Stereochemical features of the envelope protein Domain III of dengue virus reveals putative antigenic site in the five-fold symmetry axis

We bring to attention a characteristic parasitic pattern present in the dengue virus: it undergoes several intensive thermodynamic variations due to host environmental changes, from a vector's digestive tract, through the human bloodstream and intracellular medium. Comparatively, among the known dengue serotypes, we evaluate the effects that these medium variations may induce to the overall structural characteristics of the Domain III of the envelope (E) protein, checking for stereochemical congruences that could lead to the identification of immunologic relevant regions. We used molecular dynamics and principal component analysis to study the protein in solution, for all four dengue serotypes, under distinct pH and temperature. We stated that, while the core of Domain III is remarkably rigid and effectively unaffected by most of the mentioned intensive variations, the loops account for major and distinguishable flexibilities. Therefore, the rigidity of the Domain III core provides a foothold that projects specifically two of these high flexible loop regions towards the inner face of the envelope pores, which are found at every five-fold symmetry axis of the icosahedron-shaped mature virus. These loops bear a remarkable low identity though with high occurrence of ionizable residues, including histidines. Such stereochemical properties can provide very particular serotype-specific electrostatic surface patterns, suggesting a viral fingerprint region, on which other specific molecules and ions can establish chemical interactions in an induced fit mechanism. We assert that the proposed regions share enough relevant features to qualify for further immunologic and pharmacologic essays, such as target peptide synthesis and phage display using dengue patients' sera.     

Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A_2A receptor (A_2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A_2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A_2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A_2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cβ, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A_2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cβ knockdown prevents A_2AR-mediated Col1 increase. In contrast, A_2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A_2AR stimulation with CGS21680 at 0.1 μM increased Col3 expression only upon PKA blockade. A_2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.     

海洋沉积物中铁还原细菌组成及异化铁还原与产氢性质分析

【背景】一些铁还原细菌具有异化铁还原与产氢的能力,该类细菌在环境污染修复的同时能够解决能源问题。【目的】从海洋沉积物中富集获得异化铁还原菌群,明确混合菌群组成、异化铁还原及产氢性质。获得海洋沉积物中异化铁还原混合菌群组成,分析菌群异化铁还原和产氢性质。【方法】利用高通量测序技术分析异化铁还原菌群的优势菌组成,在此基础上,分析异化铁还原混合菌群在不同电子供体培养条件下异化铁还原能力和产氢性质。【结果】高通量数据表明,在不溶性氢氧化铁为电子受体和葡萄糖为电子供体厌氧培养条件下,混合菌群的优势菌属主要是梭菌(Clostridium),属于发酵型异化铁还原细菌。混合菌群能够利用电子供体蔗糖、葡萄糖以及丙酮酸钠进行异化铁还原及发酵产氢。葡萄糖为电子供体时,菌群累积产生Fe(Ⅱ)浓度和产氢量最高,分别是59.34±6.73 mg/L和629.70±11.42 mL/L。【结论】异化铁还原混合菌群同时具有异化铁还原和产氢能力,拓宽了发酵型异化铁还原细菌的种质资源,探索异化铁还原细菌在生物能源方面的应用。     

The mechanism of high affinity pentasaccharide binding to antithrombin,insights from Gaussian accelerated molecular dynamics simulations

Abstract

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from ‘static’ structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a ‘non-activated’ AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. ‘Generalized correlation’ matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.

Communicated by Ramaswamy H. Sarma     

High-efficiency protein delivery into transfection-recalcitrant cell types

Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of β-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS. This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations.     

Novel SLC20A2 mutations identified in southern Chinese patients with idiopathic basal ganglia calcification

Idiopathic basal ganglia calcification (IBGC) is a rare neuropsychiatric disorder characterized by bilateral and symmetric cerebral calcifications. Recently, SLC20A2 was identified as a causative gene for familial IBGC, and three mutations were reported in a northern Chinese population. Here, we aimed to explore the mutation spectrum of SLC20A2 in a southern Chinese population. Sanger sequencing was employed to screen mutations within SLC20A2 in two IBGC families and 14 sporadic IBGC cases from a southern Han Chinese population. Four novel mutations (c.82G > A p.D28N, c.185T > C p.L62P, c.1470_1478delGCAGGTCCT p.Q491_L493del and c.935-1G > A) were identified in two families and two sporadic cases, respectively; none were detected in 200 unrelated controls. No mutation was found in the remaining 12 patients. Different mutations may result in varied phenotypes, including brain calcification and clinical manifestations. Our study supports the hypothesis that SLC20A2 is a causative gene of IBGC and expands the mutation spectrum of SLC20A2, which facilitates the understanding of the genotype–phenotype correlation of IBGC.     

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase—A computational perspective on severe epimerase-deficiency galactosemia

UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD⁺ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (k_cat), with less substantial effects on K_m. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.     

产紫杉醇内生真菌枝状枝孢霉MD2的发酵条件优化

[目的]通过优化内生真菌枝状枝孢霉MD2的发酵条件,提高10-去乙酰巴卡亭Ⅲ (10-DAB)和紫杉醇(Taxol)的产量.[方法]采用单因素试验分析不同的培养基初始pH值、培养温度、摇床转速和培养时间对10-DAB和紫杉醇产量的影响,优化枝状枝孢霉MD2的培养条件;以YES为基本培养基,采用单因素试验和正交试验分析添加苯甲酸钠、苯丙氨酸、丝氨酸和甘氨酸4种前体物对10-DAB和紫杉醇产量的影响,优化枝状枝孢霉MD2的培养基组分.[结果]优化后发酵条件为:在初始pH为5.0的300 mL YES培养基中,添加15 mg/L苯甲酸钠、25 mg/L苯丙氨酸、5 mg/L丝氨酸、15 mg/L甘氨酸,接种1 mL枝状枝孢霉MD2的孢子悬液(107-10s个孢子/mL),28.0℃、220 r/min发酵培养12d.在此条件下,枝状枝孢霉MD2的生物量、10-DAB和紫杉醇的产量分别为15.5 g/L、471.5 μg/L和569.5 μg/L,与初始发酵条件相比,分别提高了1.3、3.6和3.4倍.[结论]首次获得了枝状枝孢霉MD2生产10-DAB和紫杉醇的较适摇瓶发酵条件,可为进一步放大发酵培养提供参考.