A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

The Chemical Nature of the Polar Functional Group of Oxidized Acyl Chain Uniquely Modifies the Physicochemical Properties of Oxidized Phospholipid-Containing Lipid Particles

Oxidative modification of phospholipids generates a variety of oxidized phospholipid (Ox-PL) species which differ considerably in their chemical compositions and molecular structures. Recent results suggest that even closely related Ox-PL species can have considerably different biological effects. However, the molecular mechanism for this is not yet clear. In truncated Ox-PLs (tOx-PLs) the fatty acyl chain is shorter in length than the parent nonoxidized phospholipid molecules and contains a polar functional group(s). In a previous study we showed that two closely related tOx-PL species having a similar polar functional group and differing only in the length of the oxidized fatty acyl chain exerts significantly different effects on the physicochemical properties of the nonoxidized phospholipid particles containing these lipids (Kar et al., Chem Phys Lipids 164:54–61, 2011). In this study we have characterized the effect of polar functional groups of oxidized fatty acyl chain on the physicochemical properties of the nonoxidized phospholipid particles containing these lipids. Our results show that Ox-PL species differing only in the chemical nature of polar functional groups in their oxidized fatty acyl chain modify the properties of nonoxidized phospholipid particles containing them in a distinctive way. These results indicate that different species of Ox-PLs induce unique changes in the physicochemical properties of lipid particles/membranes containing them and that this may lead to their different biological effects.     

An ecological perspective on marine reserves in prey–predator dynamics

This paper describes a prey–predator type fishery model with prey dispersal in a two-patch environment, one of which is a free fishing zone and other is a protected zone. The existence of possible steady states, along with their local stability, is discussed. A geometric approach is used to derive the sufficient conditions for global stability of the system at the positive equilibrium. Relative size of the reserve is considered as control in order to study optimal sustainable yield policy. Subsequently, the optimal system is derived and then solved numerically using an iterative method with Runge–Kutta fourth-order scheme. Numerical simulations are carried out to illustrate the importance of marine reserve in fisheries management. It is noted that the marine protected area enables us to protect and restore multi-species ecosystem. The results illustrate that dynamics of the system is extremely interesting if simultaneous effects of a regulatory mechanism like marine reserve is coupled with harvesting effort. It is observed that the migration of the resource, from protected area to unprotected area and vice versa, is playing an important role towards the standing stock assessment in both the areas which ultimately control the harvesting efficiency and enhance the fishing stock up to some extent.     

Stress Induced Hyperglycemia and the Subsequent Risk of Type 2 Diabetes in Survivors of Critical Illness

ObjectiveStress induced hyperglycemia occurs in critically ill patients who have normal glucose tolerance following resolution of their acute illness. The objective was to evaluate the association between stress induced hyperglycemia and incident diabetes in survivors of critical illness.DesignRetrospective cohort study.SettingAll adult patients surviving admission to a public hospital intensive care unit (ICU) in South Australia between 2004 and 2011.PatientsStress induced hyperglycemia was defined as a blood glucose ≥ 11.1 mmol/L (200 mg/dL) within 24 hours of ICU admission. Prevalent diabetes was identified through ICD-10 coding or prior registration with the Australian National Diabetes Service Scheme (NDSS). Incident diabetes was identified as NDSS registration beyond 30 days after hospital discharge until July 2015. The predicted risk of developing diabetes was described as sub-hazard ratios using competing risk regression. Survival was assessed using Cox proportional hazards regression.ConclusionsStress induced hyperglycemia identifies patients at subsequent risk of incident diabetes.     

de novo转录组学分析华南地区入侵植物五爪金龙代谢特征

为探究华南地区严重入侵植物五爪金龙(Ipomoea cairica)生物入侵的分子机制,对五爪金龙及其近缘种七爪金龙(I. digitata)和裂叶牵牛(I. nil)进行de novo转录组测序和组装,得到56551条unigenes,其中56522条得到注释,7815条GO注释,15615条COG注释,180201条KEGG数据库注释。转录组分析结果表明,五爪金龙氮代谢通路关键酶基因的表达高于对照。次生代谢关键酶(PAL、4CL、CAD、查耳酮合酶、苯基丙乙烯酮异构酶、槲皮黄3-O-甲基转移酶等)基因在五爪金龙与七爪金龙及裂叶牵牛中均得到协同性的差异表达,而这些代谢通路指导的产物合成对五爪金龙的抗逆境能力、生长、化感作用等均起关键作用。关键基因的RT-qPCR验证结果与转录组结果具有一致性。因此,这从分子生物学层面上对解释五爪金龙在华南地区的入侵机制提供了新的证据。     

Enhancement of thermal stability and inhibition of protein aggregation by osmolytic effect of hydroxyproline

A combination of spectroscopic, calorimetric, and microscopic studies to understand the effect of hydroxyproline on the thermal stability, conformation, biological activity, and aggregation of proteins has been investigated. Significantly increased protein stability and suppression of aggregation is achieved in the presence of hydroxyproline. For example, exceptional increase in the thermal stability of lysozyme up to 26.4 degrees C and myoglobin up to 31.8 degrees C is obtained in the presence of hydroxyproline. The increased thermal stability of the proteins is observed to be accompanied with significant rise of the catalytic activity. Hydroxyproline is observed to prevent lysozyme fibril formation in vitro. Fluorescence and circular dichroism studies indicate induction of tertiary structures of the studied proteins in the presence of hydroxyproline. Preferential hydration of the native state is found to be crucial for the mechanism of protein stabilization by hydroxyproline. We compared the effect of hydroxyproline to that of proline and observed similar increase in the activity and suppression of protein aggregation. The results demonstrate the use of hydroxyproline as a protein stabilizer and in the prevention of protein aggregation and fibril formation.     

Fabrication of agar-gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications

Scaffolds of agar and gelatin were developed using a novel entrapment method where agar and gelatin molecules mutually entrapped one another forming stable cell adhesive matrices. Glutaraldehyde was used as a crosslinking agent for gelatin. Three types of hybrid matrices were prepared using agar and gelatin in different proportions in the weight ratio of 1:1, 2:1, and 3:1. Surface characterization of dry scaffolds was carried out by scanning electron microscope. Swelling studies were carried out in phosphate buffer saline (PBS) at physiological pH 7.4. The integral stability of the scaffolds was evaluated by estimating the released disintegrated gelatin from them in PBS at pH 7.4. The attachment kinetics of the cells was evaluated by culturing mouse fibroblast cell line NIH 3T3 on films. The cytocompatibility of these matrices was determined by studying growth kinetics of NIH 3T3 cells on them and morphology of cells was observed through optical photographs taken at various days of culture. It was found that the matrices containing agar and gelatin in 2:1 weight ratio exhibited best growth kinetics. The results obtained from these studies have suggested that the above-described method is a cheap and easy way to fabricate agar-gelatin hybrid scaffolds to grow cells which can be used in various in vitro tissue engineering applications like screening of drugs.     

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6–RACK1–ERK1/2–FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.