<Emphasis Type="Italic">Gen</Emphasis><Superscript><Emphasis Type="Italic">Htr</Emphasis></Superscript>: a tool for comparative assessment of genetic heterogeneity in microbial genomes generated by massive short-read sequencing

Background  

Microevolution is the study of short-term changes of alleles within a population and their effects on the phenotype of organisms. The result of the below-species-level evolution is heterogeneity, where populations consist of subpopulations with a large number of structural variations. Heterogeneity analysis is thus essential to our understanding of how selective and neutral forces shape bacterial populations over a short period of time. The Solexa Genome Analyzer, a next-generation sequencing platform, allows millions of short sequencing reads to be obtained with great accuracy, allowing for the ability to study the dynamics of the bacterial population at the whole genome level. The tool referred to as Gen ^Htr was developed for genome-wide heterogeneity analysis.     

Polyethylenimine/small interfering RNA‐mediated knockdown of vascular endothelial growth factor in vivo exerts anti‐tumor effects synergistically with Bevacizumab

Background

RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA‐based therapies and, recently, the novel branched PEI F25‐LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.

Methods

In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25‐LMW/siRNA‐mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti‐tumorigenic efficacies of: (i) PEI/siRNA‐mediated VEGF targeting; (ii) treatment with the monoclonal anti‐VEGF antibody Bevacizumab (Avastin®); and (iii) a combination of both.

Results

Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side‐effects of PEI F25‐LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti‐tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed.

Conclusions

PEI F25‐LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti‐tumor therapy, and PEI/siRNA‐mediated, therapeutic VEGF knockdown exerts anti‐tumor effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Pore forming polyalkylpyridinium salts from marine sponges versus synthetic lipofection systems: distinct tools for intracellular delivery of cDNA and siRNA

Background  

Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS), which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N ⁴,N ⁹-dioleoylspermine (LipoGen)). DNA condensation was evaluated and HEK 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow.     

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

Background  

The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.     

Interfering ribonucleic acids that suppress expression of multiple unrelated genes

Background  

Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of VEGF-A and ICAM-1; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Crucial role of zebrafish <Emphasis Type="Italic">prox1</Emphasis>in hypothalamic catecholaminergic neurons development

Background  

Prox1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development.     

Para‐psychobiotic Lactobacillus gasseri CP2305 ameliorates stress‐related symptoms and sleep quality

Aims

To confirm the stress‐relieving effects of heat‐inactivated, enteric‐colonizing Lactobacillus gasseri CP2305 (paraprobiotic CP2305) in medical students taking a cadaver dissection course.

Methods and Results

Healthy students (21 males and 11 females) took paraprobiotic CP2305 daily for 5 weeks during a cadaver dissection course. The General Health Questionnaire and the Pittsburgh Sleep Quality Index were employed to assess stress‐related somatic symptoms and sleep quality respectively. The aggravation of stress‐associated somatic symptoms was observed in female students (P = 0·029). Sleep quality was improved in the paraprobiotic CP2305 group (P = 0·038), particularly in men (P = 0·004). Among men, paraprobiotic CP2305 shortened sleep latency (P = 0·035) and increased sleep duration (P = 0·048). Diarrhoea‐like symptoms were also effectively controlled with CP2305 (P = 0·005) in men. Thus, we observed sex‐related differences in the effects of paraprobiotic CP2305. In addition, CP2305 affected the growth of faecal Bacteroides vulgatus and Dorea longicatena, which are involved in intestinal inflammation.

Conclusions

CP2305 is a potential paraprobiotic that regulates stress responses, and its beneficial effects may depend on specific cell component(s).

Significance and Impact of the Study

This study characterizes the effects of a stress‐relieving para‐psychobiotic in humans.     

A meta‐analysis of the association between Helicobacter pylori (H. pylori) infection and hyperemesis gravidarum

Background

Hyperemesis gravidarum remains a common, distressing, and significant yet poorly understood disorder during pregnancy. The association between maternal Helicobacter pylori (H. pylori) infection and hyperemesis gravidarum has been increasingly recognized and investigated. This study thus aimed to provide an updated review and meta‐analysis of the topic.

Methods

Using the search terms (H. pyloriOR Helicobacter ORHelicobacter pyloriOR infection) AND (pregnancy OR emesis OR hyperemesis gravidarum OR nausea OR vomiting), a preliminary search on the PubMed, Ovid, Web of Science, Google Scholar, and WanFang database yielded 372 papers published in English between January 1st, 1960 and June 1st, 2017.

Results

A total of 38 cross‐sectional and case‐control studies, with a total of 10 289 patients were eligible for review. Meta‐analysis revealed a significant association between H. pylori infection and hyperemesis gravidarum during pregnancy, with a pooled odds ratio of 1.348 (95% CI: 1.156‐1.539, P < .001). Subgroup analysis found that serologic and stool antigen tests were comparable methods of detecting H. pylori as they yielded similar odds ratios.

