No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’-phosphorylated guide DNA (gDNA) from 2.5 ng/ μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.This work was supported from the National Collaborative Research Infrastructure (NCRIS) through the Australian Phenomics Network in Australi

Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstratedthat heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shownto be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intraislet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxideinduced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/ heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.: This work was supported by a National Health and Medical Research Council of Australia (NHMRC; https://www.nhmrc.gov.au/)/Juvenile Diabetes Research Foundation (JDRF) Special Program Grant in Type 1 Diabetes (#418138), The Canberra Hospital Private Practice Fund (http:// www.health.act.gov.au/research-publications/research/ppf-major-grants), JDRF nPOD Research Grant (#25-2010-716; http://www.jdrf.org), JDRF Research Grant (#47-2012-746) and NHMRC Project Grant (#1043284

Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models.

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and -poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.This work was funded by Biotechnology and Biological Sciences Research Council (http://www.bbsrc.ac.uk) grant BB/K004514/1. D.P.W. was funded by a Wellcome Trust (https://wellcome.ac.uk) Infection and Immunity PhD rotation studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Antimicrobial and antimutagenic activity of probiotics

Pojavom, antibiotik-rezistentnih bakterija, koncept probiotika kao prirodnog načina sprječavanja rasta patogenih bakterija postaje vrlo zanimljiv. Probiotici su definirani kao živi mikrobni dodatak hrani koji osigurava povoljan učinak na domaćina, te poboljšava ravnotežu mikroflore njegovog probavnog sustava. U humanoj prehrani najčešće se kao probiotici koriste bakterije Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum. Bifidobacterium longum i kvasac Saccharomyces boulardii. Probiotici proizvode organske kiseline od kojih su octena, mliječna i pirogrožđana u najvećoj količini. Neki istraživači vjeruju da je mliječna kiselina jedina značajna antimikrobna tvar. Prikazana je antimutagena aktivnost octene, maslačne, mliječne i pirogrožđane kiseline koje proizvode probiotici, prema 8 mutagenih ili promutagenih tvari.With the emergence of antibiotic-resistant bacteria, the concept of probiotics as a natural way of suppressing pathogens has attracted much attention. Probiotics are defined as a "live microbial good supplement, which provide beneficial effects on the host by improving its intestinal microbial balance." Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, Bifidobacterium longum and the yeast Saccharomyces boulardii are the most commonly organisms used as probiotics in human diet. Probiotic bacteria produce several organic acids with acetic, lactic and pyruvic acid being the major ones. Several researches belive that lactic acid is the only antimicrobial agent of importance. The antimutagenic activity of acetic, butyric, lactic and pyruvic acids, produced by probiotics, versus 8 mutagens or promutagens is reported

The Somatic Genomic Landscape of Chromophobe Renal Cell Carcinoma

We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations

Multiplatform Analysis of 12 Cancer Types Reveals Molecular Classification within and across Tissues of Origin

Recent genomic analyses of pathologically-defined tumor types identify “within-a-tissue” disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head & neck, and a subset of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multi-platform classification, while correlated with tissue-of-origin, provides independent information for predicting clinical outcomes. All datasets are available for data-mining from a unified resource to support further biological discoveries and insights into novel therapeutic strategies

Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

Contains fulltext : 172380.pdf (publisher's version ) (Open Access

The Relative Concentrations of Nutrients and Toxins Dictate Feeding by a Vertebrate Browser, the Greater Glider Petauroides volans

Although ecologists believe that vertebrate herbivores must select a diet that allows them to meet their nutritional requirements, while avoiding intoxication by plant secondary metabolites, this is remarkably difficult to show. A long series of field and laboratory experiments means that we have a good understanding of the factors that affect feeding by leaf-eating marsupials. This knowledge and the natural intraspecific variation in Eucalyptus chemistry allowed us to test the hypothesis that the feeding decisions of greater gliders (Petauroides volans) depend on the concentrations of available nitrogen (incorporating total nitrogen, dry matter digestibility and tannins) and of formylated phloroglucinol compounds (FPCs), potent antifeedants unique to Eucalyptus. We offered captive greater gliders foliage from two species of Eucalyptus, E . viminalisand E. melliodora, which vary naturally in their concentrations of available nitrogen and FPCs. We then measured the amount of foliage eaten by each glider and compared this with our laboratory analyses of foliar total nitrogen, available nitrogen and FPCs for each tree offered. The concentration of FPCs was the main factor that determined how much gliders ate of E. viminalis and E. melliodora, but in gliders fed E. viminalis the concentration of available nitrogen was also a significant influence. In other words, greater gliders ate E. viminalis leaves with a particular combination of FPCs and available nitrogen that maximised the nutritional gain but minimised their ingestion of toxins. In contrast, the concentration of total nitrogen was not correlated with feeding. This study is among the first to empirically show that browsing herbivores select a diet that balances the potential gain (available nutrients) and the potential costs (plant secondary chemicals) of eating leaves. The major implication of the study is that it is essential to identify the limiting nutrients and relevant toxins in a system in order to understand feeding behaviour

Variation in the nuclear effects of infection by different human rhinovirus serotypes.

Human rhinovirus (HRV) is a positive sense RNA virus, which, despite replicating in the cytoplasm, has a significant impact on nuclear transport and nuclear localisation of host proteins. A number of studies have identified differences between HRV serotypes, with respect to host response, protease activity and replicative ability. Here we report the sero-specific effects of two group-A human rhinovirus serotypes, the minor group HRV2 and the major group HRV16, on nuclear transport and nuclear protein localisation. Using Western analysis, immunofluorescence and real time PCR, we show that HRV2 replicates at a faster rate than HRV16, which correlates with earlier production of viral proteases and disruption of host nuclear transport. There is significant variation in the nuclear effects of different rhinovirus species, which in turn may impact disease progression and patient response