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung. Together, these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury, particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.

Nasal Infection of Enterovirus D68 Leading to Lower Respiratory Tract Pathogenesis in Ferrets (Mustela putorius furo).

Data from EV-D68-infected patients demonstrate that pathological changes in the lower respiratory tract are principally characterized by severe respiratory illness in children and acute flaccid myelitis. However, lack of a suitable animal model for EV-D68 infection has limited the study on the pathogenesis of this critical pathogen, and the development of a vaccine. Ferrets have been widely used to evaluate respiratory virus infections. In the current study, we used EV-D68-infected ferrets as a potential animal to identify impersonal indices, involving clinical features and histopathological changes in the upper and lower respiratory tract (URT and LRT). The research results demonstrate that the EV-D68 virus leads to minimal clinical symptoms in ferrets. According to the viral load detection in the feces, nasal, and respiratory tracts, the infection and shedding of EV-D68 in the ferret model was confirmed, and these results were supported by the EV-D68 VP1 immunofluorescence confocal imaging with α2,6-linked sialic acid (SA) in lung tissues. Furthermore, we detected the inflammatory cytokine/chemokine expression level, which implied high expression levels of interleukin (IL)-1a, IL-8, IL-5, IL-12, IL-13, and IL-17a in the lungs. These data indicate that systemic observation of responses following infection with EV-D68 in ferrets could be used as a model for EV-D68 infection and pathogenesis.

Clinical, demographic and psychological characteristics of infertile male smokers in Northeast China.

OBJECTIVES: To investigate clinical, demographic and psychological characteristics of infertile male smokers in northeast China. METHODS: Serum and semen samples were collected from infertile men. Semen analysis was performed according to conventional procedures. Serum follicle-stimulating hormone, luteinizing hormone and testosterone levels were quantified. Psychological anxiety and depression were evaluated by the self-rating anxiety scale (SAS) and self-rating depression scale (SDS), respectively. RESULTS: Both SDS and SAS scores were significantly higher in smokers (n = 704) than in nonsmokers (n = 372); in addition, sperm viability and motility were significantly lower in smokers than in nonsmokers. Spearman's correlation coefficient analysis revealed significant positive correlations between duration of smoking and SDS and SAS scores, and between cigarettes smoked per day and SDS and SAS scores. CONCLUSIONS: Cigarette smoking has a negative effect on sperm viability and motility, and is associated with increased SDS and SAS scores.

Production of recombinant protein G through high-density fermentation of engineered bacteria as well as purification.

Recombinant Streptococcus Protein G (PG) is a cell wall protein, which, when combined with mammal immunoglobulin, is used in separating antibody technology. High-density fermentation technologies using an engineered recombinant PG-producing bacteria as well as PG separation and purification technologies have a direct impact on the availability and application of PG. Through primary and secondary seed cultivation, a recombinant E. coli strain was subjected to high-density fermentation with controlled feed supplement concentration under stimulation with isopropyl ß-D-1-thiogalactopyranoside. The present study investigated the effect of factors including inoculum size, oxygen levels, pH and the cultivating method on the fermentation process, as well as the effect of the separation and purification technologies, including ultrasonication, nickel column affinity chromatography, Sephadex G-25 gel filtration chromatography and diethylaminoethanol-sepharose fast flow ion exchange chromatography on the yield and purity of PG. The efficiency of extraction was detected using SDS-PAGE. High-density fermentation yielded 80-150 g/l of bacteria and 1 g PG was obtained from one liter broth. The present study delivered a highly efficient novel method via which PG can be obtained at a high concentration and a purity >95%.

Photodynamic therapy in spinal metastases: a qualitative analysis of published results.