Limitations

Although the studies did not have high heterogeneity (I² = 28%), publication bias was observed, and interstudy discrepancies in the diagnostic criteria adopted for hyperemesis gravidarum limit the reliability of findings. Also, 15 of the included studies were from the same country (Turkey), which could limit the generalizability of current findings. The prevalence of H. pylori infection varies throughout the world, and there may also be pathogenic differences as most strains of H. pylori in East Asia carry the cytotoxin‐associated gene A gene.

Conclusion

H. pylori infection was associated with an increased likelihood of hyperemesis gravidarum during pregnancy. Given the high prevalence of H. pylori infections worldwide, detecting H. pylori infection and the eradication of maternal H. pylori infection could be part of maternal hyperemesis gravidarum management. Further confirmation with robust longitudinal studies and mechanistic investigations are needed.     

Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model

Introduction  

The etiology of degenerative disc disease is unknown. Several investigators have reported the presence of proteolytic enzymes, such as the matrix metalloproteinase (MMP) and ADAMTS (a disintegrin and metalloprotease with thrombospondin-like repeats) families, in degenerated human discs. Glasson and colleagues recently reported that a significant reduction occurs in the severity of cartilage destruction in ADAMTS5 knockout mice compared with wild-type mice. The purpose of this study was to evaluate the suppressive effects of injections of ADAMTS5 small interference RNA (siRNA) oligonucleotide on intervertebral disc degeneration in the rabbit anular needle-puncture model.     

Hyaluronic acid complexed to biodegradable poly L‐arginine for targeted delivery of siRNAs

Background

Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively‐charged HA and cationic poly L ‐arginine (PLR) for siRNA delivery systems.

Methods

Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA.

Results

Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP‐mediated delivery of siRNAs was highest in WM266.4 cells followed by B16F10 cells and COS‐7 cells, in parallel with CD44 surface densities of these cell lines. TC₅₀ values (i.e. the HP concentrations at which 50% of cells were viable after treatment) were used as indicators of cytotoxicity. HP101 showed TC₅₀ values that were 2‐fold and 23‐fold higher than those of HP110 and PLR, respectively. After delivery into cells, siRNA exerted target‐specific RNA interference effects on mRNA and protein levels. Three days after treatment of red fluorescent protein (RFP)‐expressing B16F10 cells with RFP‐specific siRNA complexed to HP101, cellular fluorescence signals were reduced. Intratumoral administration of RFP‐specific siRNA via HP101 delivery significantly reduced the expression of RFP in tumor tissues.

Conclusions

HP101 may function as a biocompatible polymeric carrier of siRNAs and have possible application to localized siRNA delivery in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting

Background  

Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance), that calculates the relative abundance of genes in cDNA libraries.     

A longitudinal study on the occurrence of <Emphasis Type="Italic">Cryptosporidium</Emphasis> and <Emphasis Type="Italic">Giardia</Emphasis> in dogs during their first year of life

Background  

The primary aim of this study was to obtain more knowledge about the occurrence of Cryptosporidium and Giardia in young dogs in Norway.     

Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology

Background  

Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs.     

Beneficial effect of Burdock complex on asymptomatic Helicobacter pylori‐infected subjects: A randomized,double‐blind placebo‐controlled clinical trial

Background

Burdock complex (BC) constitutes of burdock (Arctium lappa), angelica (Angelica sinensis), gromwell (Lithospermum erythrorhizon), and sesame (Sesamum indicum) oil, which are commonly used in traditional Chinese medicine (TCM) for treating various disorders. This study intended to examine the anti‐H. pylori activity of BC on AGS cell model as well as in asymptomatic H. pylori‐infected subjects.

Materials and Methods

AGS cell incubated with H. pylori and treated with BC to evaluate the minimum inhibition concentration (MIC), cell viability (MTT) anti‐adhesion activity, and inflammatory markers. In case of clinical trial, H. pylori‐positive subjects (urea breath test [UBT] >10%, n = 36) were enrolled and requested to intake BC (n = 19) or placebo (n = 17) for 8 weeks. Antioxidant capacity, total phenol, UBT, inflammatory markers were analyzed at the initial, 4th, 8th, and 10th weeks. Moreover, the endoscopic examination was carried out on baseline and 10th week.

Results

In vitro studies showed that BC treatment significantly inhibited (P < .05) the inflammatory markers and adhesion of H. pylori to AGS cell. However, H. pylori‐infected subject ingested with BC for 8 weeks significantly decreased (P < .05) the UBT value, inflammatory markers with improved antioxidant activity, and phenolic levels as compared to placebo. Also, consumption of BC considerably healed the ulcer wound.

Conclusion

Overall, the BC could attenuate H. pylori infection by inhibiting H. pylori adhesion and subsequent inflammatory response on the gastric epithelial cell (AGS) as well as clinically ameliorated UBT, antioxidant capacity, and alleviated inflammation to display its anti‐H. pylori activity.     

<Emphasis Type="Italic">In vitro</Emphasis> bioassay as a predictor of <Emphasis Type="Italic">in vivo</Emphasis> response

Background  

There is a substantial discrepancy between in vitro and in vivo experiments. The purpose of the present work was development of a theoretical framework to enable improved prediction of in vivo response from in vitro bioassay results.