Enusha Karunasena

Host responses to the pathogen Mycobacterium avium subsp. Paratuberculosis and beneficial microbes exhibit host sex specificity

ABSTRACT Differences between microbial pathogenesis in male and female hosts are well characterized in disease conditions connected to sexual transmission. However, limited biological insight is available on variances attributed to sex specificity in host-microbe interactions, and it is most often a minimized variable outside these transmission events. In this work, we studied two gut microbes—a pathogen, Mycobacterium avium subsp. paratuberculosis, and a probiotic, Lactobacillus animalis NP-51—and the interaction between each agent and the male and female gastrointestinal systems. This trial was conducted in BALB/c mice (n = 5 per experimental group and per sex at a given time point), with analysis at four time points over 180 days. Host responses to M. avium subsp. paratuberculosis and L. animalis were sensitive to sex. Cytokines that were significantly different (P ≤ 0.05) between the sexes included interleukin-1α/β (IL-1α/β), IL-17, IL-6, IL-10, IL-12, and gamma interferon (IFN-γ) and were dependent on experimental conditions. However, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and IL-13/23 showed no sex specificity. A metabolomics study indicated a 0.5- to 2.0-fold (log2 scale) increase in short-chain fatty acids (butyrate and acetate) in males and greater increases in o-phosphocholine or histidine from female colon tissues; variances distinct to each sex were observed with age or long-term probiotic consumption. Two genera, Staphylococcus and Roseburia, were consistently overrepresented in females compared to males; other species were specific to one sex but fluctuated depending on experimental conditions. The differences observed suggest that male and female gut tissues and microbiota respond to newly introduced microorganisms differently and that gut-associated microorganisms with host immune system responses and metabolic activity are supported by biology distinct to the host sex.

Next in line in next-generation sequencing: are we there yet?

Next-generation sequencing (NGS) technologies have provided us with an opportunity to explore genomes with unprecedented detail. Alongside these advancements, NGS has its own challenges (much like all technology) creating a plethora of analytical and biological considerations before these methods can be standardized. Meanwhile, encouraging single-cell sequencing has become a recent trend among scientists who are optimistic that this approach will better characterize cancer (and other similarly complex diseases) [1,2]. The technology necessary to sequence a complete genome from a ‘single’ cell has been available, but now, combined with growing popularity and mainstream acceptance, these methods are soon to become pedestrian tools, among a list of many existing NGS technologies. Will this technique be the missing link to scaffold data between genome sequencing methods, which currently contribute volumes of data but with nominal insight into therapies? To answer these questions we have chosen to review current trends in sequencing technology and relative to novel therapies.

Effects of the probiotic Lactobacillus animalis in murine Mycobacterium avium subspecies paratuberculosis infection

BackgroundMAP is a suspected zoonotic pathogen and the causative agent of Johne’s Disease in cattle and other ruminant animals. With over

Standardizing Next-Generation Sequencing Experiments and Analysis Methods

1 billion dollars in loss to the dairy industry due to Johne’s Disease, efforts to eliminate or reduce MAP from cattle are of importance. The purpose of this study was to determine if daily intake of probiotics could eliminate or reduce Johne’s Disease associated symptoms and pathogenesis by MAP. Post infection, animals are often asymptomatic carriers with limited shedding of the pathogen, proving early detection to be difficult. Disease and symptoms often appear 3–4 years after infection with antibiotic treatment proving ineffective. Symptoms include chronic gastrointestinal inflammation leading to severe weight-loss from poor feed and water intake cause a wasting disease. These symptoms are similar to those found in individuals with Crohn’s Disease (CD); MAP has been implicated by not proven to be the causative agent of CD. Probiotics administered to livestock animals, including dairy and beef cattle have demonstrated improvements in cattle performance and health. Our objectives included determining the benefits of Lactobacillus animalis (strain name: NP-51) in MAP infected BALB/c mice by evaluating systemic and gastrointestinal response by the host and gut microbiota. Male and female animals were fed 1×106 CFU/g probiotics in sterile, powdered mouse chow daily and infected with 1 × 107 CFU/ml MAP and compared to controls. Animals were evaluated for 180 days to assess acute and chronic stages of disease, with sample collection from animals every 45 days. MAP concentrations from liver and intestinal tissues were examined using real time-PCR methods and the expression of key inflammatory markers were measured during MAP infection (interferon-gamma [IFN-Υ], Interleukin-1α, IL-12, IL-10, IL-6, and Tumor necrosis factor alpha [TNF-α]).ResultsOur results demonstrate administration of probiotics reduces production of IFN-Υ and IL-6 while increasing TNF-α and IL-17 in chronic disease; healthful immune responses that reduce chronic inflammation associated to MAP infection.ConclusionsWe observed that the immune system’s response in the presence of probiotics to MAP contributes towards host health by influencing the activity of the immune system and gut microbial populations.