The current study was to perform qualitative comparison of photodynamic therapy (PDT), based on previously published articles on spinal disease distribution status before and after treatment. Spinal metastasis, the migration of primary cancer cells and establishment of secondary tumors in the spine. We electronically searched CENTRAL (The Cochrane Library 2012), MEDLINE, EMBASE, CINAHL and AMED (from their beginning to December 31, 2012) to identify published studies assessing the effectiveness of PDT in spinal metastases. Our inclusion criteria resulted in only 4 articles, all in mice models. Due to study limitations and sparse data, the quality of evidence for all outcomes was low. Our analyses shows that effects on stereological and mechanical properties observed at the 1-week time point post-PDT are maintained at a longer 6-week time point, with combined PDT + bisphosphonate treatment being the most beneficial in terms of bone enhancement. Additionally, the combination of PDT + radiation therapy also demonstrated significant increases in stereological parameters, suggesting that previous radiation therapy treatment does not preclude the bone-enhancing effects of PDT and in fact may be synergistic in the longer term. The bone-enhancing effects of PDT in combination with conventional treatments, and its ability to destroy metastatic human breast cancer cells within bone, present PDT as an attractive novel treatment for spinal metastasis. The positive results from these preclinical studies might motivate future clinical translation of PDT for spinal metastasis.

Generation of broadband 17-µJ mid-infrared femtosecond pulses at 3.75 µm by silicon carbide crystal.

In this contribution, we report the generation of 17-µJ mid-infrared (MIR) pulses with duration of 70 fs and bandwidth of 550 nm centered at 3.75 µm at 1-kHz repetition rate, by a two-stage femtosecond optical parametric amplifier utilizing 4H-silicon carbide crystal as the nonlinear medium. The crystal is selected as it processes orders of magnitude higher damage threshold than traditional MIR nonlinear crystals, and it supports extreme broad parametric bandwidth. With its distinguished features such as MIR central wavelength, ultra-broad bandwidth, self-stable carrier-envelope phase, and potential for energy scaling, this kind of MIR source holds promise for new approaches to extreme short isolated attosecond pulse generation as well as MIR spectroscopy applications.

The overexpression of epithelial cell adhesion molecule (EpCAM) in glioma.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in various neoplasms as a tumor-associated antigen and absent in natural brain. However, little is known about EpCAM's expression in gliomas. To investigate the expression of EpCAM in gliomas and understand the correlation of EpCAM expression with malignancy, proliferation, angiogenesis, and prognosis, we studied the expression of EpCAM in 98 glioma samples by immunohistochemistry and by western blotting (N = 12). Correlative analysis of EpCAM overexpression with microvessel density (MVD), Ki-67 expression, age, and gender were made. Survival data was analyzed with Kaplan-Meier method and Cox Proportional Hazard Model. Immunohistochemistry results showed EpCAM was widely expressed in glioma (90.8 %). The overexpression rate of WHO grade IV gliomas was significantly higher EpCAM overexpression correlated significantly with Ki-67 expression and MVD. Western blot analysis also revealed a stepwise increase in EpCAM expression from WHO II to IV glioma. The overall survival of WHO III and IV glioma patients with EpCAM overexpression was obviously lower than that without EpCAM overexpression. EpCAM overexpression was an independent prognostic factor for overall survival in glioma patients. This study firstly shows that EpCAM overexpression correlates significantly with malignancy (WHO grades), proliferation (Ki67), angiogenesis (MVD), and prognosis in gliomas. EpCAM may participate in tumorgenesis of gliomas.

Overexpression of EpCAM and Trop2 in pituitary adenomas.

