Hexokinase from grape berries: its prokaryotic expression, polyclonal antibody preparation and biochemical property analyses

Two full-length hexokinase (HXK, EC 2.7.1.1) cDNAs, VvHXK1 with 1,413 bp and VvHXK2 with 1,458 bp were cloned from grape berries (Vitis vinifera L. Cabernet Sauvignon). VvHXK1 and VvHXK2 genes sequence from grape berries were deposited in GenBank under the accession number JN118544 and JN118545, respectively. The homology of the amino acid of VvHXK1 or VvHXK2 was very similar to ‘Pinot Noir’ grape HXK sequence, their similarties were 99.36 % and 98.97 %, respectively. More intuitive phylogenetic tree showed that the homology of amino acid sequence VvHXK1 with melon CmHXK1 was 86 %, and VvHXK2 homology with rice OsHXK3 was 83 %. The HXK proteins were successfully expressed in plasmid pET-30a (+) vectors in Escherichia coli BL21 (DE3) pLysS. The expressed proteins were purified using Ni-NTA agarose column and used to produce HXK1 antibody and conducted HXK protein blotting analysis. The results,suggested that one polypeptide band of about 51 kDa HXK protein can be detected in grape berries, HXK protein level was the highest during early grape berry development, but the lowest from 50d to 60d during development. Biochemical analysis of two hexokinase isozymes indicated that glucose was the optimal substrate of HXK, The isoelectric points of the two isozymes were 5.8 and 5.6, respectively. And the optimum pH was about 8.0. These results provide a substantial basis for the further studies of functions of grape HXKs to manipulate sugar content of grape berries.     

高蛋白食物和繁殖对布氏田鼠能量摄入和产热的影响

为探讨高蛋白食物和繁殖对布氏田鼠食物摄入和产热等特征的效应,将成年雌性布氏田鼠分为非繁殖对照食物组、非繁殖高蛋白组、繁殖对照食物组和繁殖高蛋白组。对照食物蛋白含量为17.7%,高蛋白食物的蛋白含量为36.6%。实验过程中测定动物的体重、食物摄入量、静止代谢率(RMR)、身体成分、内脏器官重量、褐色脂肪组织(BAT)解偶联蛋白1(UCP1)含量、血清瘦素和催乳素水平等。结果发现:高蛋白食物明显抑制布氏田鼠的体重,但动物在妊娠期和哺乳期,这种抑制作用消失。高蛋白食物明显抑制非繁殖组动物的干物质摄入、摄入能和消化能,但对哺乳期动物没有影响。高蛋白食物提高了非繁殖期和繁殖期动物的消化率,降低了血清瘦素浓度,但仅提高了繁殖期动物肾脏的重量,而降低了盲肠的重量。RMR、UCP1含量和血清催乳素浓度等则不受高蛋白食物的影响。繁殖期动物的体重、能量摄入、RMR和血清催乳素浓度等均高于非繁殖动物。这些结果表明,食物蛋白含量可影响布氏田鼠的能量代谢和产热特征等,且在繁殖期和非繁殖期有不同的反应方式。     

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus

It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44?kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2–ΔSak and ΔSak–ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29?kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic.     

建立洁净区微生物数据库与无菌药品GMP生产过程控制的探讨

目的 建立洁净区微生物数据库,为追溯洁净区微生物污染来源提供依据,为无菌药品生产过程控制提供有力指导.方法 对乙型脑炎减毒活疫苗生产车间洁净区环境和操作人员进行微生物负载检测,并对该车间注射用水、纯化水系统样品进行微生物限度检测,鉴别研究相应分离菌,建立洁净区微生物数据库.同时对乙型脑炎减毒活疫苗中间产品进行无菌检查检测,并对阳性结果分离菌进行鉴别分析.然后根据建立的洁净区微生物数据库对阳性结果举例进行溯源探讨分析.结果 洁净区环境和人员主要存在里拉/藤黄微球菌、人葡萄球菌、表皮葡萄球菌以及科氏葡萄球菌科氏亚种等革兰阳性菌和蜡样芽胞杆菌等,水系统中则主要存在少动鞘氨醇单胞菌和铫子芽胞杆菌等.中间产品无菌阳性结果主要存在里拉/藤黄微球菌、蜡样芽胞杆菌以及奇异变形菌等,经调查分析,分别为操作过程中经环境偶然带入或试验动物操作不慎带入.结论 建立洁净区微生物数据库是追溯产品微生物污染来源的有效方法,能够为无菌药品GMP生产过程制定有效的控制措施提供依据,并使其更有针对性.     

Confidence interval of intrinsic optimum temperature estimated using thermodynamic SSI model

The intrinsic optimum temperature for the development of ectotherms is one of the most important factors not only for their physiological processes but also for ecological and evolutional processes. The Sharpe–Schoolfield–Ikemoto (SSI) model succeeded in defining the temperature that can thermodynamically meet the condition that at a particular temperature the probability of an active enzyme reaching its maximum activity is realized. Previously, an algorithm was developed by Ikemoto (Tropical malaria does not mean hot environments. Journal of Medical Entomology, 45, 963–969) to estimate model parameters, but that program was computationally very time consuming. Now, investigators can use the SSI model more easily because a full automatic computer program was designed by Shi et al. (A modified program for estimating the parameters of the SSI model. Environmental Entomology, 40, 462–469). However, the statistical significance of the point estimate of the intrinsic optimum temperature for each ectotherm has not yet been determined. Here, we provided a new method for calculating the confidence interval of the estimated intrinsic optimum temperature by modifying the approximate bootstrap confidence intervals method. For this purpose, it was necessary to develop a new program for a faster estimation of the parameters in the SSI model, which we have also done.     

