Unique substrate recognition by botulinum neurotoxins serotypes A and E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. BoNT serotype A and serotype E cleave SNAP25 at residues 197-198 and 180-181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156-202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167-186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.     

Aquaporin-2 expression in human endometrium correlates with serum ovarian steroid hormones

The aim of the present study was to examine the expression of aquaporin-2 (AQP2), a member of the water channel family aquaporins (AQPs), in human uterine endometrium and its modulation of ovarian steroid hormone at the proliferative and secretory phases. Western blot, immunohistochemistry, and RT-PCR were employed in the present study. Western blot revealed a 29-kDa band that represented AQP2 in human endometrium. The expression of AQP2 in endometrium was confirmed by RT-PCR and immunohistochemical results. The immunohistochemical analysis demonstrated that AQP2 was prominent in luminal and glandular epithelial cells of endometrium. The levels of endometrial AQP2 expression changed during the menstrual cycle and were higher in the secretory endometrium than in the proliferative endometrium. A significantly high level of AQP2 was detected at the mid-secretory phase. There was a positive correlation between the levels of the endometrial AQP2 expression and the concentrations of the serum 17beta-estradiol (E2) or/and progesterone (P4). These data for the first time corroborate that AQP2 is expressed in human endometrium and that the expression of AQP2 in human endometrium might be regulated by E2 or/and P4. The changed expression of AQP2 at different phases of the menstrual cycle may be essential to reproductive physiology in human. The high level of endometrial AQP2 expression was observed at the mid-secretory phase, the time of embryo implantation, suggesting that AQP2 might play physiological roles in the uterine receptivity.     

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny,Physiology, and Niche Adaptation on Human Skin

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin’s carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.     

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.     

Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons

Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer’s disease (AD). Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons). We found that Okadaic acid (OA) induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), mitochondria membrane potential (MMP) and Glutathione peroxidase (GSH-Px). It up-regulated malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS). In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β) and mitogen activated protein kinase (MAPK) were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD.     

Microbial metabolism and necromass mediated fertilization effect on soil organic carbon after long-term community incubation in different climates

Understanding the effects of changing climate and long-term human activities on soil organic carbon (SOC) and the mediating roles of microorganisms is critical to maintain soil C stability in agricultural ecosystem. Here, we took samples from a long-term soil transplantation experiment, in which large transects of Mollisol soil in a cold temperate region were translocated to warm temperate and mid-subtropical regions to simulate different climate conditions, with a fertilization treatment on top. This study aimed to understand fertilization effect on SOC and the role of soil microorganisms featured after long-term community incubation in warm climates. After 12 years of soil transplantation, fertilization led to less reduction of SOC, in which aromatic C increased and the consumption of O-alkyl C and carbonyl C decreased. Soil live microbes were analyzed using propidium monoazide to remove DNAs from dead cells, and their network modulization explained 60.4% of variations in soil labile C. Single-cell Raman spectroscopy combined with D₂O isotope labeling indicated a higher metabolic activity of live microbes to use easily degradable C after soil transplantation. Compared with non-fertilization, there was a significant decrease in soil α- and β-glucosidase and delay on microbial growth with fertilization in warmer climate. Moreover, fertilization significantly increased microbial necromass as indicated by amino sugar content, and its contribution to soil resistant C reached 22.3%. This study evidentially highlights the substantial contribution of soil microbial metabolism and necromass to refractory C of SOC with addition of nutrients in the long-term.Subject terms: Microbial ecology, Biodiversity     

探讨我国森林野生动物红外相机监测规范

野生动物多样性是生物多样性监测与保护管理评价的关键指标, 因此对野生动物进行长期监测是中国森林生物多样性监测网络(CForBio)等大尺度生物多样性监测研究计划的一个重要组成部分。2011年以来, CForBio网络陆续在多个森林动态监测样地开展以红外相机来监测野生动物多样性。随着我国野生动物红外相机监测网络的初步形成, 亟待建立和执行基于红外相机技术的统一监测规范。基于3年来在我国森林动态监测样地红外相机监测的进展情况, 以及热带生态评价与监测网络针对陆生脊椎动物(兽类和鸟类)所提出的红外相机监测规范, 本文从监测规范和监测注意事项等方面探讨了我国森林野生动物红外相机监测的现状和未来。     

初代和继代培养葡萄试管苗的内源IAA、ZRs和ABA含量变化及其与生根的关系

葡萄品种皇家秋天与藤稔初代试管苗在第10天(根原基发生)时内源IAA和ABA含量达最低点后上升;ZRs含量达最高值后下降.皇家秋天继代试管苗的IAA在第8天(根原基发生)时达最低点,而后上升达稳定值;ZRs含量达最高值后下降;ABA含量降至最低点后急剧上升,在第10天(生根时)达最高值,持续4 d后又下降.藤稔继代苗在第6天(根原基发生)时IAA含量最低,后上升,持续到第12天后又下降;ZRs含量达最高值后快速下降,至第12天达稳定状态;ABA含量达最低值后缓慢上升,第10天(生根)达最高点后又下降.     

关节镜下清理术联合腓骨截骨术治疗膝关节骨性关节炎的疗效及对炎性因子的影响

目的:探讨采用关节镜下清理术联合腓骨截骨术治疗膝关节骨性关节炎(KOA)的疗效及对炎性因子的影响。方法:选取2015年3月～2017年12月期间右江民族医学院附属河池医院收治的KOA患者99例,根据手术方式的不同将患者分为A组(n=46,关节镜下清理术治疗)和B组(n=53,关节镜下清理术联合腓骨截骨术治疗),比较两组患者手术时间、术中出血量、住院时间,比较两组术前、术后6个月、术后1年疼痛情况、膝关节功能恢复情况、膝关节相关角,比较两组术前、术后1个月炎性因子变化,记录两组术后并发症发生情况。结果:B组手术时间长于A组(P0.05),两组术中出血量、住院时间比较无差异(P0.05)。B组术后6个月、术后1年视觉疼痛模拟(VAS)评分低于A组,美国特种外科医院(HSS)、膝关节评分及美国膝关节协会(KSS)评分则高于A组(P0.05)。两组患者术后1个月白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)均较术前降低,且B组低于A组(P0.05)。B组术后6个月、术后1年膝关节相关角[股骨干与股骨双髁连线夹角(F角)、股骨胫骨角(FT角)、胫股关节间隙角(JS角)]均低于术前以及A组同时间点(P0.05)。两组术后并发症发生率比较差异无统计学意义(P0.05)。结论:KOA患者经关节镜下清理术、腓骨截骨术联合治疗后,疗效显著,可有效改善患者疼痛及膝关节功能,降低炎性因子水平,手术方案安全且可改善患者下肢力线。     

Crystal structure of human Gadd45 reveals an active dimer

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45, revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45 identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.