Treatment with epigenetic agents profoundly inhibits tumor growth in leiomyosarcoma

To the Editor: The past few years have seen the emergence of new strategies for high-throughput DNA sequencing that have invigorated life science research. Because of these technological advances, the available sequencing data continue to expand. Additionally, cost reductions have made access to sequences of entire genomes easier for established laboratories and even easier for small research groups. A lack of consensus exists, however, on how best to design and analyze next-generation sequencing (NGS) studies and how to compare these data to previous and future work. Comparing different sequencing platforms and analysis methods, and thus their interpretations, is becoming more complicated. The main purpose of such studies is somehow getting lost in the process of experimental design as biologists confront new territory with NGS work, which requires handling more complex and powerful statistical methods and produces large data sets. Every new tool or method is being compared with existing methods, and investigators are using statistical arguments to propose that the new methods are better. Kiezun et al. (1) have recently …

Updating microbial genomic sequences: improving accuracy & innovation

Leiomyosarcomas are rare mesenchymal neoplasms characterized by a smooth muscle differentiation pattern. Due to the extremely poor prognosis in patients, the development of novel chemotherapeutic regimens remains critically important. In this study, multiple leiomyosarcoma cell lines, SK-UT1, SK-LMS1, and MES-SA were treated with varying doses of the DNA Methyltransferase Inhibitors (DNMTi) 5-azacitidine (Aza), 5-aza-2-deoxycytidine (DAC), and guadecitabine (SGI-110). The effect of these epigenetic modulators was measured using both in-vitro and in-vivo models. Of the three epigenetic modulators, Guadecitabine was the most effective at decreasing cell survival in LMS cell lines. SK-UT1 was found to be the more sensitive to all three epigenetic modulators, while SK-LMS1 and MES-SA were more resistant. The contrast in sensitivity seen was also represented by the increase in apoptosis in Aza and guadecitabine. In parallel with Aza, guadecitabine was observed to also arrest the cell cycle. Treatment with guadecitabine led to a decrease in growth across the spectrum of sensitivity in LMS cell lines, both in a delayed in vitro and in vivo model; in parallel experiments, apoptotic pathways were activated in sensitive and less sensitive lines. Additional studies are required to explore potential therapeutic applications and mechanisms for leiomyosarcoma treatment.

A novel epigenetic modulating agent sensitizes pancreatic cells to a chemotherapy agent

BackgroundMany bacterial genome sequences completed using the Sanger method may contain assembly errors due in-part to low sequence coverage driven by cost.FindingsTo illustrate the need for re-sequencing of pre-nextgen genomes and to validate sequenced genomes, we conducted a series of experiments, using high coverage sequencing data generated by a Illumina Miseq sequencer to sequence genomic DNAs of Bacteroides fragilis NCTC 9343, Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150, Vibrio cholerae O1 biovar El Tor str. N16961, Bacillus halodurans C-125 and Caulobacter crescentus CB15, which had previously been sequenced by the Sanger method during the early 2000’s.ConclusionsThis study revealed a number of discrepancies between the published assemblies and sequence read alignments for all five bacterial species, suggesting that the continued use of these error-containing genomes and their genetic information may contribute to false conclusions and/or incorrect future discoveries when they are used.

Microsatellite genotyping reveals a signature in breast cancer exomes

Pancreatic ductal adenocarcinoma (PDAC) is expected to be the second leading cause of cancer mortality by 2030. PDAC remains resistant to the majority of systemic chemotherapies. In this paper, we explore if epigenetic sensitization can improve chemotherapy response in PDAC. Multiple PDAC cell lines were tested with serial concentrations of the epigenetic modulators 5-azacitidine (Aza) and guadecitabine (SGI-110). Guadecitabine was effective at inhibiting the expression of DNA Methyltransferase 1 (DNMT1) and in decreasing cell viability at nanomolar concentrations. We also report that guadecitabine has increased efficacy following a delay period or as we reference, a ‘rest period’. Sensitization with guadecitabine improved response to the chemotherapeutic agent–Irinotecan- as measured by decreased cell viability and accompanied by an increase in caspase activity. Additional studies are needed to understand the mechanism of action.