An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7: gene cloning, expression and characterization

A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50°C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

DDX39 as a predictor of clinical prognosis and immune checkpoint therapy efficacy in patients with clear cell renal cell carcinoma

DEAD-box protein 39 (DDX39) has been demonstrated to be a tumorigenic gene in multiple tumor types, but its role in the progression and immune microenvironment of clear cell renal cell cancer (ccRCC) remains unclear. The aim of the present study was to investigate the role of DDX39 in the ccRCC tumor progression, immune microenvironment and efficacy of immune checkpoint therapy.The DDX39 expression level was first detected in tumors in the public data and then verified in ccRCC samples from Changzheng Hospital. The prognostic value of DDX39 expression was assessed in the Cancer Genome Atlas (TCGA) and ccRCC patients from Changhai Hospital. The role of DDX39 in promoting ccRCC was analyzed by bioinformatic analysis and in vitro experiments. The association between DDX39 expression and immune cell infiltration and immune inhibitory markers was analyzed, and its value in predicting the immune checkpoint therapy efficacy in ccRCC were evaluated in the public database. DDX39 expression was elevated in Oncomine, GEO and TCGA ccRCC databases, as well as in Changzheng ccRCC samples. In TCGA ccRCC patients, increased DDX39 expression predicted worse overall survival (OS) (p<0.0001) and progression-free interval (PFI) (p<0.0001), and was shown as an independent predictive factor for OS (p=0.002). These findings were consistent with those from Changhai ccRCC patients. In addition, GO and GSEA analysis identified DDX39 as a pro-ccRCC gene. In vitro experiments confirmed the role of DDX39 in promoting ccRCC cell. Finally, DDX39 was found to be positively correlated with a variety of immune inhibitory markers, and could predict the adverse efficacy of immune checkpoint therapy in TIDE analysis. In conclusion, Increased DDX39 in ccRCC patients predicted worse clinical prognosis, promoted ccRCC cell proliferation, migration and invasion, and also predicted adverse efficacy of immune checkpoint therapy.     

基于旅行费用法的衢州市柯城区“一村万树”工程生态旅游服务价值评估

生态旅游服务价值作为生态系统生产总值（Gross Ecosystem Product,GEP）的重要组成部分,是生态产品价值实现的重要途径。以衢州市柯城区"一村万树"实施区为研究区域,采用国际上普遍认可的旅行费用区间法（Travel Cost Interval Analysis,TCIA）进行了问卷调查,开展了生态旅游服务的价值核算;同时基于计量经济学原理构建了游客随机效用模型（Random Utility Model,RUM）,探究了不同自然地理属性对游客生态旅游选址决策的影响。结果表明：（1）2018年"一村万树"实施区的生态旅游服务总价值约19.29亿元,单位面积生态旅游价值量为8.37万元/hm²;（2）2018年"一村万树"实施区的游客人均游憩价值为713.89元,只占整个柯城区人均游憩价值（1204.58元）的59.3%,说明其还有较大的生态产品价值实现提升空间;（3）柯城区游客人均游憩价值远高于当地文化旅游局提供的621.80元的旅游人均消费支出,说明游客时间成本和消费者剩余蕴含了丰富的潜在价值,需要在评估时予以考虑;（4）随机效用模型结果显示,目的地旅行费用与游客访问呈负相关,森林和湿地面积均与游客访问呈正相关,且湿地的相对贡献远高于森林。研究结果对"一村万树"实施区有针对性地进行生态保护、管理和开发具有指导意义,对提高GEP核算的准确性和探索生态产品价值实现的机制具有借鉴意义。     

Plasma membrane biophysical properties linked to the antiproliferative effect of interferon-alpha

