Temporal Dynamics Of The Skin Microbiome In Disease

The skin is colonized by communities of bacteria, fungi, and viruses, collectively referred to as the skin microbiome. These microbial communities are shaped by the topology and diseases of the skin. Dysbiosis of the cutaneous microbiome has been associated with several ailments of the skin including atopic dermatitis, acne, rosacea, psoriasis, and chronic wounds. However, our understandings of the processes by which these microbes initiate, maintain, or modulate skin diseases is lacking. Moreover, previous research on the topic has largely been limited by cross-sectional study designs, neglecting the natural dynamism of microbial communities. Here we present a comprehensive analysis of the temporal dynamics of the skin microbiome in various diseases. In the first section, we characterize the diversity and dynamics of both bacterial and fungal communities colonizing chronic wounds and its associations with clinical outcomes. In a study of 100 subjects with diabetic foot ulcers, we sampled the wound microbiota in 2-week intervals until healing, amputation of 26 weeks of follow-up. We demonstrate the high levels of community instability in chronic wounds and expose the positive association between wound healing community instability. We also reveal the effect of antibiotic perturbation on the microbiota. The fungal component was found to have associations with various bacteria and clinical outcomes. Our results should inform the design of future studies and provides evidence that microbial dynamics may be an effective biomarker for identifying high-risk ulcers. The second section investigates the body-site specific effects of psoriasis on the skin microbiome and how it responds to therapy. We reveal these patterns in a study of 114 subjects, across 6 body sites, and over 112 weeks of follow-up. The effect of psoriatic lesions was found to be mild and body-site specific. In contrast, ustekinumab treatment was found to induce moderate shifts in microbial composition, including an increase in atypical skin bacteria and inter-individual heterogeneity. These results suggest that the effect of psoriasis lesions is secondary to the effect the broad effects of the immune environment. Together the work presented in this thesis represents a significant advancement in our understanding of the microbial dynamics of the skin and their associations with human health

Bacterial community development in experimental gingivitis.

Current knowledge of the microbial composition of dental plaque in early gingivitis is based largely on microscopy and cultural methods, which do not provide a comprehensive description of oral microbial communities. This study used 454-pyrosequencing of the V1-V3 region of 16S rRNA genes (approximately 500 bp), and bacterial culture, to characterize the composition of plaque during the transition from periodontal health to gingivitis. A total of 20 healthy volunteers abstained from oral hygiene for two weeks, allowing plaque to accumulate and gingivitis to develop. Plaque samples were analyzed at baseline, and after one and two weeks. In addition, plaque samples from 20 chronic periodontitis patients were analyzed for cross-sectional comparison to the experimental gingivitis cohort. All of the healthy volunteers developed gingivitis after two weeks. Pyrosequencing yielded a final total of 344,267 sequences after filtering, with a mean length of 354 bases, that were clustered into an average of 299 species-level Operational Taxonomic Units (OTUs) per sample. Principal coordinates analysis (PCoA) plots revealed significant shifts in the bacterial community structure of plaque as gingivitis was induced, and community diversity increased significantly after two weeks. Changes in the relative abundance of OTUs during the transition from health to gingivitis were correlated to bleeding on probing (BoP) scores and resulted in the identification of new health- and gingivitis-associated taxa. Comparison of the healthy volunteers to the periodontitis patients also confirmed the association of a number of putative periodontal pathogens with chronic periodontitis. Taxa associated with gingivitis included Fusobacterium nucleatum subsp. polymorphum, Lachnospiraceae [G-2] sp. HOT100, Lautropia sp. HOTA94, and Prevotella oulorum, whilst Rothia dentocariosa was associated with periodontal health. Further study of these taxa is warranted and may lead to new therapeutic approaches to prevent periodontal disease.BBSRC Industrial Case Studentship ref no. BB/G01714X/1 in collaboration with GlaxoSmithKline

