Human Thromboxane A2 Receptor Genetic Variants: In Silico,In Vitro and “In Platelet” Analysis

Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote (hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C⁶⁸S, V⁸⁰E, E⁹⁴V, A¹⁶⁰T, V¹⁷⁶E, and V²¹⁷I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C⁶⁸S) to normal (V²¹⁷I), with most variants demonstrating functional alteration in binding, expression or activation (V⁸⁰E, E⁹⁴V, A¹⁶⁰T, and V¹⁷⁶E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and “in platelet” evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.     

Genome Sequence of the Aerobic Bacterium Bacillus sp. Strain FJAT-13831

Bacillus sp. strain FJAT-13831 was isolated from the no. 1 pit soil of Emperor Qin''s Terracotta Warriors in Xi''an City, People''s Republic of China. The isolate showed a close relationship to the Bacillus cereus group. The draft genome sequence of Bacillus sp. FJAT-13831 was 4,425,198 bp in size and consisted of 5,567 genes (protein-coding sequences [CDS]) with an average length of 782 bp and a G+C value of 36.36%.     

Reduction in leaf size at higher altitudes across 39 broad-leaved herbaceous species on the northeastern Qinghai-Tibetan Plateau

青藏高原东北部39种阔叶草本植物叶大小随海拔增加而减少 种间或种内的叶大小随环境变化存在很大的差异，但这些差异如何随海拔变化一直都在争论。我们在青藏高原东北缘的冷龙岭和达坂山，沿海拔3200–4400 m的山坡上选取生长在开阔环境下的39种阔叶草本植物，观测了叶大小、叶长、叶宽和比叶重。研究结果表明，随海拔增加叶片显著减小，而且叶片面积的减小主要受叶片长度的影响，即随海拔增加叶长度减小明显。此外，叶片面积与海拔之间的关系随物种、叶倾角和叶表面特征而不同。利用局地环境观测数据驱动的能量平衡模型分析发现：叶温能更密切地追随气温变化，叶大小变化对叶温的影响在高海拔更为强烈。同时，基于上述能量平衡的计算结果，我们认为青藏高原东北部阔叶草本植物的海拔分布上限大约为5400 m。     

Isolation and Characterization of Anti-Inflammatory Sorbicillinoids from the Mangrove-Derived Fungus Penicillium sp. DM815

Marine derived fungus has gained increasing ground in the discovery of novel lead compounds with potent biological activities including anti-inflammation. Here, we first report the characterization of one new sorbicillinoid ( 1 ) and fourteen known compounds ( 2 – 15 ) from the ethyl acetate (AcOEt) extract of a cultured mangrove derived fungus Penicillium sp. DM815 by UV, IR, HR ESI-Q-TOF MS, and NMR spectra. We then evaluated the anti-inflammatory effects of eleven sorbicillinoids ( 1 – 11 ) using cultured macrophage RAW264.7 cells. The results show that compound 9 , and to a lesser degree compound 5 , significantly inhibited the Gram-negative bacteria lipopolysaccharide (LPS)-induced upregulation of the inducible nitric oxide synthase (iNOS). Consistently, compounds 5 and 9 significantly reduced the level of nitric oxide (NO), the product of iNOS, induced by LPS. We further show that these two compounds dose-dependently inhibited LPS-triggered iNOS expression and NO production, but had no effect on proliferation of RAW264.7 cells in the presence of LPS. In conclusion, our study identifies novel and known sorbicillinoids as potent anti-inflammatory agents, holding the promise of developing novel anti-inflammation treatment in the future.     

Seminal Plasma and Semen Amyloids Enhance Cytomegalovirus Infection in Cell Culture

Among the modes of transmission available to the cytomegalovirus (CMV) is sexual transmission, primarily via semen. Both male-to-female (M-F) and male-to-male (M-M) sexual transmission significantly contribute toward the spread of CMV infections in the global population. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa, thereby initiating viral replication. Both semen and seminal plasma (SP) can enhance HIV-1 infection in cell culture, and two amyloid fibrils, semen-derived enhancer of viral infection (SEVI) and amyloids derived from the semenogelins (SEM amyloids), have been identified as seminal factors sufficient to enhance HIV-1 infection (J. Munch et al., Cell 131:1059–1071, 2007; N. R. Roan et al., Cell Host Microbe 10:541–550, 2011; F. Arnold et al., J. Virol. 86:1244–1249, 2012). Whether SP, SEVI, or SEM amyloids can enhance other viral infections has not been extensively examined. In this study, we found that SP, SEVI, and SEM amyloids strongly enhance both human CMV (HCMV) and murine CMV infection in cell culture. SEVI and SEM amyloids increased infection rates by >10-fold, as determined by both flow cytometry and fluorescence microscopy. Viral replication was increased by 50- to 100-fold. Moreover, viral growth curve assays showed that SP, SEVI, and SEM amyloids sped up the kinetics of CMV replication such that the virus reached its replicative peak more quickly. Finally, we discovered that SEM amyloids and SEVI counteracted the effect of anti-gH in protecting against CMV infection. Collectively, the data suggest that semen enhances CMV infection through interactions between semen amyloid fibrils and viral particles, and these interactions may prevent HCMV from being neutralized by anti-gH antibody.     

