大熊猫精液和包皮菌群组成及潜在致病菌调查

[目的] 大熊猫是我国国家一级保护动物,其种群面临着传染病和栖息地破碎化等持续威胁,其中生殖系统的细菌感染和菌群失衡会影响大熊猫生殖健康,严重者可导致流产,是引起大熊猫繁殖障碍的原因之一。本研究对精液与包皮分泌物样本的菌群组成情况及分离培养潜在致病菌开展研究。[方法] 通过采集13份大熊猫包皮分泌物和12份精液样本,采用16S rRNA扩增子测序技术、细菌培养及PCR鉴定的方法,确定样本中的细菌种类。[结果] 菌群组成分析结果显示,在门水平上,厚壁菌门（Firmicutes）的细菌丰度在大熊猫包皮与精液中均为最高;在属水平上,不同时期的雄性大熊猫包皮的菌群可能会发生改变,棒状杆菌属（Corynebacterium）和Dolosicoccus是Ⅰ期包皮样本中最丰富的微生物菌群,相对丰度分别为15.45%和12.40%;链球菌属（Streptococcus）和埃希氏菌属（Escherichia）是Ⅱ期包皮样本中最丰富的微生物菌群,相对分度分别为37.94%和9.68%;拟杆菌属（Bacteroides）和普雷沃氏菌属（Prevotella）是精液样本中最丰富的微生物菌群,相对丰度分别为14.40%和12.88%。菌群多样性分析结果显示,精液样品高于Ⅰ期包皮样品和Ⅱ期包皮样品,Ⅰ期包皮样品和Ⅱ期包皮样品之间无显著差异。通过细菌分离培养得到肺炎克雷伯菌（Klebsiella pneumonia）在内的多种潜在性致病菌。[结论] 本研究分析了大熊猫精液和不同时期包皮分泌物的菌群组成,其优势菌属存在差异,大熊猫包皮与精液中存在潜在性致病菌,这可能对大熊猫的生殖系统健康带来威胁,其致病性有待进一步研究。     

稀土-落叶松单宁配合物的合成及抗真菌活性研究

以稀土(Re~(3+))和落叶松单宁(LT)为原料,采用液相合成法合成了5种廉价的稀土-落叶松单宁(Re~(3+)-LT)配合物,并通过红外光谱、X射线光电子能谱、紫外光谱以及配位数测定确定了配合物的结构.采用牛津杯法、琼脂稀释法测定配合物对黑曲霉、红曲霉、白腐菌、毛霉4种真菌的抑制作用.在抑菌方面,5种配合物对上述4种真菌均具有较强的抑制作用,其抑菌活性大小顺序为Ce~(3+)-LTGd~(3+)-LTLa~(3+)-LTNd~(3+)-LTYb~(3+)-LT,其中Ce~(3+)-LT对4种真菌的最小抑菌浓度分别为:1.6、1.6、0.8和1.6 g·L~(-1);Yb~(3+)-LT对4种真菌的最小抑菌浓度分别为:3.2、1.6、3.2和3.2 g·L~(-1).在杀菌方面,Yb~(3+)-LT的杀菌活性最强,其对4种真菌的最小杀菌浓度分别为:6.4、3.2、3.2和6.4 g·L~(-1).此外,尽管Nd~(3+)-LT和Gd~(3+)-LT具有较强的抑菌活性,但对黑曲霉和毛霉的杀菌作用较弱.     

Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images

Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.     

Characterization of long non-coding RNAs and MEF2C-AS1 identified as a novel biomarker in diffuse gastric cancer

Previous studies proved that long noncoding RNAs (lncRNAs) play important role in human cancer. However, the knowledge of genome scale expression of lncRNAs and their potential biological function in gastric cancer is still lacking. Next generation RNA sequencing (RNA-seq) was performed on tumor tissues and matched adjacent normal tissues of six diffuse gastric cancer (DGC) patients. Then we performed a comprehensive analysis on lncRNAs and mRNA. Fifty-eight lncRNAs were upregulated and 54 lncRNAs were downregulated in diffuse gastric cancer tissue compared with adjacent tissue. The numbers of up- and downregulated mRNAs were 306 and 161, respectively. In addition, we inferred the function of lncRNAs by construction of a co-expression network for deregulated mRNAs and lncRNAs. Co-expressed genes of MEF2C-AS1 and FENDRR were enriched to RAS and TGF-beta signaling pathway. MEF2C-AS1 and FENDRR expression were re-evaluated by Real-time Quantitative PCR in 42 DGC patients' tumor and normal tissues, and other 46 DGC patents' and 21 healthy controls' plasma. Validation data showed MEF2C-AS1 and FENDRR were significantly downregulated in tumor tissues compared with normal tissues. And decreased FENDRR are associated with aggressive tumor characteristics including more advanced stage (P = .030), poor differentiation (P = .043) and lymphatic metastasis (P = .001). The expression level MEF2C-AS1 was significantly lower in DGC patients' plasma than that in healthy controls' plasma. In gastric cancer cell lines, knock-down of MEF2C-AS1 or FENDRR reduced the protein levels of FAT3, NTN1 and LYVE1 (the co-expressed genes), which were related with gastric cancer cell proliferation and invasion by previous studies. In addition, knock-down of MEF2C-AS1 or FENDRR promoted aggressive tumor behaviors in in-vitro assays. In this study, we provide a valuable resource of lncRNAs which might play important roles in the function of oncogenes or tumor suppressors affecting the development and progression of diffuse gastric cancer.     

Partial purification of α-amylase from culture supernatant of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in aqueous two-phase systems

A study was made of the partition and purification of -amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)—citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of -amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of -amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.     

A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations

Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 A resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process.     

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study

Background

Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.

Methods and Findings

The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001).

Conclusions

The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.     

Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies

Recent molecular cytogenetic data have shown that the constitution of complex chromosome rearrangements (CCRs) may be more complicated than previously thought. The complicated nature of these rearrangements challenges the accurate delineation of the chromosomal breakpoints and mechanisms involved. Here, we report a molecular cytogenetic analysis of two patients with congenital anomalies and unbalanced de novo CCRs involving chromosome 17p using high-resolution array-based comparative genomic hybridization (array CGH) and fluorescent in situ hybridization (FISH). In the first patient, a 4-month-old boy with developmental delay, hypotonia, growth retardation, coronal synostosis, mild hypertelorism, and bilateral club feet, we found a duplication of the Charcot-Marie–Tooth disease type 1A and Smith-Magenis syndrome (SMS) chromosome regions, inverted insertion of the Miller-Dieker lissencephaly syndrome region into the SMS region, and two microdeletions including a terminal deletion of 17p. The latter, together with a duplication of 21q22.3-qter detected by array CGH, are likely the unbalanced product of a translocation t(17;21)(p13.3;q22.3). In the second patient, an 8-year-old girl with mental retardation, short stature, microcephaly and mild dysmorphic features, we identified four submicroscopic interspersed 17p duplications. All 17 breakpoints were examined in detail by FISH analysis. We found that four of the breakpoints mapped within known low-copy repeats (LCRs), including LCR17pA, middle SMS-REP/LCR17pB block, and LCR17pC. Our findings suggest that the LCR burden in proximal 17p may have stimulated the formation of these CCRs and, thus, that genome architectural features such as LCRs may have been instrumental in the generation of these CCRs.