Epidemiological and Immunological Features of Obesity and SARS-CoV-2

Obesity is a key correlate of severe SARS-CoV-2 outcomes while the role of obesity on risk of SARS-CoV-2 infection, symptom phenotype, and immune response remain poorly defined. We examined data from a prospective SARS-CoV-2 cohort study to address these questions. Serostatus, body mass index, demographics, comorbidities, and prior COVID-19 compatible symptoms were assessed at baseline and serostatus and symptoms monthly thereafter. SARS-CoV-2 immunoassays included an IgG ELISA targeting the spike RBD, multiarray Luminex targeting 20 viral antigens, pseudovirus neutralization, and T cell ELISPOT assays. Our results from a large prospective SARS-CoV-2 cohort study indicate symptom phenotype is strongly influenced by obesity among younger but not older age groups; we did not identify evidence to suggest obese individuals are at higher risk of SARS-CoV-2 infection; and remarkably homogenous immune activity across BMI categories suggests immune protection across these groups may be similar

SARS-CoV-2 antibodies protect against reinfection for at least 6 months in a multicentre seroepidemiological workplace cohort

Identifying the potential for Severe Acute Respiratory Syndrome : Coronavirus 2 (SARS-CoV-2) reinfection is crucial for understanding possible long-term epidemic dynamics. We analysed longitudinal PCR and serological testing data from a prospective cohort of 4,411 United States employees in 4 states between April 2020 and February 2021. We conducted a multivariable logistic regression investigating the association between baseline serological status and subsequent PCR test result in order to calculate an odds ratio for reinfection. We estimated an odds ratio for reinfection ranging from 0.14 (95% CI: 0.019 to 0.63) to 0.28 (95% CI: 0.05 to 1.1), implying that the presence of SARS-CoV-2 antibodies at baseline is associated with around 72% to 86% reduced odds of a subsequent PCR positive test based on our point estimates. This suggests that primary infection with SARS-CoV-2 provides protection against reinfection in the majority of individuals, at least over a 6-month time period. We also highlight 2 major sources of bias and uncertainty to be considered when estimating the relative risk of reinfection, confounders, and the choice of baseline time point and show how to account for both in reinfection analysis

Optimizing virtual reality for visual behavior experiments in tethered walking flies

When shown a rotating grating pattern, Drosophila melanogaster moves in the direction of the pattern. This is known as the optomotor response. It is a robust response and has been observed over multiple experimental conditions. Here, I obtain replicable data of the robust optomotor response of flies on an inexpensive fly-on-ball setup, thus validating the setup. I also characterize the intensity of the stimuli at which flies show optomotor responses on the setup. Characterizing the setup increases the replicability of experiments by having multiple fly-on-ball setups using a low budget. Experimenters have used virtual reality environments to solve neuroscience problems. LED arenas and projectors have been used to show virtual reality environments to study behavior. Here, I used a single tablet to stimulate flies visually and study their responses on the inexpensive setup. I attempted to optimize the virtual reality of two tablets to replicate optomotor experiments on the inexpensive fly-on-ball setup. This increases the field of view of the stimuli on the fly eye and opens up a repertoire of experiments that researchers can do on inexpensive setups to study visual circuitry. In the single tablet setup, I obtained symmetric responses in the case when I placed the tablet in the front and asymmetric responses when I placed it at the side of the fly. When the tablet was placed in the front of the fly, the stimuli were lateral to the fly, while when the tablet was at the side, the stimuli had a component along the anteroposterior axis of the fly. The result of asymmetric responses corroborates previous literature, where it has been shown that flies have a greater optomotor response to stimuli moving from the front-to-back than those moving from the back-to-front. In the two-tablet setup, I obtained unexpected results. I obtained asymmetric responses where I expected symmetric responses and symmetric responses where I expected asymmetric responses. Three plausible factors of the cause of these unexpected results are reflections between the two tablets, differences in brightness levels between the tablets, and a larger distance of the stimulus in the two-tablet case than in the one-tablet case. After minimizing reflections and standardizing brightness levels in both the tablets, I still obtained a few unexpected responses of low magnitude. In order to see if distance of the tablets affected the optomotor responses, I changed the distance between the tablet and the fly in the single tablet case when it is placed in the front. This experiment revealed that optomotor responses decreased when the tablet is placed farther away from the fly.</p

