The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

Background

High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.

Methods

Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.

Results

No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.

Conclusions

Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.     

Landscape Genetics of Schistocephalus solidus Parasites in Threespine Stickleback (Gasterosteus aculeatus) from Alaska

The nature of gene flow in parasites with complex life cycles is poorly understood, particularly when intermediate and definitive hosts have contrasting movement potential. We examined whether the fine-scale population genetic structure of the diphyllobothriidean cestode Schistocephalus solidus reflects the habits of intermediate threespine stickleback hosts or those of its definitive hosts, semi-aquatic piscivorous birds, to better understand complex host-parasite interactions. Seventeen lakes in the Cook Inlet region of south-central Alaska were sampled, including ten in the Matanuska-Susitna Valley, five on the Kenai Peninsula, and two in the Bristol Bay drainage. We analyzed sequence variation across a 759 bp region of the mitochondrial DNA (mtDNA) cytochrome oxidase I region for 1,026 S. solidus individuals sampled from 2009-2012. We also analyzed allelic variation at 8 microsatellite loci for 1,243 individuals. Analysis of mtDNA haplotype and microsatellite genotype variation recovered evidence of significant population genetic structure within S. solidus. Host, location, and year were factors in structuring observed genetic variation. Pairwise measures revealed significant differentiation among lakes, including a pattern of isolation-by-distance. Bayesian analysis identified three distinct genotypic clusters in the study region, little admixture within hosts and lakes, and a shift in genotype frequencies over time. Evidence of fine-scale population structure in S. solidus indicates that movement of its vagile, definitive avian hosts has less influence on gene flow than expected based solely on movement potential. Observed patterns of genetic variation may reflect genetic drift, behaviors of definitive hosts that constrain dispersal, life history of intermediate hosts, and adaptive specificity of S. solidus to intermediate host genotype.     

The uses of Kenyan aloes: an analysis of implications for names,distribution and conservation

Background

The genus Aloe is renowned for its medicinal and cosmetic properties and long history of use. Sixty-three Aloe species occur in Kenya, of which around 50 % are endemic. Several species of aloes are threatened with extinction and knowledge about their use is of major importance for sound conservation strategies. The main aims of this study were to assess the biocultural value of Aloe in Kenya by documenting local uses of aloes and evaluating how the vernacular names reflect the relative importance in different ethnic groups.

Methods

Ethnobotanical and ethnotaxonomical data were collected using field observations and semi-structured interviews. Information was collected by interviewing 63 respondents from nine different ethnic groups, representing different ages, gender and occupations. Statistical analyses were performed using R version 3.1.2.

Results

A total of 19 species of Aloe were found in the study area, of which 16 were used. On the generic level Aloe was easily distinguished. At species level, the local and scientific delimitation were almost identical for frequently used taxa. Aloe secundiflora, with 57 unique use records was the most important species. The two most frequently mentioned Aloe treatments, were malaria and poultry diseases. In our study area neither age nor gender had a significant influence on the level of knowledge of Aloe use. Finally, no correlation was found between extent of use and people’s perception of decrease in local aloe populations. The aloes are highly appreciated and are therefore propagated and transported over large areas when people relocate.

Conclusion

Biocultural value is reflected in the ethnotaxonomy of Aloe in Kenya. Different ethnic groups recognise their most-valued Aloe at the genus level as “the aloe” and add explanatory names for the other species, such as the “spotted aloe” and the “one-legged aloe”. Widespread species of Aloe have the highest number of uses. There is no obvious correlation with high use and decrease in abundance of aloes locally, and we found no compelling evidence for local uses causing devastating damage to populations of the 19 species in use, whereas habitat loss and commercial harvesting appear to be of urgent concern for these important plants.

     

Chemotherapy-Related Amenorrhea and Menopause in Young Chinese Breast Cancer Patients: Analysis on Incidence,Risk Factors and Serum Hormone Profiles

Purpose

In this prospective cross-sectional study on young premenopausal breast cancer patients, the objectives were to: determine the incidences of chemotherapy-related amenorrhea (CRA) and menopause (CRM); identify associated factors; and assess plasma levels of estradiol (E2) and follicular stimulating hormone (FSH) among patients who developed menopause.

