k-gram方法识别microRNA前体

MicroRNAs(miRNAs)是动植物中较短的参与调控基因表达的功能性非编码RNA序列.第一个miRNA是通过实验手段发现的,然而通过实验手段识别miRNA在技术上仍然具有很大的挑战性和不完整性.因此,miRNA基因识别需要寻求计算方法来弥补实验方法的不足.提出了一个全新的miRNA前体的识别方法.在构造识别模型中,把初级序列和序列二级结构相结合,采用k-gram方法把序列信息映射到高维特征空间中,然后通过特征选取方法提取特征,并用这些特征为miRNA前体的识别构造了基于SVM的识别模型.同时,采用隐马尔可夫模型(HMM)的学习方法进行了比较.实验结果表明,该方法是有效的,可以达到较高的敏感性和特异性.     

利用人工microRNA实现基因沉默

MicroRNA是一组长度约为21 nt的非编码蛋白质的短序列RNA,能通过碱基互补配对的方式指导降解靶基因mRNA或抑制靶基因的翻译。MicroRNA的主要功能是调控基因的表达,在生物体的生长、发育及疾病发生中扮演着重要的角色。本文介绍了利用microRNA实现基因沉默的作用原理,人工合成microRNA,构建转基因载体,实现对目的基因的沉默及这种工具在生命科学领域的应用前景。     

A Mixed-Method Approach for Quantifying Illegal Fishing and Its Impact on an Endangered Fish Species

Illegal harvest is recognized as a widespread problem in natural resource management. The use of multiple methods for quantifying illegal harvest has been widely recommended yet infrequently applied. We used a mixed-method approach to evaluate the extent, character, and motivations of illegal gillnet fishing in Lake Hovsgol National Park, Mongolia and its impact on the lake’s fish populations, especially that of the endangered endemic Hovsgol grayling (Thymallus nigrescens). Surveys for derelict fishing gear indicate that gillnet fishing is widespread and increasing and that fishers generally use 3–4 cm mesh gillnet. Interviews with resident herders and park rangers suggest that many residents fish for subsistence during the spring grayling spawning migration and that some residents fish commercially year-round. Interviewed herders and rangers generally agree that fish population sizes are decreasing but are divided on the causes and solutions. Biological monitoring indicates that the gillnet mesh sizes used by fishers efficiently target Hovsgol grayling. Of the five species sampled in the monitoring program, only burbot (Lota lota) showed a significant decrease in population abundance from 2009–2013. However, grayling, burbot, and roach (Rutilus rutilus) all showed significant declines in average body size, suggesting a negative fishing impact. Data-poor stock assessment methods suggest that the fishing effort equivalent to each resident family fishing 50-m of gillnet 11–15 nights per year would be sufficient to overexploit the grayling population. Results from the derelict fishing gear survey and interviews suggest that this level of effort is not implausible. Overall, we demonstrate the ability for a mixed-method approach to effectively describe an illegal fishery and suggest that these methods be used to assess illegal fishing and its impacts in other protected areas.     

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.Download video file.^{(114M, mov)}     

MicroRNA Targeted Therapeutic Approach for Pancreatic Cancer

Pancreatic cancer remains the fourth leading cause of cancer-related death in the US and is expected to be the second leading cause of cancer-related death by 2030. Therefore, it is important to better understand the molecular pathogenesis, phenotypes and features of pancreatic cancer in order to design novel molecularly targeted therapies for achieving better therapeutic outcome of patients with pancreatic cancer. Recently, the roles of microRNAs (miRNAs) in the development and progression of pancreatic cancer became a hot topic in the scientific community of pancreatic cancer research. By conducting miRNA expression profiling, the aberrant expression of miRNAs was revealed in the serum and in cancer tissues from patients with pancreatic cancer. These aberrantly expressed miRNAs are critically correlated with the disease stage, drug resistance, and survival of pancreatic cancer patients. Hence, targeting these tiny molecules, the specific miRNAs, could provide an efficient and optimal approach in the therapy of pancreatic cancer. Indeed, the pre-clinical and in vivo experiments showed that nanoparticle delivery of synthetic oligonucleotides or treatment with natural agents could be useful to modulate the expression of miRNAs and thereby inhibit pancreatic cancer growth and progression, suggesting that targeting miRNAs combined with conventional anti-cancer therapeutics could be a novel therapeutic strategy for increasing drug sensitivity and achieving better therapeutic outcome of patients diagnosed with pancreatic cancer.     

