Generating Phenotypic Diversity in a Fungal Biocatalyst to Investigate Alcohol Stress Tolerance Encountered during Microbial Cellulosic Biofuel Production

peer-reviewedConsolidated bioprocessing (CBP) of lignocellulosic biomass offers an alternative route to renewable energy. The crop pathogen Fusarium oxysporum is a promising fungal biocatalyst because of its broad host range and innate ability to co-saccharify and ferment lignocellulose to bioethanol. A major challenge for cellulolytic CBP-enabling microbes is alcohol inhibition. This research tested the hypothesis that Agrobacterium tumefaciens - mediated transformation (ATMT) could be exploited as a tool to generate phenotypic diversity in F. oxysporum to investigate alcohol stress tolerance encountered during CBP. A random mutagenesis library of gene disruption transformants (n=1,563) was constructed and screened for alcohol tolerance in order to isolate alcohol sensitive or tolerant phenotypes. Following three rounds of screening, exposure of select transformants to 6% ethanol and 0.75% n-butanol resulted respectively in increased (≥11.74%) and decreased (≤43.01%) growth compared to the wild –type (WT). Principal component analysis (PCA) quantified the level of phenotypic diversity across the population of genetically transformed individuals and isolated candidate strains for analysis. Characterisation of one strain, Tr. 259, ascertained a reduced growth phenotype under alcohol stress relative to WT and indicated the disruption of a coding region homologous to a putative sugar transporter (FOXG_09625). Quantitative PCR (RT-PCR) showed FOXG_09625 was differentially expressed in Tr. 259 compared to WT during alcohol-induced stress (P<0.05). Phylogenetic analysis of putative sugar transporters suggests diverse functional roles in F. oxysporum and other filamentous fungi compared to yeast for which sugar transporters form part of a relatively conserved family. This study has confirmed the potential of ATMT coupled with a phenotypic screening program to select for genetic variation induced in response to alcohol stress. This research represents a first step in the investigation of alcohol tolerance in F. oxysporum and has resulted in the identification of several novel strains, which will be of benefit to future biofuel research.Funding provided through the Irish Department of Agriculture and Food's Research Stimulus Fund (Project Code RSF 07 513

Introducing new diagnostics into STI control programmes: the importance of programme science.

Many innovative diagnostic technologies will become commercially available over the next 5-10 years. These tests can potentially transform the diagnosis of sexually transmitted infections but their introduction into control programmes can be hampered by health system constraints, and political, cultural, socioeconomic and behavioural factors. We used the introduction of syphilis rapid tests to illustrate the importance of programme science to address the gap between accruing evidence of acceptable test performance and the complexity of programme design, implementation and evaluation of test deployment to address public health needs and improve patient-important outcomes

Understanding the role of chromatin remodeling in the regulation of circadian transcription in Drosophila.

Circadian clocks enable organisms to anticipate daily changes in the environment and coordinate temporal rhythms in physiology and behavior with the 24-h day-night cycle. The robust cycling of circadian gene expression is critical for proper timekeeping, and is regulated by transcription factor binding, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Recently, it has become clear that dynamic alterations in chromatin landscape at the level of histone posttranslational modification and nucleosome density facilitate rhythms in transcription factor recruitment and RNAPII activity, and are essential for progression through activating and repressive phases of circadian transcription. Here, we discuss the characterization of the BRAHMA (BRM) chromatin-remodeling protein in Drosophila in the context of circadian clock regulation. By dissecting its catalytic vs. non-catalytic activities, we propose a model in which the non-catalytic activity of BRM functions to recruit repressive factors to limit the transcriptional output of CLOCK (CLK) during the active phase of circadian transcription, while the primary function of the ATP-dependent catalytic activity is to tune and prevent over-recruitment of negative regulators by increasing nucleosome density. Finally, we divulge ongoing efforts and investigative directions toward a deeper mechanistic understanding of transcriptional regulation of circadian gene expression at the chromatin level

‘Role and Ritual: Function and Performance in Domestic Spaces of Elite Eighteenth-Century English Houses, 1750-1800’

This thesis offers an, albeit necessary brief, historical analysis of the eighteenth-century incorporation of ritual practises and sociabilities enacted in a few elite interior environments of what I consider to be six of the most interesting English houses, either newly constructed or extended during the course of the eighteenth century. Two of which houses are now demolished in whole, as is the case with Bulstrode Park, situated in Gerrard’s Cross, Buckinghamshire, or part, as is the case with Norfolk House, located in St. James’s Square, central London. It is useful to note that before the start of the research for this thesis began it was assumed on the part of the author that eighteenth-century domestic space were increasingly segmented for inflexible and separate activities. There follows a focused discussion of the varying types of display, ritual and sociable interaction made manifest in; the ground floor spaces of the Entrance Hall, Library, State Bedchamber and Etruscan Dressing Room at Osterley House and Great Hall and Long Gallery of Syon House; the Music and Ball Room of Norfolk House; and, finally, the first floor Dressing Rooms of Bulstrode Park, No. 23 Hill Street and Montagu House in Portman Square. Discussion has a concern to establish the ways in which such material display and sociabilities may have served their owners for a variety of ends. Such interior (and exterior) commissions of assimilated materiality and resulting sociabilities are found to serve their male householder, or as is discovered, female head of house, in a number of different, distinctive and often adaptable ways. Analysis of the specific spatial environments in each elite household discussed reveals that different types of social rituals were often adopted depending upon the individual interests of a patron, their gender and nature of relationship to the head of household. Often divergent strands of cultural influence overlapping forms of ritual practise were incorporated in modernised, areas within a grand household, like that of Bulstrode Park’s Dressing Room, which could be tailored to a surprising number of functions and usages, according to the nature of the relationship with the householder. It also emerges, somewhat surprisingly, that strong and educated female patrons included in this thesis incorporated most successfully different cultural forms of practise and unusual methodologies of aesthetic display within the most private spatial areas in their residences. Again, illustrated most notably with the case of Bulstrode Park, that enabled these women to effectively, and creatively, portray a memorable image and identity individual to themselves

The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila.

Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription

Insurance impacts survival for children, adolescents, and young adults with bone and soft tissue sarcomas.

BackgroundWhile racial/ethnic survival disparities have been described in pediatric oncology, the impact of income has not been extensively explored. We analyzed how public insurance influences 5-year overall survival (OS) in young patients with sarcomas.MethodsThe University of California San Francisco Cancer Registry was used to identify patients aged 0-39 diagnosed with bone or soft tissue sarcomas between 2000 and 2015. Low-income patients were defined as those with no insurance or Medicaid, a means-tested form of public insurance. Survival curves were computed using the Kaplan-Meier method and compared using log-rank tests and Cox models. Causal mediation was used to assess whether the association between public insurance and mortality is mediated by metastatic disease.ResultsOf 1106 patients, 39% patients were classified as low-income. Low-income patients were more likely to be racial/ethnic minorities and to present with metastatic disease (OR 1.96, 95% CI 1.35-2.86). Low-income patients had significantly worse OS (61% vs 71%). Age at diagnosis and extent of disease at diagnosis were also independent predictors of OS. When stratified by extent of disease, low-income patients consistently had significantly worse OS (localized: 78% vs 84%, regional: 64% vs 73%, metastatic: 23% vs 30%, respectively). Mediation analysis indicated that metastatic disease at diagnosis mediated 15% of the effect of public insurance on OS.ConclusionsLow-income patients with bone and soft tissue sarcomas had decreased OS regardless of disease stage at presentation. The mechanism by which insurance status impacts survival requires additional investigation, but may be through reduced access to care

Postmastectomy Breast Reconstruction With the Totally Autologous Latissimus Dorsi Flap in the Thin, Small-Breasted Woman: Give It More Thought!

Introduction: Thin women have fewer autologous tissue breast reconstructive options than their higher body mass index counterparts—due to a lack of adequate donor sites. They are therefore usually offered expander/implant techniques. The total autologous latissimus dorsi flap is generally used in “well-padded” individuals, as they have enough fat on their back on which a completely autologous reconstruction could be based. When implant-based reconstruction is contraindicated (for instance due to planned adjuvant radiotherapy) or unacceptable to the patient, the total autologous latissimus dorsi flap can provide adequate tissue volume by utilizing the additional back fat deposits even in the thin, small-breasted patient. This option is often overlooked by many surgeons. Our case series assesses indications and patient and surgeon satisfaction with the cosmetic outcome of this technique. Methods: The oncological and clinical details of 6 patients with breast cancer who underwent total autologous latissimus dorsi myocutaneous flap immediate breast reconstruction by a single surgeon over an 8-year period were reviewed. An objective assessment of satisfaction with the cosmetic result was made by whether any additional surgical interventions (ipsilateral fat grafting/implant augmentation or contralateral liposuction/ reduction) were needed or not. A subjective assessment of breast symmetry by the surgeon using photographic records was also undertaken. The aesthetic outcomes were also objectively quantified using the BCCT.core software, initially developed for assessing the results of breast conservation surgery. Results: All 6 patients had small breasts and a low or normal body mass index. The mastectomies were performed for invasive carcinoma (n = 3) and extensive high-grade ductal carcinoma in situ (n = 3). Four had axillary surgery (2 sentinel lymph node biopsies and 2 axillary clearances), and 3 received adjuvant radiotherapy. All were happy with their reconstructive outcomes, and none suffered major postoperative complications or disease recurrence. None requested or needed any subsequent ipsilateral adjustment or contralateral symmetrizing procedures. Subjectively, the reconstructions provided acceptable or excellent cosmetic results. The cosmetic results were categorized as excellent or good on the BCCT.core scoring system. Conclusion: This underutilized method of totally autologous breast reconstruction in thinner patients with lower body mass indexes yielded good, well-accepted cosmetic results without recourse to adjustment procedures, contralateral balancing surgery, or complex microvascular surgery. We recommend that the total autologous latissimus dorsi flap should be given more consideration when planning immediate breast reconstruction in this challenging group of thin, small-breasted patients

Edge-based compartmental modelling of an SIR epidemic on a dual-layer static-dynamic multiplex network with tunable clustering

The duration, type and structure of connections between individuals in real-world populations play a crucial role in how diseases invade and spread. Here, we incorporate the aforementioned heterogeneities into a model by considering a dual-layer static-dynamic multiplex network. The static network layer affords tunable clustering and describes an individual’s permanent community structure. The dynamic network layer describes the transient connections an individual makes with members of the wider population by imposing constant edge rewiring. We follow the edge-based compartmental modelling approach to derive equations describing the evolution of a susceptible - infected - recovered (SIR) epidemic spreading through this multiplex network of individuals. We derive the basic reproduction number, measuring the expected number of new infectious cases caused by a single infectious individual in an otherwise susceptible population. We validate model equations by showing convergence to pre-existing edge-based compartmental model equations in limiting cases and by comparison with stochastically simulated epidemics. We explore the effects of altering model parameters and multiplex network attributes on resultant epidemic dynamics. We validate the basic reproduction number by plotting its value against associated final epidemic sizes measured from simulation and predicted by model equations for a number of setups. Further, we explore the effect of varying individual model parameters on the basic reproduction number. We conclude with a discussion of the significance and interpretation of the model and its relation to existing research literature. We highlight intrinsic limitations and potential extensions of the present model and outline future research considerations, both experimental and theoretical