Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.     

THE ULTRASTRUCTURE OF THE ENAMEL IN THE GIANT PANDA OF PLEISTOCENE

<正> Enamel ultrastructures in the molar teeth of the giant panda, including Ailuropoda microta of the Early Pleistocene, Ailuropoda melanoleuca bacont of the Middle and Late Pleistocene and a living form, Ailuropoda rnelanoleuca, have been investigated by scanning electron microscopy. Transverse and longitudinal sections of enamel were made in order co evaluate shape, size and arrangement of the prisms. The sections were etched then with 0.074 M H_3PO_4 for 30-60 sec. Our investigations have shown certain features of the enamel which allow us to recognize differences among Ailuropoda on the basis of examination of large areas of the enamel. The results are summarized below.     

Identification, characterization, and localization of a novel kidney polycystin-1-polycystin-2 complex

The functions of the two proteins defective in autosomal dominant polycystic kidney disease, polycystin-1 and polycystin-2, have not been fully clarified, but it has been hypothesized that they may heterodimerize to form a "polycystin complex" involved in cell adhesion. In this paper, we demonstrate for the first time the existence of a native polycystin complex in mouse kidney tubular cells transgenic for PKD1, non-transgenic kidney cells, and normal adult human kidney. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 differing in their sensitivity to endoglycosidase H (Endo H) were found; in contrast, native polycystin-2 was fully Endo H-sensitive. Using highly specific antibodies to both proteins, we show that polycystin-2 associates selectively with two species of full-length polycystin-1, one Endo H-sensitive and the other Endo H-resistant; importantly, the latter could be further enriched in plasma membrane fractions and co-immunoprecipitated with polycystin-2. Finally, a subpopulation of this complex co-localized to the lateral cell borders of PKD1 transgenic kidney cells. These results demonstrate that polycystin-1 and polycystin-2 interact in vivo to form a stable heterodimeric complex and suggest that disruption of this complex is likely to be of primary relevance to the pathogenesis of cyst formation in autosomal dominant polycystic kidney disease.     

The mechanism of myoblast deformation in response to cyclic strain - A cytomechanical study

Mechanical strain is one of the important epigenetic factors that cause deformation and differentiation of skeletal muscles. This research was designed to investigate how myoblast deformation occurs after cyclic strain loading. Myoblasts were passaged three times and harvested; various cyclic strains (2.5kPa, 5kPa and 10kPa) were then loaded using a pulsatile mechanical system. The adaptive response of the myoblasts was observed at different time points (0.5h, 1h, 6h and 12h) post-loading. At the early stage of cyclic strain loading (<1h), almost no visible morphological changes were observed in the myoblasts. The actin cytoskeleton showed a disordered arrangement and a weak fluorescence expression; there was little expression of talin. At 6h and 12h post-loading, the myoblasts changed their orientation to parallel (in the 2.5kPa and 5kPa groups) or perpendicular (in the 10kPa group) to the direction of strain. Fluorescence expression of both the actin cytoskeleton and talin was significantly increased. The results suggest that cyclic strain has at least two ways to regulate adaptation of myoblasts: (1) by directly affecting actin cytoskeleton at an early stage post-loading to cause depolymerization; and (2) by later chemical signals transmitted from the extracellular side to intracellular side to initiate repolymerization.     

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.     

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

2011年、2013年和2016年医院内获得性血流感染常见病原菌分布及其耐药性分析

动态监测2011年、2013年和2016年我国不同地区医院内获得性血流感染病原菌分布及耐药进展趋势。从全国10个城市回顾性收集血流感染病原菌非重复性株,采用琼脂稀释法或微量肉汤稀释法进行药物敏感性试验,采用Whonet 5.6软件对药敏试验结果进行分析。收集的2 248株血流感染病原菌中革兰阴性杆菌为1 657株 (占73.7%),革兰阳性球菌为591株 (占26.3%)。分离率排名前五的病原菌依次为大肠埃希菌 (32.6%,733株/2 248株)、肺炎克雷伯菌 (14.5%,327株/2 248株)、金黄色葡萄球菌 (10.0%,225株/2 248株)、鲍曼不动杆菌 (8.7%,196株/ 2 248株) 和铜绿假单胞菌 (6.2%,140株/2 248株)。血流感染分离的革兰阴性杆菌对抗菌药物体外敏感率较高的抗菌药物依次为粘菌素 (96.5%,1 525株/1 581株,不包括天然耐药菌株)、替加环素 (95.6%,1 375株/1 438株,不包括天然耐药菌株)、头孢他啶/克拉维酸 (89.2%,1 112株/1 246株)、阿米卡星 (86.4%,1 382株/1 599株) 和美罗培南 (85.7%,1 376株/1 605株);革兰阳性球菌对抗菌药物体外敏感率较高的抗菌药物依次为替加环素、替考拉宁和达托霉素 (敏感率均为100.0%)、万古霉素和利奈唑胺 (敏感率均为99.7%)。2011年、2013年和2016年产超广谱β-内酰胺酶肠杆菌科细菌分离率分别为50.6% (206株/407株)、49.8% (136株/273株) 和38.9% (167株/429株);碳青霉烯不敏感肠杆菌科细菌分离率分别为2.2% (9株/408株)、4.0% (16株/402株) 和3.9% (17株/ 439株);多重耐药鲍曼不动杆菌分离率分别为76.4% (55株/72株)、82.7% (43株/52株) 和87.5% (63株/72株),多重耐药铜绿假单胞菌分离率分别为9.8% (5株/51株)、20.0% (7株/35株) 和13.0% (7株/54株);甲氧西林耐药金黄色葡萄球菌的分离率分别为51.9% (41株/79株)、29.7% (19株/64株) 和31.7% (26株/82株)。屎肠球菌和粪肠球菌中高水平庆大霉素耐药株分离率分别为43.2% (48株/111株) 和40.9% (27株/66株)。碳青霉烯不敏感肠杆菌科细菌中肺炎克雷伯菌居首位,占57.1% (24株/42株) 。肠杆菌科细菌中分离出30株替加环素不敏感株,其中肺炎克雷伯菌占76.7% (23株/30株);分离出粘菌素耐药肠杆菌科细菌39株,其中大肠埃希菌、阴沟肠杆菌和肺炎克雷伯菌分别占43.6% (17株/39株)、35.9% (14株/39株) 和15.4% (6株/39株)。医院获得性血流感染病原菌主要为革兰阴性杆菌 (以大肠埃希菌和肺炎克雷伯菌为主),其对替加环素、粘菌素和碳青霉烯类药物的敏感率较高;革兰阳性球菌中分离率最高的为金黄色葡萄球菌,其次为屎肠球菌,这两种细菌对替加环素、达托霉素、利奈唑胺、万古霉素和替考拉宁的敏感率较高。粘菌素耐药肠杆菌科细菌、替加环素不敏感肠杆菌科细菌、利奈唑胺或万古霉素不敏感革兰阳性球菌的分离,警示临床高度关注,仍需动态监测耐药进展趋势。