The Role of Rigid Residues in Modulating TEM-1 β-Lactamase Function and Thermostability

The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in β-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A β-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on β-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of β-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.     

Cervid Prion Protein Polymorphisms: Role in Chronic Wasting Disease Pathogenesis

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal’s susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrP^C) to convert into infectious prions (PrP^Sc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrP^C and PrP^Sc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrP^C to PrP^Sc conversion and determining PrP^C stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal’s genetics in CWD can help to predict, contain, or prevent transmission of CWD.     

Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca²⁺-dependent ion channels and/or Ca²⁺-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.     

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin’s influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.     

The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.     

Reaction process of monazite and bastnaesite mixed rare earth minerals calcined by CaO-NaCl-CaCl2

The decomposition reactions of monazite and bastnaesite mixed rare earth minerals calcined by CaO-NaCl-CaCl2 were studied by means of TG-DTA and XRD. The results show that the process of the minerals decomposed by CaO involves two steps. The first step occurs in the temperature range of 425-540 ℃, and the main reactions are bastnaesite decomposition, i.e. REOF reacts with CaO to produce RE2O3 and CaF2, and Ce2O3 is oxidized to CeO2. During this step, CaCO3 is formed at about 500 ℃. The second step takes place in the temperature range of 610-700 ℃, and the reactions are monazite decomposition into RE2O3, Ca5F（PO4）3 and Ca3（PO4）2 by CaO and CaF2. In this process, the decomposition ability is improved because CaO from CaCO3 decomposing has high chemical activity. In calcining process, the new formed Ca5F（PO4）3 restrains fluorine that can escape in form of gaseous compound. The decomposition ratio of the mixed rare earth minerals reaches 90.8% at 700 ℃.     

H-PSMA/PVA/PP复合膜的制备及油-水乳液分离性能

通过两步法对聚丙烯（PP）膜进行了亲水改性:首先,通过在碱性条件下水解苯乙烯-马来酸酐共聚物（PSMA）制备了水解的苯乙烯-马来酸酐共聚物（H-PSMA）;然后,以聚乙烯醇（PVA）为亲水性改性剂,戊二醛（GA）为交联剂,将H-PSMA/PVA/GA复合材料浸涂在PP基底膜上,制备了亲水性水解苯乙烯-马来酸酐共聚物/聚乙烯醇/聚丙烯（H-PSMA/PVA/PP）复合膜。利用傅里叶变换红外光谱（FT-IR）分析表征了H-PSMA和H-PSMA/PVA/GA的结构;通过测试复合膜表面的静态接触角,探究了PVA和GA含量及成膜溶液的质量浓度等对膜表面润湿性能的影响;最后,测试了复合膜的油水分离效率和重复利用率。结果表明:当GA、PVA和H-PSMA的质量比为0.225∶1∶1,成膜溶液的质量浓度为0.03 g/mL时,所制备的H-PSMA/PVA/PP复合物膜对水包油乳液（O/W）表现出优异的分离性能,初次油水分离效率为99.40%,当重复分离10次后,油水乳液的分离效率仍达到98.83%以上。该复合膜的制备工艺具有安全性好、成本低和环境友好的特点。     

Selective leaching of vanadium from V-Ti magnetite concentrates by pellet calcification roasting-H_2SO_4 leaching process

A novel method of pellet calcification roasting-H_2 SO_4 leaching was proposed to efficiently separate and extract vanadium(V) from vanadium-titanium(V-Ti) magnetite concentrates.The leaching rate of V is as high as 88.98%,while the leaching rate of impurity iron is only 1.79%.Moreover,the leached pellets can be used as raw materials for blast furnace ironmaking after secondary roasting.X-ray photoelectron spectroscopy(XPS) and scanning electron microscopy with energy dispersive X-ray spectrometry(SEMEDS) analyses showed that V~(3+) was oxidized to V~(5+) after roasting at 1200℃,and V~(5+) was then leached by H_2 SO_4.X-ray diffraction(XRD) analyses and single factor experiment revealed a minimal amount of dissolved Fe_2 O_3 during H_2 SO_4 leaching.Therefore,a high separation degree of V and iron(Fe) from V-Ti magnetite concentrate was achieved through H_2 SO_4 leaching.Compared with the traditional roastingleaching process,this process can achieve a high selectivity of V and Fe,and has excellent prospects for industrial production.     

剩余污泥资源化提取蛋白质方法的优选

剩余污泥中蛋白质的资源化利用是目前研究的热点,污泥预处理则是实现污泥中蛋白质释放的重要途径。为了进一步提高剩余污泥中蛋白质的溶出效果,选取热碱预处理、超声碱联合预处理、溶菌酶预处理对污泥进行溶胞,以蛋白质提取浓度为主要指标进行参数优化,并利用等电点法对粗提取蛋白进行纯化回收。结果表明：溶胞效果热碱预处理（pH值13、温度140℃、时间1.5 h,2 062.98 mg/L）>超声碱联合预处理（497.76 mg/L）>溶菌酶预处理（269.95 mg/L）,且在pH值为3时热碱预处理蛋白质纯化回收率可达62.42%。试验结果表明：热碱预处理在提取效果方面较另外两种方法优势明显,具有良好的利用前景。