Triterpene-Based Carboxamides Act as Good Inhibitors of Butyrylcholinesterase

A set of overall 40 carboxamides was prepared from five different natural occurring triterpenoids including oleanolic, ursolic, maslinic, betulinic, and platanic acid. All of which were derived from ethylene diamine holding an additional substituent connected to the ethylene diamine group. These derivatives were evaluated regarding their inhibitory activity of the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) employing Ellman&rsquo;s assay. We further determined the type of inhibition and inhibition constants. Carboxamides derived from platanic acid have been shown to be potent and selective BChE inhibitors. Especially the mixed-type inhibitor (3&beta;)-N-(2-pyrrolidin-1-ylethyl)-3-acetyloxy-20-oxo-30-norlupan-28-amide (35) showed a remarkably low Ki of 0.07 &plusmn; 0.01 &micro;M (Ki&prime; = 2.38 &plusmn; 0.48 &micro;M) for the inhibition of BChE

Point-of-care Ultrasound in Morbidity and Mortality Cases in Emergency Medicine: Who Benefits the Most?

Introduction: Point-of-care ultrasound (POCUS) is an essential tool in the timely evaluation of an undifferentiated patient in the emergency department (ED). Our primary objective in this study was to determine the perceived impact of POCUS in high-risk cases presented at emergency medicine (EM) morbidity and mortality (M&amp;M) conferences. Additionally, we sought to identify in which types of patients POCUS might be most useful, and which POCUS applications were considered to be highest yield.Methods: This was a retrospective survey of cases submitted to M&amp;M at an EM residency program that spans two academic EDs, over one academic year. Postgraduate year 4 (PGY) residents who presented M&amp;M cases at departmental sessions were surveyed on perceived impacts of POCUS on individual patient outcomes. We evaluated POCUS use and indications while the POCUS was used.Results: Over the 12-month period, we reviewed 667 cases from 18 M&amp;M sessions by 15 PGY-4 residents and a supervising EM attending physician who chairs the M&amp;M committee. Of these cases, 75 were selected by the M&amp;M committee for review and presentation. POCUS was used in 27% (20/75) of the cases and not used in 73% (55/75). In cases where POCUS was not used, retrospective review determined that if POCUS had been used it would have “likely prevented the M&amp;M” in 45% (25/55). Of these 25 cases, the majority of POCUS applications that could have helped were cardiac (32%, 8/25) and lung (32%, 8/25) ultrasound. POCUS was felt to have greatest potential in identifying missed diagnoses (92%, 23/25), and decreasing the time to diagnosis (92%, 23/25). Patients with cardiopulmonary chief complaints and abnormal vital signs were most likely to benefit. There were seven cases (35%, 7/20, 95% CI 15-59%) in which POCUS was performed and thought to have possibly adversely affected the outcome of the M&amp;M.Conclusion: POCUS was felt to have the potential to reduce or prevent M&amp;M in 45% of cases in which it was not used. Cardiac and lung POCUS were among the most useful applications, especially in patients with cardiopulmonary complaints and in those with abnormal vital signs

Redefining the Chronic-Wound Microbiome: Fungal Communities Are Prevalent, Dynamic, and Associated with Delayed Healing

Chronic nonhealing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial biofilms in the wound bed are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic-wound microbiome have almost exclusively focused on bacteria, omitting what we hypothesize are important fungal contributions to impaired healing and the development of complications. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with poor outcomes, and form mixed fungal-bacterial biofilms. We longitudinally profiled 100, nonhealing diabetic-foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The “mycobiome” was highly heterogeneous over time and between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. The proportions of the phylum Ascomycota were significantly greater (P = 0.015) at the beginning of the study in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while taxa identified as allergenic filamentous fungi were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that interkingdom biofilms formed between yeasts and coisolated bacteria. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clinical outcomes