We sought to investigate the expression of EpCAM and Trop2 in Pituitary adenomas (PAs) and study the correlation of protein expression with invasiveness, proliferation, clinical functioning, recurrence/progression, and some other factors. We investigated the expression of EpCAM and Trop2 in 74 samples of PAs by immunohistochemistry and made correlative analysis of protein overexpression with clinicopathological parameters. Follow-up data was analyzed for recurrence/progression with Kaplan-Meier method and Multivariate Cox regression analysis. Immunohistochemistry results showed that overexpression rates of EpCAM and Trop2 were 51/74 (68.9%) and 43/74 (58.1%), respectively. For both EpCAM and Trop2, PAs with invasiveness showed a higher overexpression rate than PAs without invasiveness (PEpCAM = 0.001; PTrop2 = 0.006). Nonfunctional Pituitary adenomas (NFPAs) demonstrated a higher EpCAM overexpression than functional Pituitary adenomas (FPAs) (P = 0.026). Both EpCAM and Trop2 overexpression correlated significantly with expression of proliferation factor Ki-67 (PEpCAM = 0.011; PTrop2 = 0.000), but not with gender and age. Follow-up analysis revealed that Trop2 overexpression was a significantly predictive factor for recurrence/progression by means of Kaplan-Meier method d (P = 0.028) and Multivariate Cox regression analysis (P = 0.025). This study reveals that both EpCAM and Trop2 overexpression in PAs correlate significantly with invasiveness and proliferation. EpCAM presents a potential target for differential diagnosis and immunotherapy for NFPAs. Follow-up analysis shows that Trop2 is a predictive factor for recurrence/progression for PAs.

[Relationship of motor deficits and imaging features in metastatic epidural spinal cord compression].

OBJECTIVE: To explore the relationship of motor deficits of the lower extremities with the imaging features of malignant spinal cord compression (MESCCs). METHODS: From July 2006 through December 2008, 56 successive MESCC patients were treated at our department. All were evaluated by magnetic resonance imaging and computed tomography and were scored according to motor deficits Frankel grading on admission. Imaging assessment factors of main involved vertebrae were level of vertebral metastatic location, epidural space involvement, vertebral body involvement, lamina involvement, posterior protrusion of posterior wall, pedicle involvement, continuity of main involved vertebrae, fracture of anterior column, fracture of posterior wall, location in upper thoracic spine and/or cervicothoracic junction. RESULTS: Occurrence was the same between paralytic state of MESCCs and epidural space involvement of imaging features. Multiple regression equation showed that paralytic state had a linear regression relationship with imaging factors of lamina involvement (X1), posterior protrusion of posterior wall (X2), location in upper thoracic spine and/or cervicothoracic junction (X7) of main involved vertebrae. The optimal regression equation of paralytic state (Y) and imaging feature (X) was Y = -0.009 +0.639X, + 0.149X, +0.282X. Lamina involvement of main involved vertebrae has a greatest influence upon paralytic state of MESCC patients. CONCLUSIONS: Imaging factors of lamina involvement, posterior protrusion of posterior wall, location in upper thoracic spine and/or cervicothoracic junction of main involved vertebrae can predict the paralytic state of MESCC patients. MESCC with lamina involvement is more easily encroached on epidural space.

Expression of hepatocyte growth factor and its receptor c-Met in human pituitary adenomas.

Hepatocyte growth factor (HGF) and its receptor c-Met have been known as key determinants of growth and angiogenesis in some brain tumors like gliomas, meningiomas, and schwannomas. But little is known about their expression in pituitary adenomas. In this study, the expression of HGF and c-Met in pituitary adenomas of different histology types was investigated by immunohistochemistry, and correlative analysis of their expression with microvessel density (MVD), Ki-67 expression, and other clinicopathologic factors was made. The results showed that the expression of HGF and c-Met exists in 98% (64 of 65) and 92% (60 of 65) pituitary adenomas, respectively, and co-expression of them existed in 91% (59 of 65) adenomas. HGF had significant correlation with MVD (Spearman's correlation coefficient, r = .31, P = .01) and Ki-67 (r = .32, P = .01). c-Met had significant correlation with MVD (r = .30, P = .02) and Ki-67 (r = .38, P = .00). HGF and c-Met expression had no significant correlation with age or extrasellar extension. There were no significant differences in HGF and c-Met expression between pituitary adenomas of different histology types. The results indicate that HGF and c-Met are widely expressed in pituitary adenomas, and their expression correlates with MVD and Ki-67 expression.

[A new monoterpene glycoside from active fraction (DSS-A-N-30) of Danggui Shaoyao San].