H2S catalysed by CBS regulates testosterone synthesis through affecting the sulfhydrylation of PDE

Testosterone deficiency resulted in increased mortality in men. Our previous work found that hydrogen sulphide (H₂S) significantly alleviated the spermatogenesis disorder. To investigate whether H₂S could regulate testosterone synthesis and the relative signalling pathways. Disorder model of testosterone synthesis was constructed in vitro and in vivo. The cell viability was detected using CCK-8 method. The concentration of H₂S and testosterone were examined using ELISA kits. The relative mRNA and protein expression of CBS, PDE4A, PDE8A and proteins related to testosterone synthesis were detected by RT-qPCR and western blotting. PAS staining was used to detect the inflammatory status of testis. The sulfhydryl level of PDE4A and PDE8A was determined by Biotin Switch Technique. CBS overexpression inhibited while knockdown promoted LPS + H₂O₂ induced injury in testosterone synthesis of MLTC-1 cells, though regulating the level of H₂S. The LPS + H₂O₂ induced inhibition on cAMP and p-PKA was recovered by CBS overexpression, while addition of the specific inhibitor of PKA had opposite effects. CBS overexpression alleviated the inflammation status in testis and promoted the expression of StAR, P450scc, P450c17 and 3β-HSD. CBS could also exhibit its protective role through promoting sulfhydrylation of PDE4A and PDE8A. H₂S catalysed by CBS could recover testosterone synthesis in vitro and in vivo through inhibiting PDE expression via sulfhydryl modification and activating cAMP/PKA pathway.     

Spatio-Temporal Patterns in Rhizosphere Oxygen Profiles in the Emergent Plant Species Acorus calamus

Rhizosphere oxygen profiles are the key to understanding the role of wetland plants in ecological remediation. Though in situ determination of the rhizosphere oxygen profiles has been performed occasionally at certain growing stages within days, comprehensive study on individual roots during weeks is still missing. Seedlings of Acorus calamus, a wetland monocot, were cultivated in silty sediment and the rhizosphere oxygen profiles were characterized at regular intervals, using micro-optodes to examine the same root at four positions along the root axis. The rhizosphere oxygen saturation culminated at 42.9% around the middle part of the root and was at its lowest level, 3.3%, at the basal part of the root near the aboveground portion. As the plant grew, the oxygen saturation at the four positions remained nearly constant until shoot height reached 15 cm. When shoot height reached 60 cm, oxygen saturation was greatest at the point halfway along the root, followed by the point three-quarters of the way down the root, the tip of the root, and the point one-quarter of the way down. Both the internal and rhizosphere oxygen saturation steadily increased, as did the thickness of stably oxidized microzones, which ranged from 20 µm in younger seedlings to a maximum of 320 µm in older seedlings. The spatial patterns of rhizosphere oxygen profiles in sediment contrast with those from previous studies on radial oxygen loss in A. calamus that used conventional approaches. Rhizosphere oxygen saturation peaked around the middle part of roots and the thickness of stably oxidized zones increased as the roots grew.     

Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA

RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called ‘universal stem-loop primer’ (USLP) with 8 random nucleotides instead of a specific sequence at the 3′ end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV<2.5%). The sensitivity of the USLP is up to 10³ copies/µl miR. As compared with the TSLP, USLP saved 75% the cost of primers and 60% of the test time. The USLP method is a simple, rapid, precise, sensitive, and cost-effective approach that is suitable for screening miR profiles.     

The influence of different aphid prey species on the biology and life table parameters of Propylaea japonica

The influence of three aphid prey species – Aphis craccivora Koch, Megoura viciae Buckton and Rhopalosiphum maidis (Fitch) – on the biology of Propylaea japonica (Thunberg) was studied under laboratory conditions. The development, survivorship, longevity, reproduction and life table parameters of P. japonica differed significantly among different treatments. The shortest developmental period of P. japonica (from first-instar larvae to adult) was observed on A. craccivora, whereas the longest was observed on M. viciae. The highest survivorship was observed on A. craccivora, and the lowest survivorship was observed on M. viciae. The highest sex ratio, fecundity and the shortest pre-oviposition period were observed when A. craccivora was used as prey. The longevities of P. japonica females and males did not differ significantly when reared on different aphid species. The highest values of net reproductive rate, intrinsic rate of increase and finite rate of increase were observed on A. craccivora. The results suggest that A. craccivora is a suitable prey for P. japonica among the three aphid species tested and can serve as a diet for the mass rearing of P. japonica under laboratory conditions for possible use in integrated pest management.