High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli

Hepatocyte growth factor (HGF) is an effective anti-fibrotic factor because of its bioactivity in inhibiting fibrosis-related proteins in the development of hepatic fibrosis. However, high-level production of bioactive mature form HGF is difficult because of its complex structure. Here, we report a non-fusion protein expression system to obtain truncated variant of N-terminal hairpin and first kringle domains of HGF (tvNK1) in Escherichia coli to determine its anti-fibrotic effects on hepatic stellate cells (HSCs). Under the selected conditions of cultivation and isopropyl-β-D-1-thiogalactopyranoside induction, the expression level of tvNK1 accounted for approximately 65 % of the total cellular protein and 50 % of fusion protein in the supernatant of whole cell lysates. The recombinant protein could be purified in one step with Ni²⁺-affinity chromatograph. Finally, about 65 mg recombinant tvNK1 was obtained from 1 l fermentation culture with no <95 % purity. In vitro, the final purified tvNK1 was shown to inhibit the proliferation of HSCs and decrease the mRNA and protein expression levels of fibrosis-related COL1A1 and α-smooth muscle actin genes.     

Disease pathways at the Rat Genome Database Pathway Portal: genes in context-a network approach to understanding the molecular mechanisms of disease

Background

Biological systems are exquisitely poised to respond and adjust to challenges, including damage. However, sustained damage can overcome the ability of the system to adjust and result in a disease phenotype, its underpinnings many times elusive. Unraveling the molecular mechanisms of systems biology, of how and why it falters, is essential for delineating the details of the path(s) leading to the diseased state and for designing strategies to revert its progression. An important aspect of this process is not only to define the function of a gene but to identify the context within which gene functions act. It is within the network, or pathway context, that the function of a gene fulfills its ultimate biological role. Resolving the extent to which defective function(s) affect the proceedings of pathway(s) and how altered pathways merge into overpowering the system's defense machinery are key to understanding the molecular aspects of disease and envisioning ways to counteract it. A network-centric approach to diseases is increasingly being considered in current research. It also underlies the deployment of disease pathways at the Rat Genome Database Pathway Portal. The portal is presented with an emphasis on disease and altered pathways, associated drug pathways, pathway suites, and suite networks.

Results

The Pathway Portal at the Rat Genome Database (RGD) provides an ever-increasing collection of interactive pathway diagrams and associated annotations for metabolic, signaling, regulatory, and drug pathways, including disease and altered pathways. A disease pathway is viewed from the perspective of networks whose alterations are manifested in the affected phenotype. The Pathway Ontology (PW), built and maintained at RGD, facilitates the annotations of genes, the deployment of pathway diagrams, and provides an overall navigational tool. Pathways that revolve around a common concept and are globally connected are presented within pathway suites; a suite network combines two or more pathway suites.

Conclusions

The Pathway Portal is a rich resource that offers a range of pathway data and visualization, including disease pathways and related pathway suites. Viewing a disease pathway from the perspective of underlying altered pathways is an aid for dissecting the molecular mechanisms of disease.

     

Comparison of Medial Temporal Measures between Binswanger's Disease and Alzheimer's Disease

Binswanger''s disease (BD) is a common cause of vascular dementia in elderly patients; however, few studies have investigated the medial temporal lobe (MTL) atrophy in BD, and the differences in the atrophic patterns between BD and Alzheimer''s disease (AD) remain largely unknown. Such knowledge is essential for understanding the pathologic basis of dementia. In this study, we collected structural magnetic resonance imaging (MRI) data from 16 normal controls, 14 patients with AD and 14 patients with BD. The volumes of the hippocampus and amygdala, and morphologic parameters (volume, surface area, cortical thickness and mean curvature) of the entorhinal cortex (ERC) and perirhinal cortex (PRC) were calculated using an automated approach. Volume reduction of the hippocampus, amygdala and ERC, and disturbance of the PRC curvature was found in both AD and BD patients compared with the controls (p<0.05, uncorrected). There were no significant differences among all the structural measures between the AD and BD patients. Finally, partial correlation analyses revealed that cognitive decline could be attributed to ERC thinning in AD and volume reduction of PRC in BD. We conclude that AD and BD exhibit similar atrophy patterns in the medial temporal cortices and deep gray matter but have distinct pathologic bases for cognitive impairments. Although atrophy of the MTL structures is a sensitive biomarker for AD, it is not superior for discrimination between AD and BD.     

Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~ 50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.     

