Biology subjects

Carter, D. A.

Publications and source records attributed to Carter, D. A..

8 recordsLinked to original sources

bioRxiv2024

Bisphosphonates synergistically enhance the antifungal activity of azoles in dermatophytes and other pathogenic molds

Superficial infections of the skin, hair and nails by fungal dermatophytes are the most prevalent of human mycoses, and many infections are refractory to treatment. As current treatment options are limited, recent research has explored drug synergy with azoles for dermatophytoses. Bisphosphonates, which are approved to treat osteoporosis, can synergistically enhance the activity of azoles in diverse yeast pathogens but their activity has not been explored in dermatophytes or other molds. Market bisphosphonates risedronate, alendronate and zoledronate (ZOL) were evaluated for antifungal efficacy and synergy with three azole antifungals: fluconazole (FLC), itraconazole (ITR), and ketoconazole (KET). ZOL was the most active bisphosphonate tested, displaying moderate activity against nine dermatophyte species (MIC range 64-256 {micro}g/mL), and was synergistic with KET in 88.9% of these species. ZOL was also able to synergistically improve the anti-biofilm activity of KET and combining KET and ZOL prevented the development of antifungal resistance. Rescue assays in Trichophyton rubrum revealed that the inhibitory effects of ZOL alone and in combination with KET were due to the inhibition of squalene synthesis. Fluorescence microscopy using membrane- and ROS-sensitive probes demonstrated that ZOL and KET:ZOL compromised membrane structure and induced oxidative stress. Antifungal activity and synergy between bisphosphonates and azoles were also observed in other clinically relevant molds, including species of Aspergillus and Mucor. These findings indicate that repurposing bisphosphonates as antifungals is a promising strategy for revitalising certain azoles as topical antifungals, and that this combination could be fast-tracked for investigation in clinical trials. ImportanceFungal infections of the skin hair and nails, generally grouped together as "tineas" are the most prevalent infectious disease globally. These infections, caused by fungal species known as dermatophytes, are generally superficial, but can in some cases become aggressive. They are also notoriously difficult to resolve, with few effective treatments and rising levels of drug resistance. Here we report a potential new treatment that combines azole antifungals with bisphosphonates. Bisphosphonates are approved for the treatment of low bone density diseases, and in fungi they inhibit the biosynthesis of the cell membrane, which is also the target of azoles. Combinations were synergistic across the dermatophyte species and prevented the development of resistance. We extended the study to molds that cause invasive disease, finding synergy in some problematic species. We suggest bisphosphonates could be repurposed as synergents for tinea treatment, and that this combination could be fast-tracked for use in clinical therapy.

microbiology↗

bioRxiv2023

An effective sanitizer for fresh produce production: In situ plasma activated water treatment inactivates pathogenic bacteria and maintains the quality of cucurbit fruit

The effect of plasma activated water (PAW) generated with a dielectric barrier discharge diffusor (DBDD) system on microbial load and organoleptic quality of cucamelons was investigated and compared to the established sanitizer, sodium hypochlorite (NaOCl). Pathogenic serotypes of Escherichia coli, Salmonella enterica, and Listeria monocytogenes were inoculated onto the surface of cucamelons (6.5 log CFU g-1) and into the wash water (6 log CFU mL-1). PAW treatment involved 2 minutes in situ with water activated at 1500 Hz and 120 V, and air as the feed gas; NaOCl treatment was a wash with 100 ppm total chlorine; and the control treatment was a wash with tap water. PAW treatment produced a 3 log CFU g-1 reduction of pathogens on the cucamelon surface without negatively impacting quality or shelf life. NaOCl treatment reduced the pathogenic bacteria on the cucamelon surface by 3-4 log CFU g-1, however, this treatment also reduced fruit shelf life and quality. Both systems reduced 6 log CFU ml-1 pathogens in the wash water to below detectable limits. The critical role of superoxide anion radical ({middle dot}O2-) in the antimicrobial power of DBDD-PAW was demonstrated through a scavenger assay, and chemistry modelling confirmed that {middle dot}O2- generation readily occurs in DBDD-PAW generated with the employed settings. Modelling of the physical forces produced during plasma treatment showed that bacteria likely experience strong local electric fields and polarization. We hypothesize that these physical effects synergise with reactive chemical species to produce the acute antimicrobial activity seen with the in situ PAW system. ImportancePlasma activated water (PAW) is an emerging sanitizer in the fresh food industry, where food safety must be achieved without a thermal kill step. Here we demonstrate PAW generated in situ to be a competitive sanitizer technology, providing a significant reduction of pathogenic and spoilage micro-organisms while maintaining the quality and shelf life of the produce item. Our experimental results are supported by modelling of the plasma chemistry and applied physical forces, which show that the system can generate highly reactive superoxide radicals and strong electric fields that combine to produce potent antimicrobial power. In situ PAW has promise in industrial applications as it only requires low power (12 W), tap water and air. Moreover, it does not produce toxic by-products or hazardous effluent waste, making it a sustainable solution for fresh food safety.