Iron Is a Sensitive Biomarker for Inflammation in Multiple Sclerosis Lesions

MRI phase imaging in multiple sclerosis (MS) patients and in autopsy tissue have demonstrated the presence of iron depositions in white matter lesions.The accumulation of iron in some but not all lesions suggests a specific, potentially disease-relevant process, however; its pathophysiological significance remains unknown.Here, we explore the role of lesional iron in multiple sclerosis using multiple approaches: immunohistochemical examination of autoptic MS tissue, an in vitro model of iron-uptake in human cultured macrophages and ultra-highfield phase imaging of highly active and of secondary progressive MS patients.Using Perls'' stain and immunohistochemistry, iron was detected in MS tissue sections predominantly in non-phagocytosing macrophages/microglia at the edge of established, demyelinated lesions. Moreover, iron-containing macrophages but not myelin-laden macrophages expressed markers of proinflammatory (M1) polarization.Similarly, in human macrophage cultures, iron was preferentially taken up by non-phagocytosing, M1-polarized macrophages and induced M1 (super) polarization. Iron uptake was minimal in myelin-laden macrophages and active myelin phagocytosis led to depletion of intracellular iron.Finally, we demonstrated in MS patients using GRE phase imaging with ultra-highfield MRI that phase hypointense lesions were significantly more prevalent in patients with active relapsing than with secondary progressive MS.Taken together, our data provide a basis to interpret iron-sensitive GRE phase imaging in MS patients: iron is present in non-phagocytosing, M1-polarized microglia/macrophages at the rim of chronic active white matter demyelinating lesions. Phase imaging may therefore visualize specific, chronic proinflammatory activity in established MS lesions and thus provide important clinical information on disease status and treatment efficacy in MS patients.     

Immunogenic and Invasive Properties of Brucella melitensis 16M Outer Membrane Protein Vaccine Candidates Identified via a Reverse Vaccinology Approach

Brucella is the etiologic agent of brucellosis, one of the most common and widely distributed zoonotic diseases. Its highly infectious nature, the insidious, systemic, chronic, debilitating aspects of the disease and the lack of an approved vaccine for human use in the United States are features that make Brucella a viable threat to public health. One of the main impediments to vaccine development is identification of suitable antigens. In order to identify antigens that could potentially be used in a vaccine formulation, we describe a multi-step antigen selection approach. We initially used an algorithm (Vaxign) to predict ORF encoding outer membrane proteins with antigenic determinants. Differential gene expression during acute infection and published evidence for a role in virulence were used as criteria for down-selection of the candidate antigens that resulted from in silico prediction. This approach resulted in the identification of nine Brucella melitensis outer membrane proteins, 5 of which were recombinantly expressed and used for validation. Omp22 and Hia had the highest in silico scores for adhesin probability and also conferred invasive capacity to E. coli overexpressing recombinant proteins. With the exception of FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice, and humans. BtuB, Hia and FlgK stimulated a mixed Th1–Th2 response in splenocytes from immunized mice while BtuB and Hia elicited NO release from splenocytes of S19 immunized mice. The results support the applicability of the current approach to the identification of antigens with immunogenic and invasive properties. Studies to assess immunogenicity and protective efficacy of individual proteins in the mouse are currently underway.     

Enhancing Specific-Antibody Production to the ragB Vaccine with GITRL That Expand Tfh,IFN-γ+ T Cells and Attenuates Porphyromonas gingivalis Infection in Mice

The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ⁺ T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ⁺ T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ⁺ T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ⁺ T cells and antibody production to P. gingivalis.     

Common Oncogenic Mutations Are Infrequent in Oral Squamous Cell Carcinoma of Asian Origin

Objectives

The frequency of common oncogenic mutations and TP53 was determined in Asian oral squamous cell carcinoma (OSCC).

Materials and Methods

The OncoCarta^™ panel v1.0 assay was used to characterize oncogenic mutations. In addition, exons 4-11 of the TP53 gene were sequenced. Statistical analyses were conducted to identify associations between mutations and selected clinico-pathological characteristics and risk habits.

Results

Oncogenic mutations were detected in PIK3CA (5.7%) and HRAS (2.4%). Mutations in TP53 were observed in 27.7% (31/112) of the OSCC specimens. Oncogenic mutations were found more frequently in non-smokers (p = 0.049) and TP53 truncating mutations were more common in patients with no risk habits (p = 0.019). Patients with mutations had worse overall survival compared to those with absence of mutations; and patients who harbored DNA binding domain (DBD) and L2/L3/LSH mutations showed a worse survival probability compared to those patients with wild type TP53. The majority of the oncogenic and TP53 mutations were G:C > A:T and A:T > G:C base transitions, regardless of the different risk habits.

Conclusion

Hotspot oncogenic mutations which are frequently present in common solid tumors are exceedingly rare in OSCC. Despite differences in risk habit exposure, the mutation frequency of PIK3CA and HRAS in Asian OSCC were similar to that reported in OSCC among Caucasians, whereas TP53 mutations rates were significantly lower. The lack of actionable hotspot mutations argue strongly for the need to comprehensively characterize gene mutations associated with OSCC for the development of new diagnostic and therapeutic tools.