Organic carbon burial in lake sediments in the middle and lower reaches of the Yangtze River Basin, China

Lakes are important in the global and regional carbon cycle, and lake sediments potentially store substantial quantities of organic carbon. The middle and lower reaches of the Yangtze River basin (MLYB) are some of the largest agricultural areas in China with an extremely high density of lakes and rivers. The lakes in the region have undergone dramatic changes over the past several decades. In this study, six cores from five lakes (the macrophyte-dominated: Shijiuhu Lake and Honghu Lake; the algae-dominated: Chaohu Lake, Taihu Lake, and Nanyihu Lake) in the MLYB were collected from 2002 A.D. to 2008 A.D. Mass accumulation rates (MARs) of sediment derived from ²¹⁰Pb and ¹³⁷Cs along with total organic carbon content (TOC) were used to determine organic carbon accumulation rates (OC ARs) over the last 100 years. The TOC in the five lakes exhibited a significant increase since the mid or late 20th century, which was consistent with the increase in the lake water trophic status due to nutrient input. The average organic carbon accumulation rates for the Taihu Lake, Nanyihu Lake, Chaohu Lake, Shijiuhu Lake, and Honghu Lake were calculated to be 16.6, 28.9, 9.8, 25.4, and 113.2 g C m^?2 year^?1, respectively, over the past 100 years. Based on the average OC AR of 32.1 g C m^?2 year^?1 from the five lakes, carbon burial in lake sediments may be as much as 6.8 × 10¹³ g C in the MLYB over the past 100 years.     

马尾松根生产苗与常规苗外生菌根真菌多样性特征

以湖南省林业科学院龙伏试验基地3年生常规方法培养的马尾松苗Pinus massoniana（以下简称常规苗）和3年生马尾松根生产（root production method,RPM）苗根、根际土为研究对象,采用 Illumina MiSeq 测序技术研究其根系及根际土壤外生菌根真菌（ectomycorrhizal fungi,ECMF）群落结构特征,旨在探明其土壤微生境的差异,进而为人工接种菌根真菌及改良常规苗的土壤微生境奠定基础。测序共获得170 148条ECMF序列,划分为20个OTUs（operational taxonomic units,OTU）,隶属于2门、3纲、7目、8科、11属。Chao1丰富度指数、Ace丰富度指数、Simpson多样性指数与Shannon-Wiener多样性指数均表现为常规苗根际土的OTUs丰富度高于RPM苗根际土,并且根样OTUs丰富度低于根际土样（P<0.05）。不同样品的ECMF优势属占比也不同,RPM苗根样中占比最大的为Amphinema（47.89%）,常规苗根样为Tomentella（70.60%）;RPM苗土样中占比最大的为Tylospora（62.33%）,常规苗土样为Tomentella（55.69%）。冗余分析表明,土壤pH值对ECMF的影响程度最大,其次为速效磷和有机质;同时,不同理化因子对群落多样性指数及优势属的影响也存在差异。     

Dopamine D1 Receptor in the Nucleus Accumbens Modulates the Emergence from Propofol Anesthesia in Rat

Neurochemical Research - It has been reported that systemic activation of D1 receptors promotes emergence from isoflurane-induced unconsciousness, suggesting that the central dopaminergic system is...     

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Δψm collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-κB activation and intracellular Ca²⁺ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.     

Shedding of syndecan-1 by stromal fibroblasts stimulates human breast cancer cell proliferation via FGF2 activation

The cell surface heparan sulfate proteoglycan syndecan-1 is induced in stromal fibroblasts of breast carcinomas and participates in a reciprocal feedback loop, which stimulates carcinoma cell growth in vitro and in vivo. To define the molecular mechanism of carcinoma growth stimulation, a three-dimensional co-culture model was developed that combines T47D breast carcinoma cells with immortalized human mammary fibroblasts in collagen gels. By silencing endogenous syndecan-1 induction with short interfering RNA and expressing mutant murine syndecan-1 constructs, it was determined that carcinoma cell mitogenesis required proteolytic shedding of syndecan-1 from the fibroblast surface. The paracrine growth signal was mediated by the syndecan-1 heparan lfate chains rather than the ectodomain of the core protein and required fibroblast growth factor 2 and stroma-derived factor 1. This paracrine pathway may provide an opportunity for the therapeutic disruption of stromaepithelial signaling.     

Late marine isotope stage 3 palaeoclimate for East Asia: A data–model comparison

Climate prediction under impact of anthropological greenhouse-gas emission is impossible to validate, but it can be inferred from past climate and modeling. In East Asia, a general warm–wet period at late Marine Isotope Stage 3, ca. 30–40 ky BP has been identified based upon extensive geological records; this provides an arid/humid reference for possible future warming caused by human activities. Based upon syntheses of geological evidence and the AGCM + SSiB modeling, this paper presents climate simulations focused on 35 ky BP, using forcing of insolation, glaciation and land surface conditions for East Asia. Results of the simulation can be compared with geological records and show that (1) the climate patterns of 35 ky BP were warm–wet conditions in northern China, but with warm–dry conditions in southern China compared to today; (2) mean annual temperatures were higher in most mid-low latitude areas, mainly contributed to by increased winter temperatures, suggesting that insolation has generated significant climate effects through the coupling in atmospheric circulation with land surface patterns; (3) Quaternary ice sheets in the Northern Hemisphere played an important role in temperature decrease at the mid-high-latitudes, and also enhanced the south–north temperature gradients, which in turn, increased moisture transport from low to high latitudes and increased monsoonal precipitation over the Tibetan Plateau; and (4) Vegetation changes in East Asia resulted from an increased temperature in the low latitudes, extended rain-belt northwards into China, and an enlarged area of increased precipitation inland.