Mechanism of nucleic acid unwinding by SARS-CoV helicase

The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.     

Cyclase‐associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells

Adenylyl cyclase‐associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K‐mTOR/SREBP‐1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.     

The endosperm development and the variations of structures of embryo sacs: unravelling the low fecundity of Zephyranthes candida (Amaryllidaceae)

To unravel a low fecundity in Zephyranthes candida (Lindl.) Herb., the development of the endosperm was studied using conventional paraplast section technique. The results show that the endosperm develops normally and comprises four major stages viz. syncytial, cellularization, differentiation and maturation. Both proliferation of antipodal cells and their close contact with the primary endosperm nucleus were observed, which should favor transportation of nutrients and accelerate development of embryo and endosperm. In Z. candida, at least four events of nuclear migration occurred during the course of embryogenesis and endosperm development. The 12.7% structurally and functionally abnormal ovules, along with the 22.3% collapsed and aborted ovules observed accounts for the low fecundity to some extent.     

Drought leads to reproductive quiescence in smooth-billed anis: Phenotypic evidence for opportunistic breeding and reproductive readiness

Previous studies have suggested that the smooth-billed ani (Crotophaga ani, Linnaeus, 1758) breeds opportunistically following unpredictable rainfall in drought areas. To obtain proof of this phenomenon, the present study described and compared reproductive morphology and cell proliferation in the gonads of free-living smooth-billed anis during a wet season (April to June 2012) and the following dry season (July to September 2012) in a semiarid area using light and electron microscopy (transmission and scanning) and the AgNOR method. The morphological findings indicated distinct levels of reproductive activity related to seasonal changes. Morphological and morphometric analyses of the gonads confirmed intense gametogenic activity during the wet season, whereas gonadal involution occurred after rainfall ceased. The sizes of the testes and ovaries were significantly reduced compared to those in the wet season. The volumetric fraction of the seminiferous tubules in the testis decreased considerably, and no preovulatory follicles were detected in the ovary in the dry season. Moreover, the AgNOR count in the gonads revealed a significant decline in cell recruitment for gametogenesis after rainfall ceased. The histological findings indicated partial gonadal activation throughout the dry season. The analysis of the seminiferous epithelium confirmed the early testicular recrudescence phase, and sporadic postovulatory follicles indicated random ovulation during this time. The excurrent ducts and the oviduct also underwent remarkable involution in the dry season. Taken together, these findings confirm opportunistic breeding by smooth-billed anis in a semiarid habitat and suggest that gonadal recrudescence has been established as a reproductive strategy to cope with unexpected precipitation events.     

An Economic Evaluation of Venous Thromboembolism Prophylaxis Strategies in Critically Ill Trauma Patients at Risk of Bleeding

Background

Critically ill trauma patients with severe injuries are at high risk for venous thromboembolism (VTE) and bleeding simultaneously. Currently, the optimal VTE prophylaxis strategy is unknown for trauma patients with a contraindication to pharmacological prophylaxis because of a risk of bleeding.

Methods and Findings

Using decision analysis, we estimated the cost effectiveness of three VTE prophylaxis strategies—pneumatic compression devices (PCDs) and expectant management alone, serial Doppler ultrasound (SDU) screening, and prophylactic insertion of a vena cava filter (VCF)—in trauma patients admitted to an intensive care unit (ICU) with severe injuries who were believed to have a contraindication to pharmacological prophylaxis for up to two weeks because of a risk of major bleeding. Data on the probability of deep vein thrombosis (DVT) and pulmonary embolism (PE), and on the effectiveness of the prophylactic strategies, were taken from observational and randomized controlled studies. The probabilities of in-hospital death, ICU and hospital discharge rates, and resource use were taken from a population-based cohort of trauma patients with severe injuries (injury severity scores >12) admitted to the ICU of a regional trauma centre. The incidence of DVT at 12 weeks was similar for the PCD (14.9%) and SDU (15.0%) strategies, but higher for the VCF (25.7%) strategy. Conversely, the incidence of PE at 12 weeks was highest in the PCD strategy (2.9%), followed by the SDU (1.5%) and VCF (0.3%) strategies. Expected mortality and quality-adjusted life years were nearly identical for all three management strategies. Expected health care costs at 12 weeks were Can$55,831 for the PCD strategy, Can$55,334 for the SDU screening strategy, and Can$57,377 for the VCF strategy, with similar trends noted over a lifetime analysis.

