E-box元件修饰端粒酶核心启动子序列及体外转录活性分析

人类端粒酶启动子(hTERT启动子)在肿瘤基因治疗中的有效性已经得到了证实. 然而,hTERT启动子有限的肿瘤靶向转录活性困扰着它的临床应用.早期研究已经揭示,核心hTERT启动子上的-34位E-box元件与该启动子的肿瘤靶向转录活性有关.为进一步探索核心hTERT启动子序列3′端富余E-box元件是否能提高启动子的肿瘤靶向转录能力,用化学合成方法在野生型hTERT(WT-hTERT)核心启动子片段(编码蛋白起始子ATG上游-268 bp～-10 bp)的3′端接入3个E-box序列, 构建成修饰型hTERT(Mod-hTERT)启动子. 然后,分别用WT-hTERT和Mod-hTERT启动子去调控增强型绿色荧光蛋白(EGFP)及荧光素酶报告基因在293FT、HepGⅡ、SGC7901、U2OS、以及原代培养人成纤维细胞(PHF)中表达. 结果表明, 在Mod-hTERT启动子的各实验组细胞中,能够在端粒酶阳性的293FT、HepGⅡ及 SGC7901细胞组中观测到EGFP的表达,而在端粒酶阴性的U2OS及PHF细胞组中没有观测到EGFP的表达;在端粒酶阳性的293FT、HepGⅡ和SGC7901细胞株中,Mod-hTERT启动子调控下的荧光素酶活性要高于WT-hTERT启动子组（P＜0.01）; 而在端粒酶阴性的U2OS细胞组中,Mod-hTERT启动子调控下的荧光素酶活性则低于WT-hTERT启动子组（P＜0.01); 在PHF细胞组中,Mod-hTERT启动子组与WT-hTERT启动子组的荧光素酶活性差异不显著(P＞0.05).研究提示,在3′端增加E-box元件可以提高核心hTERT启动子序列的肿瘤靶向转录活性.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Frequent coexistence of anti-topoisomerase I and anti-U1RNP autoantibodies in African American patients associated with mild skin involvement: a retrospective clinical study

Introduction  

The presence of anti-topoisomerase I (topo I) antibodies is a classic scleroderma (SSc) marker presumably associated with a unique clinical subset. Here the clinical association of anti-topo I was reevaluated in unselected patients seen in a rheumatology clinic setting.     

MicroRNAs in systemic rheumatic diseases

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length. miRNAs have been shown to regulate gene expression and thus influence a wide range of physiological and pathological processes. Moreover, they are detected in a variety of sources, including tissues, serum, and other body fluids, such as saliva. The role of miRNAs is evident in various malignant and nonmalignant diseases, and there is accumulating evidence also for an important role of miRNAs in systemic rheumatic diseases. Abnormal expression of miRNAs has been reported in autoimmune diseases, mainly in systemic lupus erythematosus and rheumatoid arthritis. miRNAs can be aberrantly expressed even in the different stages of disease progression, allowing miRNAs to be important biomarkers, to help understand the pathogenesis of the disease, and to monitor disease activity and effects of treatment. Different groups have demonstrated a link between miRNA expression and disease activity, as in the case of renal flares in lupus patients. Moreover, miRNAs are emerging as potential targets for new therapeutic strategies of autoimmune disorders. Taken together, recent data demonstrate that miRNAs can influence mechanisms involved in the pathogenesis, relapse, and specific organ involvement of autoimmune diseases. The ultimate goal is the identification of a miRNA target or targets that could be manipulated through specific therapies, aiming at activation or inhibition of specific miRNAs responsible for the development of disease.     

Dilution-to-Extinction Culturing of Psychrotolerant Planktonic Bacteria from Permanently Ice-covered Lakes in the McMurdo Dry Valleys,Antarctica

Lakes in the McMurdo Dry Valleys of Antarctica are characterized by a permanent ice cover and little or no anthropogenic influence. Although bacterial cultures have been obtained from these habitats, recent culture-independent studies indicate that the most abundant microbes in these systems are not yet cultivated. By using dilution-to-extinction cultivation methods with sterilized and nutrient-amended lake water as media, we isolated 148 chemotrophic psychrotolerant bacterial cultures from fresh surface water of Lake Fryxell and the east lobe of Lake Bonney and the hypersaline, suboxic bottom water from the west lobes of Lake Bonney. Screening of the 16S ribosomal ribonucleic acid (rRNA) genes of the cultures by restriction fragment length polymorphism (RFLP) yielded 57 putatively pure psychrotolerant, slow growing cultures grouped into 18 clusters. The sequencing of 16S rRNA genes of randomly selected representatives of each RFLP cluster revealed that the corresponding isolates belong to the Alphaproteobacteria (six RFLP patterns), Betaproteobacteria (six RFLP patterns), Bacteroidetes (four RFLP patterns), and Actinobacteria (two RFLP patterns). Phylogenetic analysis of the sequences showed that the vast majority of the isolates were not closely related to previously described species. Thirteen of 18 RFLP patterns shared a 16S ribosomal deoxyribonucleic acid sequence similarity of 97% or less with the closest described species, and four isolates had a sequence similarity of 93% or less with the nearest described species. Phylogenetic analysis showed that these sequences were representatives of deeply branching organisms in the respective phylum. A comparison of the isolates with 16S rRNA clone libraries prepared from the same environments showed substantial overlap, indicating that dilution-to-extinction culturing in natural lake water media can help isolate some of the most abundant organisms in these perennially ice-covered lakes.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Determination of 2,4-diamino-5-benzylpyrimidines in combination with sulphadiazine in biological fluids using high-performance liquid chromatography

High-performance liquid chromatographic methods for the simultaneous analysis of tetroxoprim (TXP)/sulphadiazine (SDZ) and metioprim (MTP)/SDZ in serum and prostatic secretion (PS) are described. The detection limits in serum and PS were 50 ng TXP per ml and 100 ng SDZ per ml, and 40 ng MTP per ml and 100 ng SDZ per ml, respectively. The intra-assay coefficients of variation were in the range of 2.7–2.19%. Some preliminary data from a pharmacokinetic study in geriatric patients and a distribution study in dogs are presented. These methods enable the investigator to process a large number of TXP/SDZ and MTP/SDZ samples in one working day.