绵羊肺腺瘤病特异性PCR及套式PCR诊断方法的建立

绵羊肺腺瘤是由外源性反转录病毒JSRV引起的接触性传染的肺肿瘤性疾病。感染羊没有针对JSRV的循环抗体,但在感染羊的肺肿瘤组织、淋巴网状系统及外周血单核细胞中可检测到外源性前病毒exJSRV及JSRV转录产物。健康羊基因组中存在15~20拷贝的内源性前病毒enJSRV,内外源性前病毒的结构基因高度相似,而在U3区存在差别,因而,设计了针对exJSRVU3区的特异性引物并在国内首次建立了一步法特异性PCR检测法及套式PCR检测法。以700ng健康羊基因组DNA为背景,梯度稀释阳性质粒pJSRV-LTR作为模板,比较两种方法的敏感性,结果表明套式法的敏感性是一步法的10倍以上,套式法也是目前可用于检测感染羊血液样品的唯一方法,可望在绵羊肺腺瘤病的流行病学调查及防控方面起重要作用。     

A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.     

Partially Penetrant Postnatal Lethality of an Epithelial Specific MicroRNA in a Mouse Knockout

MicroRNAs are small noncoding RNAs thought to have pivotal roles in numerous diseases and developmental processes. However, a growing body of literature indicates that in vivo elimination of these tiny RNAs usually has little to no observable consequence, suggesting functional redundancy with other microRNAs or cellular pathways. We provide an in-depth analysis of miR-205 expression and define miR-205 as an epithelial-specific microRNA, and for the first time show that ablation of this microRNA knockout exhibits partially penetrant lethality in a constitutive mouse knockout model. Given the role of this microRNA in cancer and development, this mouse model will be an incredible reagent to study the function and mechanisms of miR-205 in epithelial tissue development and disease.     

A Facile Approach to Distinct Unusual Sucrose in Honey by Titanium Oxide Nanoparticles

In this study, we present a new approach to distinguish original honey from sucrose-overdosed one by a colloid of oxygen-deficient TiO₂ nanoparticles (NPs). Results show that the original honey does not affect the colloidal NPs while sucrose-overdosed one precipitates it in a few seconds. Our studies show that the precipitating time has a non-linear dependence on absorption peak intensity, and the settling rate increases up to 0.185 cm min^?1 by increasing sucrose in honey. Our results show that sucrose in honey can enhance the hydrodynamic radius of NPs up to 482.9 nm by suppressing zeta potential besides agglomerating the NPs and a blue shift in the absorption peak (up to 20 eV). The macroscopic behavior of the honey-solved colloid can be applied to determine the quality of honey according to the settling rate in a few minutes.

     

Specific Dot-Immunobinding Assay for Detection and Enumeration of Thiobacillus ferrooxidans

A specific and very sensitive dot-immunobinding assay for the detection and enumeration of the bioleaching microorganism Thiobacillus ferrooxidans was developed. Nitrocellulose spotted with samples was incubated with polyclonal antisera against whole T. ferrooxidans cells and then in ¹²⁵I-labeled protein A or ¹²⁵I-labeled goat antirabbit immunoglobulin G; incubation was followed by autoradiography. Since a minimum of 10³ cells per dot could be detected, the method offers the possibility of simultaneous processing of numerous samples in a short time to monitor the levels of T. ferrooxidans in bioleaching operations.     

Synergy of Homocysteine, MicroRNA, and Epigenetics: A Novel Therapeutic Approach for Stroke

Homocysteine (Hcy) is a thiol-containing amino acid formed during methionine metabolism. Elevated level of Hcy is known as hyperhomocysteinemia (HHcy). HHcy is an independent risk factor for cerebrovascular diseases such as stroke, dementia, Alzheimer’s disease, etc. Stroke, which is caused by interruption of blood supply to the brain, is one of the leading causes of death and disability in a number of people worldwide. The HHcy causes an increased carotid artery plaque that may lead to ischemic stroke but the mechanism is currently not well understood. Though mutations or polymorphisms in the key genes of Hcy metabolism pathway have been well elucidated in stroke, emerging evidences suggested epigenetic mechanisms equally play an important role in stroke development such as DNA methylation, chromatin remodeling, RNA editing, noncoding RNAs (ncRNAs), and microRNAs (miRNAs). However, there is no review available yet that describes the role of genetics and epigenetics during HHcy in stroke. The current review highlights the role of genetics and epigenetics in stroke during HHcy and the role of epigenetics in its therapeutics. The review also highlights possible epigenetic mechanisms, potential therapeutic molecules, putative challenges, and approaches to deal with stroke during HHcy.     

A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.     

