CMG2-Fc 融合蛋白突变体筛选及中和炭疽毒素活性分析

目的:筛选能有效中和炭疽毒素和抵抗炭疽毒素损伤细胞的CMG2-Fc(炭疽毒素受体II-人免疫球蛋白Fc段融合蛋白)突变体。方法:运用FoldX等计算软件分析CMG2与PA晶体学结构,设计能提高CMG2-PA亲和力的突变体分子,并与人IgG1Fc片段构成融合基因,转染CHO-S细胞并通过亲和层析获得CMG2-Fc突变体蛋白,通过亲和力检测和细胞保护实验分析各突变体中和炭疽毒素能力。结果:筛选并表达了8个CMG2-Fc突变体分子,亲和力实验显示其中E117Q突变可明显提高CMG2-Fc与PA的亲和力(KD=1.35×10-11 mol/L),细胞保护实验提示E117Q突变能有效提高CMG2-Fc中和炭疽毒素能力(CMG2-Fc(E117Q)的IC50为15 ng/μL,而wt CMG2-Fc的IC50为50ng/μL)。结论:CMG2-Fc(E117Q)突变体分子可作为拮抗炭疽毒素损伤的炭疽治疗药物分子,进行进一步研究。     

高通量血液透析治疗慢性肾衰竭尿毒症的疗效及对尿毒症毒素、免疫球蛋白及肺功能指标的影响

目的:探讨高通量血液透析(HFHD)治疗慢性肾衰竭尿毒症的疗效及对尿毒症毒素、免疫球蛋白及肺功能指标的影响。方法:选取90例于2012年1月-2017年3月期间在喀什地区第一人民医院治疗的慢性肾衰竭尿毒症患者,依据随机数字表法将其分为对照组(n=45)和观察组(n=45),对照组给予血液透析滤过(HDF)治疗,观察组给予HFHD治疗,两组均透析治疗1个月。对比两组患者透析前后症状缓解情况及尿毒症毒素、免疫球蛋白及肺功能指标水平,记录两组相关并发症的发生情况。结果:透析治疗结束后观察组患者缓解率为91.11%(41/45),高于对照组的73.33%(33/45)(P0.05)。两组患者透析后血磷(P~-)、血钾(K~+)、甲状旁腺激素(PTH)、β2-微球蛋白(β2-MG)水平明显低于透析前,血钙(Ca~(2+))水平明显高于透析前(P0.05);观察组透析后K~+、Ca~(2+)、P~-等尿毒症毒素水平与对照组比较差异无统计学意义(P0.05),观察组透析后PTH、β2-MG水平明显低于对照组(P0.05)。透析后,两组患者的免疫球蛋白Ig M、Ig A、Ig G水平均较透析前上升,且观察组高于对照组(P0.05)。透析后,两组患者残气量(RV)均低于治疗前,最大肺活量(FVC)、肺活量(VC)、肺总量(TLC)均高于治疗前,且观察组RV低于对照组,FVC、VC、TLC均高于对照组(P0.05)。观察组并发症发生率为8.89%(4/45),低于对照组的24.44%(11/45)(P0.05)。结论:HFHD治疗慢性肾衰竭尿毒症能够安全有效地清除尿毒症毒素,缓解患者的临床症状,且能够提高患者的免疫功能和肺功能。     

Multi-species okadaic acid contamination and human poisoning during a massive bloom of Dinophysis acuminata complex in southern Brazil