Methods

Eligibility criteria include Chinese stage I-III breast cancer patients, premenopausal, age ≤45 at breast cancer diagnosis, having received adjuvant chemotherapy, within 3–10 years after breast cancer diagnosis. Detailed menstrual history prior to and after adjuvant treatment was taken at study entry. Patients’ background demographics, tumor characteristics and anti-cancer treatments were collected. The rates of CRA and CRM were determined. Analysis was conducted to identify factors associated with CRM. For postmenopausal patients, levels of E2 and FSH were analyzed.

Results

286 patients were recruited; the median time from breast cancer diagnosis to study entry was 5.0 years. 255 patients (91.1%) developed CRA. Of these, 66.7% regained menstruation. At the time of study entry, 137 (48.9%) had developed CRM, amongst whom 84 were age ≤45. On multivariate analysis, age was the only associated factor. Among patients with CRM, the median FSH was 41.0 IU/L; this was significantly lower in those who were taking tamoxifen compared to those who were not (20.1 vs. 59.7 IU/L, p<0.0001). The E2 level was <40 pmol/L; there was no difference between those who were still on tamoxifen or not.

Conclusion

After adjuvant chemotherapy, the majority of young Chinese breast cancer patients developed CRA; ~50% developed CRM, with 61% at age ≤45. Age at diagnosis is the only factor associated with CRM. FSH level may be affected by tamoxifen intake.     

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background

Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).

Methods

We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.

Results

Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).

Conclusions

Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.

Key Messages

Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.

Trial Registration

Clinical Trials.gov NCT02284074     

Impact of Diet-Induced Obesity and Testosterone Deficiency on the Cardiovascular System: A Novel Rodent Model Representative of Males with Testosterone-Deficient Metabolic Syndrome (TDMetS)

Introduction

Current models of obesity utilise normogonadic animals and neglect the strong relationships between obesity-associated metabolic syndrome (MetS) and male testosterone deficiency (TD). The joint presentation of these conditions has complex implications for the cardiovascular system that are not well understood. We have characterised and investigated three models in male rats: one of diet-induced obesity with the MetS; a second using orchiectomised rats mimicking TD; and a third combining MetS with TD which we propose is representative of males with testosterone deficiency and the metabolic syndrome (TDMetS).

Methods

Male Wistar rats (n = 24) were randomly assigned to two groups and provided ad libitum access to normal rat chow (CTRL) or a high fat/high sugar/low protein “obesogenic” diet (OGD) for 28 weeks (n = 12/group). These groups were further sub-divided into sham-operated or orchiectomised (ORX) animals to mimic hypogonadism, with and without diet-induced obesity (n = 6/group). Serum lipids, glucose, insulin and sex hormone concentrations were determined. Body composition, cardiovascular structure and function; and myocardial tolerance to ischemia-reperfusion were assessed.

Results

OGD-fed animals had 72% greater fat mass; 2.4-fold greater serum cholesterol; 2.3-fold greater serum triglycerides and 3-fold greater fasting glucose (indicative of diabetes mellitus) compared to CTRLs (all p<0.05). The ORX animals had reduced serum testosterone and left ventricle mass (p<0.05). In addition to the combined differences observed in each of the isolated models, the OGD, ORX and OGD+ORX models each had greater CK-MB levels following in vivo cardiac ischemia-reperfusion insult compared to CTRLs (p<0.05).

Conclusion

Our findings provide evidence to support that the MetS and TD independently impair myocardial tolerance to ischemia-reperfusion. The combined OGD+ORX phenotype described in this study is a novel animal model with associated cardiovascular risk factors and complex myocardial pathology which may be representative of male patients presenting with TDMetS.     

Collagen Promotes Higher Adhesion,Survival and Proliferation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM) proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A) levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.