OBJECTIVE: To study the chemical constituents of an active fraction (DSS-A-N-30) from Danggui Shaoyao San. METHOD: DSS-A-N30 was prepared by macroporous resin chromatography, the compound was isolated by column chromatography on silica gel and RPC-18, the structure was elucidated by spectroscopic methods. RESULT: A new monoterpene glycoside was isolated and identified from DSS-A-N-30. CONCLUSION: The new monoterpene glycoside was identified as 4"-hydroxyl-albiflorin.

[Diagnosis and treatment of non-specific granulomatous prostatitis: a report of 32 cases].

OBJECTIVE: To discuss the diagnosis and treatment of non-specific granulomatous prostatitis (NSGP). METHODS: Thirty-two cases of NSGP were diagnosed by puncture biopsy under transrectal ultrasound (TRUS) and treated with antibiotics and other medicines from September, 2000 to May, 2006. RESULTS: Pathomorphologically, NSGP was basically characterized by granuloma with vessels or grand alveoli in the center. The mean follow-up was 24 months. Urination irritation and obstruction were improved. Q(max) was increased to 15.0-24.0 ml/s, and in 3 cases of urinary retention, to 12.0, 14.5 and 16.5 ml/s, respectively. Digital rectal examination (DRE) indicated a reduced size and softened texture of the prostate induration. PSA was decreased to 1.3-11.5 microg/L. Four cases experienced relapse but were cured after retreated. No prostate cancer was observed. CONCLUSION: NSGP can be definitely diagnosed by puncture biopsy under TRUS and effectively relieved by antibiotics with the alpha-receptor blocker. In case of serious obstruction complicated by urinary retention, transurethral electrotomy can be considered.

[Transrectal ultrasound: an applicable diagnostic approach to chronic prostatitis].

OBJECTIVE: To assess the application value of transrectal ultrasound (TRUS) in the diagnosis of chronic prostatitis. METHODS: TRUS and examination of prostatic secretion (EPS) were used in the diagnosis of 3 500 cases of chronic prostatitis from September, 2000 to May, 2006. RESULTS: Lower resonance of the inner gland, low-level echo, uneven echo light spots, incomplete outlines and unsmooth borderlines were found in 2279 cases (65.1%), and the enlarged prostate in 1 084 cases (31.0%), with clear integrated amicula and enhanced echogenic spots at the juncture of the external and inner gland. No obvious changes were noted in 137 cases (4.0%), and in another 391 cases (11.2%) were detected alteration of the acoustic image of cystospermitis and blurred margins and uneven echoes of the seminal vesicle. The WBC count in EPS was < 10/HP in 132 cases (3.8%), 10-19/HP in 2 156 cases (61.6%) and > or =20/HP in 1212 cases (34.6%). CONCLUSION: TRUS, as a diagnostic means for chronic prostatitis, can be easily performed and causes little pain and therefore is readily accepted by patients. Combined with EPS, TRUS can provide more definite diagnostic evidence, and for those who are afraid of pain and reject EPS, it is a desirable alternative in the diagnosis of chronic prostatitis.

[Modification of in situ cryopreservation of human bone marrow mesenchymal stem cells].

The study was aimed to evaluate if the modified in situ cryopreservation could affect the biological function of mesenchymal stem cells (MSC) in vitro. Mesenchymal stem cells from human bone marrow were isolated by standard method and characterized with their morphology, cell-surface antigen profile and differentiation repertoire in vitro. The culture-expanded MSC were cryopreserved in situ with culture medium (DMEM-LG) containing 10% D MSO and 30% selected FCS in -70 degrees C. Following recovery of cryopreservation, differentiation to adipocytes, chondrocytes, and osteoblast in vitro and cell cycle analysis were performed to investigate whether the cryopreservation would change the differentiation potential of MSC. The results showed that after recovery of cryopreservation, there was no changes detected as compared with the culture-expanded MSC in both differentiation potency and growth pattern at 12 weeks. In conclusions: this optimized short term in situ cryopreservation at -70 degrees C could retain biological characteristics of human MSC for at least 3 months, and this method may be useful for cryopreservation of hum an bone marrow MSCs.