Kinetic Studies of Ammonia Monooxygenase Inhibition in Nitrosomonas europaea by Hydrocarbons and Halogenated Hydrocarbons in an Optimized Whole-Cell Assay

The inhibitory effects of 15 hydrocarbons and halogenated hydrocarbons on NH₃ oxidation by ammonia monooxygenase (AMO) in intact cells of the nitrifying bacterium Nitrosomonas europaea were determined. Determination of AMO activity, measured as NO₂^- production, required coupling of hydroxylamine oxidoreductase (HAO) activity with NH₃-dependent NH₂OH production by AMO. Hydrazine, an alternate substrate for HAO, was added to the reaction mixtures as a source of reductant for AMO. Most inhibitors exhibited competitive or noncompetitive inhibition patterns. The competitive character generally decreased (K_iE/K_iES increased) as the molecular size of the inhibitors increased. For example, CH₄ and C₂H₄ were competitive inhibitors of NH₃ oxidation, whereas the remaining alkanes (up to C₄) and monohalogenated (Cl, Br, I) alkanes were noncompetitive. Oxidation of C₂H₅Br (noncompetitive) increased as the NH₄⁺ concentration increased up to 40 mM, whereas oxidations of inhibitors with competitive character (K_iE K_iES) were diminished at 40 mM NH₄⁺. Multichlorinated compounds produced nonlinear Lineweaver-Burk plots. Iodinated alkanes (CH₃I, C₂H₅I) and C₂Cl₄ were potent inhibitors of NH₃ oxidation. Maximum rates of NH₃, C₂H₄, and C₂H₆ oxidations were approximately equivalent, suggesting a common rate-determining step. These data support an active-site model for AMO consisting of an NH₃-binding site and a second site that binds noncompetitive inhibitors, with oxidation occurring at either site.     

A novel and universal method for microRNA RT-qPCR data normalization

Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.     

A Specific Method for the Detection of Hiochi Bacteria Using an Enzyme-linked Immunosorbent Assay System with Anti-hiochi Bacteria Monoclonal Antibodies

Ten standard strains of hiochi bacteria were selected based on the SDS-PAGE patterns of their cellular proteins. We then obtained ten hybridoma systems that secreted highly reactive monoclonal antibodies (MAbs) to the whole cells of each strain. It was apparent that these MAbs were highly reactive and specific for hiochi bacterial whole cells when using an enzyme-linked immunosorbent assay (ELISA). Three MAbs (two against the homo-fermentative hiochi lactobacilli and an MAb against the hetero-fermentative true hiochi bacilli) showed cross-reactivity to some of the other strains of lactobacilli tested. However, the other MAbs did not react with strains other than the immunogen. A sensitive ELISA method for the detection of hiochi bacteria was examined. It was possible to detect the order of 10³ cells of the ten standard strains. Using this procedure and a mixture of the ten MAbs, a detection limit of 10⁴ cells or less could be obtained for 98.2% of the hiochi bacteria isolated from sake brewing factories. Thus, this immunological technique using MAbs specific for hiochi bacteria is a sensitive and rapid detection method for hiochi bacteria, which can be used in the quality control of sake.     

Quantifying Hypoxia Impacts on an Estuarine Demersal Community Using a Hierarchical Ensemble Approach

In coastal marine ecosystems, hypoxia and anoxia are emerging as growing threats whose ecological impacts are difficult to ascertain because of the frequent lack of adequate references for comparison. We applied conventional and hierarchical ensemble analyses to evaluate the weight of evidence in support of hypoxia impacts on local densities of individual and groups of demersal fish and invertebrate species in Hood Canal, WA, which is subject to seasonal hypoxia in its southern reaches. Central to our approach was a sample design and analysis scheme that was designed specifically to consider multiple alternative hypotheses regarding factors that dictate local species’ densities. We anticipated persistent effects of hypoxia (felt even when seasonal hypoxia was absent) on species densities would be most pronounced for sessile species, but that immediate effects (felt only when seasonal hypoxia was present) would dominate for mobile species. Conventional analysis provided strong evidence that densities of sessile species were persistently reduced in the hypoxic-impacted site, but did not indicate widespread immediate density responses during hypoxic events among mobile species. The absence of strong weights of evidence for hypoxia effects was partly a consequence of alternative hypotheses that better explained spatial-temporal variation in species’ densities. The hierarchical ensemble analysis improved the precision of species-specific effect sizes, and also allowed us to make inferences about the response of aggregated groups of species. The estimated mean density reductions during hypoxic events (dissolved oxygen ~2 mg/l) ranged from 73 to 98% among mobile invertebrates, benthic, and benthopelagic fishes. The large reduction in benthic and benthopelagic species suggests substantial effects of hypoxia in Hood Canal even at oxygen levels that were marginally hypoxic. Understanding the full ecological consequence of hypoxia will require a greater knowledge on the spatial extent of distributional shifts and their effects on competitive and predator–prey interactions.     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.