mTORC1 in Thymic Epithelial Cells Is Critical for Thymopoiesis,T-Cell Generation,and Temporal Control of γδT17 Development and TCRγ/δ Recombination

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.     

Discovery of piRNAs Pathway Associated with Early-Stage Spermatogenesis in Chicken

Piwi-interacting RNAs (piRNAs) play a key role in spermatogenesis. Here, we describe the piRNAs profiling of primordial germ cells (PGCs), spermatogonial stem cells (SSCs), and the spermatogonium (Sp) during early-stage spermatogenesis in chicken. We obtained 31,361,989 reads from PGCs, 31,757,666 reads from SSCs, and 46,448,327 reads from Sp cells. The length distribution of piRNAs in the three samples showed peaks at 33 nt. The resulting genes were subsequently annotated against the Gene Ontology (GO) database. Five genes (RPL7A, HSPA8, Pum1, CPXM2, and PRKCA) were found to be involved in cellular processes. Interactive pathway analysis (IPA) further revealed three important pathways in early-stage spermatogenesis including the FGF, Wnt, and EGF receptor signaling pathways. The gene Pum1 was found to promote germline stem cell proliferation, but it also plays a role in spermatogenesis. In conclusion, we revealed characteristics of piRNAs during early spermatogonial development in chicken and provided the basis for future research.     

An Active Type I-E CRISPR-Cas System Identified in Streptomyces avermitilis

CRISPR-Cas systems, the small RNA-dependent immune systems, are widely distributed in prokaryotes. However, only a small proportion of CRISPR-Cas systems have been identified to be active in bacteria. In this work, a naturally active type I-E CRISPR-Cas system was found in Streptomyces avermitilis. The system shares many common genetic features with the type I-E system of Escherichia coli, and meanwhile shows unique characteristics. It not only degrades plasmid DNA with target protospacers, but also acquires new spacers from the target plasmid DNA. The naive features of spacer acquisition in the type I-E system of S. avermitilis were investigated and a completely conserved PAM 5’-AAG-3’ was identified. Spacer acquisition displayed differential strand bias upstream and downstream of the priming spacer, and irregular integrations of new spacers were observed. In addition, introduction of this system into host conferred phage resistance to some extent. This study will give new insights into adaptation mechanism of the type I-E systems in vivo, and meanwhile provide theoretical foundation for applying this system on the genetic modification of S. avermitilis.     

Comparative Screening of Digestion Tract Toxic Genes in Proteus mirabilis

Proteus mirabilis is a common urinary tract pathogen, and may induce various inflammation symptoms. Its notorious ability to resist multiple antibiotics and to form urinary tract stones makes its treatment a long and painful process, which is further challenged by the frequent horizontal gene transferring events in P. mirabilis genomes. Three strains of P. mirabilis {"type":"entrez-nucleotide","attrs":{"text":"C02011","term_id":"1434241"}}C02011/{"type":"entrez-nucleotide","attrs":{"text":"C04010","term_id":"1467261"}}C04010/C04013 were isolated from a local outbreak of a food poisoning event in Shenzhen, China. Our hypothesis is that new genes may have been acquired horizontally to exert the digestion tract infection and toxicity. The functional characterization of these three genomes shows that each of them independently acquired dozens of virulent genes horizontally from the other microbial genomes. The representative strain {"type":"entrez-nucleotide","attrs":{"text":"C02011","term_id":"1434241"}}C02011 induces the symptoms of both vomit and diarrhea, and has recently acquired a complete type IV secretion system and digestion tract toxic genes from the other bacteria.     

Determining an Optimal Cutoff of Serum β-Human Chorionic Gonadotropin for Assisting the Diagnosis of Intracranial Germinomas

Background

Beta (β)-human chorionic gonadotropin (β-HCG) is used to confirm the diagnosis and plan treatment of intracranial germinomas. However, the cutoff values of serum β-HCG in diagnosis of intracranial germinomas reported in the literature are inconsistent. To establish an appropriate cutoff value of serum β-HCG for diagnosis of intracranial germinomas, we retrospectively reviewed the records of intracranial tumor patients who received serum β-HCG and α-fetoprotein (AFP) tests for diagnostic purposes at our hospital from 2005 to 2014.

Methods

A total of 93 intracranial germinomas and 289 intracranial non-germ cell tumors were included in this study. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of 3 cutoffs (0.1, 0.4, and 0.5 mIU/mL) for diagnosing intracranial germinomas. The serum β-HCG level of intracranial germinoma patients was further analyzed to investigate the effect of metastasis status and tumor location on serum β-HCG level.

Results

The area under the ROC curve was 0.81 (P < .001), suggesting β-HCG is an effective marker. Of the 3 cutoff values, 0.1 mIU/mL possessed a highest sensitivity (66.67%) and good specificity (91%). Although there was no β-HCG level difference between metastatic and non-metastatic intracranial germinoma patients, the diagnostic rate of metastatic neurohypophyseal germinomas was significantly higher than that of its non-metastatic counterpart (P < .05), implying that the location of the germinoma might need to be considered when β-HCG is used as a marker to predict metastasis.

Conclusions

Determining an optimal cutoff of serum β-HCG is helpful for assisting the diagnosis of intracranial germinoma.     

The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth

Background

As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC.

Methods

Three NSCLC cell lines (A549, SK-MES-1, and NCI-H460) were treated with PF-4708671 at five different concentrations, including 0.1μM, 0.3μM, 1μM, 3μM and 10μM, and protein levels were determined by Western-blot. Then, PF-4708671’s effects were assessed both in vitro (cell proliferation, apoptosis, cell cycle distribution, and invasion) and in vivo.

Results

The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo.

Conclusion

These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.     

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type,Neuronal Subtype,and Ca2+ Signaling

Uptake of Ca²⁺ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca²⁺ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca²⁺ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca²⁺ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca²⁺, as well as diversity of mitochondrial Ca²⁺ uptake mechanisms.     

Effects of Intra-Operative Total Intravenous Anaesthesia with Propofol versus Inhalational Anaesthesia with Sevoflurane on Post-Operative Pain in Liver Surgery: A Retrospective Case-Control Study

Background

Patients receiving total intravenous anesthesia (TIVA) with propofol have been shown to experience less postoperative pain. We evaluated the post-operative analgesic effects of propofol compared with sevoflurane maintenance of anesthesia in liver surgery. This study was registered at ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT02179437","term_id":"NCT02179437"}}NCT02179437).

Methods

In this retrospective study, records of patients who underwent liver surgery between 2010 and 2013 were reviewed. Ninety-five patients anesthetized with propofol TIVA were matched with 95 patients anesthetized with sevoflurane. Numeric pain rating scale (NRS) pain scores, postoperative morphine consumption, side effects and patients’ satisfaction with pain relief were evaluated.

Results

The TIVA group reported lower NRS pain scores during coughing on postoperative days 1 and 2 but not 3 (p = 0.0127, p = 0.0472, p = 0.4556 respectively). They also consumed significantly less daily (p = 0.001 on day 1, p = 0.0231 on day 2, p = 0.0004 on day 3), accumulative (p = 0.001 on day 1, p<0.0001 on day 2 and p = 0.0064 on day 3) and total morphine (p = 0.03) when compared with the sevoflurane group. There were no differences in total duration of intravenous patient controlled analgesia (PCA) morphine use and patient satisfaction. No difference was found in reported side effects.

Conclusion

Patients anesthetized with propofol TIVA reported less pain during coughing and consumed less daily, accumulative and total morphine after liver surgery.