Conclusions

The attributable mortality due to PE in trauma patients with severe injuries is low relative to other causes of mortality. Prophylactic placement of VCF in patients at high risk of VTE who cannot receive pharmacological prophylaxis is expensive and associated with an increased risk of DVT. Compared to the other strategies, SDU screening was associated with better clinical outcomes and lower costs. Please see later in the article for Editors'' Summary     

Cardiac effects of postconditioning depend critically on the duration of index ischemia

Postconditioning (POC) is known as the phenomenon whereby brief intermittent ischemia applied at the onset of reperfusion following index ischemia limits myocardial infarct size. Whereas there is evidence that the algorithm of the POC stimulus is an important determinant of the protective efficacy, the importance of the duration of index ischemia on the outcome of the effects of POC has received little attention. Pentobarbital sodium-anesthetized Wistar rats were therefore subjected to index ischemia produced by coronary artery occlusions (CAO) of varying duration (15-120 min) followed by reperfusion, without or with postconditioning produced by three cycles of 30-s reperfusion and reocclusion (3POC30). 3POC30 limited infarct size produced by 45-min CAO (CAO45) from 45 +/- 3% to 31 +/- 5%, and CAO60 from 60 +/- 3% to 47 +/- 6% (both P < or = 0.05). In contrast, 3POC30 increased infarct size produced by CAO15 from 3 +/- 1% to 19 +/- 6% and CAO30 from 36 +/- 6 to 48 +/- 4% (both P < or = 0.05). This deleterious effect of 3POC30 was not stimulus sensitive because postconditioning with 3POC5 and 3POC15 after CAO30 also increased infarct size. The cardioprotection by 3POC30 after CAO60 was accompanied by an increased stimulation of Akt phosphorylation at 7 min of reperfusion and a 36% lower superoxide production, measured by dihydroethidium fluorescence, after 2 h of reperfusion. Consistent with these results, cardioprotection by 3POC30 was abolished by phosphatidylinositol-3-OH-kinase inhibition, as well as nitric oxide (NO) synthase inhibition. The deleterious effect of 3POC30 after CAO15 was accompanied by an increased superoxide production with no change in Akt phosphorylation and was not affected by NO synthase inhibition. In conclusion, the effect of cardiac POC depends critically on the duration of the index ischemia and can be either beneficial or detrimental. These paradoxical effects of POC may be related to the divergent effects on Akt phosphorylation and superoxide production.     

Synthesis and amine transporter affinities of novel phenyltropane derivatives as potential positron emission tomography (PET) imaging agents

A series of novel fluoroalkyl-containing tropane derivatives (6-8, 10-14, 17, and 18) were synthesized from cocaine. Novel compounds were evaluated for affinity and selectivity in competitive radioligand binding assays selective for cerebral serotonin (5-HT), dopamine (DA), and norepinephrine (NE) transporters (SERT, DAT, and NET). The nortropane-fluoroalkyl esters (7, 10, 11) were most potent for SERT (K(i): 0.18, 0.24, and 0.30 nM, respectively). Tosylate esters 17 and 18, synthesized as precursors for [(18)F]-labeled, Positron Emission Tomography (PET) imaging agents, also showed high affinity for DAT.     

大肠杆菌苹果酸合酶A的酶学和生理功能研究

苹果酸合酶是乙醛酸循环的关键酶之一。E.coli中苹果酸合酶A(malate synthase A,MSA)由aceB基因编码。根据E.coli基因组序列设计引物,利用PCR技术扩增aceB基因,并将其克隆入pET-29b(+),构建了重组表达质粒pET-MSA。经IPTG诱导,MSA在E.coliRosetta(DE3)中获得高效表达。纯化的MSA蛋白的分子量大小约为60 kDa,最适反应pH值和最适温度分别是pH值8.0、30℃。纯化的蛋白质在Mg2+存在时才能发挥最大的活性,其对乙酰辅酶A的Km和Vmax分别是8.07μM和3.6μM/min。此外构建了MSA和苹果酸合酶G(MSG)基因敲除菌株MG::ΔaceB和MG::ΔaceBΔglcB。研究发现缺少MSA的E.coli突变菌株在乙酸中的生长速率要比野生型菌株慢很多,表明MSA对大肠杆菌在乙酸中的生长起着重要作用。MSG虽然能部分补偿MSA的作用,但是包含MSA的乙醛酸旁路是更有效的乙醛酸代谢途径。     

中国首批国家公园体制试点的经验与成效、问题与建议

建立国家公园体制是建设生态文明制度的重要内容,是解决我国自然保护地发展过程中存在的重叠设置、多头管理、边界不清、权责不明、保护与发展矛盾突出等问题的重大举措。根据国家统一部署,2020年要设立一批国家公园,分级统一的管理体制基本建立。在跨时2年的资料收集、实地考察基础上,全面总结首批10个国家公园体制试点的经验与成效,梳理试点过程中存在的问题,并提出针对性建议,以期全面展现国家公园体制试点的最新进展,为国家公园深化建设提供参考和依据。结果显示,各试点区基本建立起分级统一的管理体制,创新了运行机制,保护力度持续加强,资金投入不断加大,科研合作不断深化,社会参与逐渐扩大。体制试点产生了明显的生态成效,民生改善初步显现,社会效益充分彰显。但是,仍然存在管理机构级别和类型参差不齐,法律制度不健全,资金保障长效机制未建立,人才队伍建设滞后,保护与发展的矛盾突出,空间范围不合理等问题。建议采取必要措施改进存在的问题,积极推广有效经验,加快健全国家公园体制。