The relationship of plasma membrane biophysical properties to the anti-proliferative effect of interferon-alpha (IFN-alpha) was investigated in Daudi lymphoblasts cell lines with sensitivity to growth inhibition, parallel clonal variants selected for resistance, and one revertant subclone. Lateral mobility of surface differentiation antigens (I2, CD19, CD20, and sIgM-kappa) were measured by fluorescence recovery after photobleaching (FRAP). The mean diffusion coefficients, D, values for two clones of IFN-alpha resistant Daudi cells were significantly higher (D = 8.1-11 x 10(-10) cm2/sec) than for parental sensitive cells (D = 4.9-7.4 x 10(-10) cm2/sec). Microviscosity of the plasma membranes were probed by electron spin resonance (ESR) spectrometry. These results also indicate a greater degree of molecular motional freedom in resistant cells. Treatment of sensitive lymphoblasts with IFN-alpha (100-400 U/10(6) cells) for 5-30 min consistently increased mean values of D and the degree of spin-probe motional freedom, whereas no significant differences were detected in resistant cells. The effect of IFN-alpha on the membrane potential (Em) of Daudi cells was quantitated by flow cytometry using a voltage-sensitive oxonol dye. Membrane potential of all clones was similar (-50 to -56 mV). Treatment with IFN-alpha for 8-10 min caused hyperpolarization in the sensitive cells (deltaEm up to 45 mV), but only minimal hyperpolarization in the resistant ones (deltaEm up to 7 mV). We concluded that sensitivity to IFN-alpha and treatment with IFN-alpha are related to the biophysical status of plasma membranes.     

Characterization of conformational epitope of alginate-derived polymannuronates by surface plasmon resonance

In the present study, we developed a mAb to alginate-derived polymannuronates (ADPM) and examined the antigenic epitopes using surface plasmon resonance (SPR) in conjunction with a large panel of oligomannuronate probes. We found that tetrasaccharide is the minimum-binding unit, and that increases in chain length from the tetrasaccharide to the heptsaccharide further enhance monovalent binding. A sharp increase in affinity was observed when increasing from the octasaccharide to the cosasaccharide, which is due to a further enhancement of the individual antigenic epitope combined with multivalency. Kinetic binding studies further suggested that the conformational epitope is discontinuous and infrequent and that a C6-carboxyl group is important in maintaining the conformational epitope. Moreover, CD analysis revealed there were conformational structures in epitopes. The data support our hypothesis that the conformational epitope for the mAb may be an extended helical segment of ADPM. ADPM exists mainly in linear form, but it can infrequently and spontaneously form extended helices. Although helices are not prevalent in ADPM, the immune system preferentially selects these conformational epitopes because they are unique. Together, our results indicate that the antigenic epitopes in beta-d-mannuronates are conformational and require C6-carboxyl groups.     

Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily

The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an alpha,beta-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.     

Projected timing of perceivable changes in climate extremes for terrestrial and marine ecosystems

Human and natural systems have adapted to and evolved within historical climatic conditions. Anthropogenic climate change has the potential to alter these conditions such that onset of unprecedented climatic extremes will outpace evolutionary and adaptive capabilities. To assess whether and when future climate extremes exceed their historical windows of variability within impact‐relevant socioeconomic, geopolitical, and ecological domains, we investigate the timing of perceivable changes (time of emergence; TOE) for 18 magnitude‐, frequency‐, and severity‐based extreme temperature (10) and precipitation (8) indices using both multimodel and single‐model multirealization ensembles. Under a high‐emission scenario, we find that the signal of frequency‐ and severity‐based temperature extremes is projected to rise above historical noise earliest in midlatitudes, whereas magnitude‐based temperature extremes emerge first in low and high latitudes. Precipitation extremes demonstrate different emergence patterns, with severity‐based indices first emerging over midlatitudes, and magnitude‐ and frequency‐based indices emerging earliest in low and high latitudes. Applied to impact‐relevant domains, simulated TOE patterns suggest (a) unprecedented consecutive dry day occurrence in >50% of 14 terrestrial biomes and 12 marine realms prior to 2100, (b) earlier perceivable changes in climate extremes in countries with lower per capita GDP, and (c) emergence of severe and frequent heat extremes well‐before 2030 for the 590 most populous urban centers. Elucidating extreme‐metric and domain‐type TOE heterogeneities highlights the challenges adaptation planners face in confronting the consequences of elevated twenty‐first century radiative forcing.     

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope

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