On June 2016, a major bloom of Dinophysis acuminata complex was noticed over the coast of Paraná State (PR), southern Brazil, an area unprotected by any official monitoring program. Here we report the results of an extensive sampling effort that ultimately led PR authorities to issue the first State shellfish-harvesting ban due to multi-species okadaic acid (OA) contamination. During its peak, the bloom covered an area of 201 km² (∼2.0–3.5 × 54.0 km), attaining unprecedentedly high cell densities along the shallow (<15 m) continental shelf (mean 2.2 × 10⁵, maximum 2.1 × 10⁶ cells L⁻¹) and adjacent sandy beaches (mean 2.8 × 10⁵, maximum 5.2 × 10⁶ cells L⁻¹). Only OA was detected in suspension (max. 188 ng L⁻¹). Toxin levels measured in bivalves were several times greater than the regulatory limit of 160 ng g⁻¹, reaching up to 3600 ng g⁻¹ in Crassostrea gasar, by far the highest OA concentrations ever reported in oysters worldwide, 7700 ng g⁻¹ in brown mussels, Perna perna, and lower levels in clams, Anomalocardia brasiliana, and mangrove mussels, Mytella spp. Nine cases of human intoxication were officially reported and five people were hospitalized with typical symptoms of Diarrhetic Shellfish Poisoning linked to the consumption of contaminated bivalves. All bivalves quickly converted most of the OA into its esterified form, DTX-3, and eliminated the toxins only a few weeks following the bloom, with C. gasar being the slowest-detoxifying species. Lower OA levels were accumulated in zooplankton, gastropods and several novel toxin vectors, including benthic organisms such as sand dollars Mellita quinquiesperforata and the ghost-shrimp Callichirus major, which may act as a good indicator of the presence of toxins in sandy beaches, and pelagic fish species that can serve as potential alternative sources of OA to humans (Chaetodipterus faber and Mugil liza). Monitoring toxin contamination in seafood other than bivalves is thus recommended to ensure comprehensive human health protection during massive Dinophysis blooms. Additionally, since OA was also present at low concentrations in the liver of Guiana dolphins Sotalia guianensis and penguins Spheniscus magellanicus, exposure to biotoxins should be considered in conservation actions involving threatened and near-threatened marine organisms in this region.     

Trends in Dinophysis abundance and diarrhetic shellfish toxin levels in California mussels (Mytilus californianus) from Monterey Bay,California

Diarrhetic shellfish toxins (DSTs) are produced by the marine dinoflagellate, Dinophysis, as well as select species of benthic Prorocentrum. The DSTs can bioaccumulate in shellfish and cause gastrointestinal illness when humans consume high levels of this toxin. Although not routinely monitored throughout the U.S., recent studies in Washington, Texas, and New York suggest DSTs may be widespread throughout U.S. coastal waters. This study describes a four-year time series (2013–2016) of Dinophysis concentration and DST level in California mussels (Mytilus californianus) from Santa Cruz Municipal Wharf (SCMW) in Monterey Bay, California. Results show a maximum Dinophysis concentration of 9404 cells/L during this study and suggest Dinophysis persists as a member of the background phytoplankton community throughout the year. In California mussels, DSTs were found at persistent low levels throughout the course of this study, and exceeded the FDA guidance level of 160 ng/g 19 out of 192 weeks sampled. Concentrations of Dinophysis alone are a positive but weak predictor of DST level in California mussels, and basic environmental variables (temperature, salinity, and nutrients) do not sufficiently explain variation in Dinophysis concentration at SCMW. This study demonstrates that Dinophysis in Monterey Bay are producing DSTs that accumulate in local shellfish throughout the year, occasionally reaching levels of concern.     

Identification of a α‐helical molten globule intermediate and structural characterization of β‐cardiotoxin,an all β‐sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.     

A Centipede Toxin Family Defines an Ancient Class of CSαβ Defensins

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Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.     

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.     

The use of killer sensitivity patterns for biotyping yeast strains: the state of the art, potentialities and limitations

In recent years molecular techniques have been the most useful tools for the unequivocal identification of undetermined strains at the species level. In many instances, however, a further discrimination at the strain level (biotyping) is required, such as during epidemiological investigations, in which the distribution of pathogenic microorganisms is studied, and for patent protection purposes. Although molecular methods are routinely used also for yeast biotyping, several nonmolecular techniques have been proposed. One of these, the determination of the killer sensitivity pattern (KSP) towards a panel of selected killer toxins has proven to be a good auxiliary method. Despite the plethora of studies published, the potential and limitations of the determination of KSPs have never been critically evaluated. In this review the use of this nonmolecular technique as a biotyping tool is discussed and compared with some currently used DNA-based procedures. In addition, methodological, mechanistic and ecological implications are evaluated.     

Long-time molecular dynamics simulations of botulinum biotoxin type-A at different pH values and temperatures

Botulinum neurotoxins type A (BoNT/A) are highly potent toxins, but are also useful in the treatment of illnesses. We studied the properties of BoNT/A at various temperatures and pH values in order to understand its toxicity and structure variations. The pH values of the environment of BoNT/A are obtained by changing the protonation states of certain titratable residue groups. Our results show that certain parts of the protein are active at acidic pH environments or at high temperatures. The protein is more stable in neutral environments at normal human body temperature, whereas, at high temperature, the protein is more stable in acidic environments. Also, the three domains of the protein tend to have relative motion rather than within individual domains.