microbiology↗

bioRxiv2022

Lineages derived from Cryptococcus neoformans type strain H99 support a link between the capacity to be pleomorphic and virulence

The pathogenic yeast Cryptococcus neoformans causes nearly 200,000 deaths annually in immunocompromised individuals. Cryptococcus cells can undergo substantial morphological change during mammalian infection, including increased capsule and cell size, the release of shed capsule, and the production of titan (> 10 m), micro (< 2 m) and irregular cells. We examined phenotypic variation under conditions designed to simulate in vivo stress in a collection of nine lineages derived from the C. neoformans type strain H99. These lineages are highly genetically similar but have a range of virulence levels. Strains from hypervirulent lineages had a larger average capsule size, greater variation in cell size, and an increased production of micro cells and shed capsule. We tested whether disruption of SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in the hypervirulence of some lineages, might also have a role in the production of morphological variants. Deletion of SGF29 in a lineage with intermediate virulence substantially increased its production of micro cells and released capsule, consistent with a switch to hypervirulence. We further examined SGF29 in a set of 52 clinical isolates and found loss-of-function mutations were significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 was positively correlated with cell and capsule size, suggesting it may also affect Sgf29 function. This study extends the evidence for a link between pleomorphism and virulence in Cryptococcus, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation. IMPORTANCECryptococcosis is a devastating cause of death and disease worldwide. During infection, Cryptococcus cells can undergo substantial changes to their size and shape. In this study, we used a collection C. neoformans strains that are highly genetically similar but possess differing levels of virulence to investigate how morphological variation aligns with virulence. We found hypervirulent strains on average had larger capsules and greater variation in cell size, and also produced more micro cells and shed capsule. These hypervirulent strains possessed a mutation in SGF29, which encodes a component of the SAGA complex involved in epigenetic regulation. Analysis of the SGF29 gene in a set of clinical isolates found strains with loss-of-function mutations were associated with higher patient death rates. The capacity to vary appears to be linked with virulence in Cryptococcus, and this can occur in the absence of genetic variation via epigenetic mechanisms.

microbiology↗

bioRxiv2022

Impaired spatial memory in adult vitamin D deficient BALB/c mice is associated with reductions in spine density, nitric oxide, and neural nitric oxide synthase in the hippocampus

Vitamin D deficiency is prevalent in adults and is associated with cognitive impairment. However, the mechanism by which adult vitamin D (AVD) deficiency affects cognitive function remains unclear. We examined spatial memory impairment in AVD-deficient BALB/c mice and its underlying mechanism by measuring spine density, long term potentiation (LTP), nitric oxide (NO), neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) in the hippocampus. Adult male BALB/c mice were fed a control or vitamin D deficient diet for 20 weeks. Spatial memory performance was measured using an active place avoidance (APA) task, where AVD-deficient mice had reduced latency entering the shock zone compared to controls. We characterised hippocampal spine morphology in the CA1 and dentate gyrus (DG) and made electrophysiological recordings in the hippocampus of behaviourally naive mice to measure LTP. We next measured NO, as well as glutathione, lipid peroxidation and oxidation of protein products and quantified hippocampal immunoreactivity for nNOS and eNOS. Spine morphology analysis revealed a significant reduction in the number of mushroom spines in the CA1 dendrites but not in the DG. There was no effect of diet on LTP. However, hippocampal NO levels were depleted whereas other oxidation markers were unaltered by AVD deficiency. We also showed a reduced nNOS, but not eNOS, immunoreactivity. Finally, vitamin D supplementation for 10 weeks to AVD-deficient mice restored nNOS immunoreactivity to that seen in in control mice. Our results suggest that lower levels of NO, reduced nNOS immunostaining contribute to hippocampal-dependent spatial learning deficits in AVD-deficient mice.

animal behavior and cognition↗

bioRxiv2021

The Antimicrobial Efficacy Of Plasma Activated Water Is Modulated By Reactor Design And Water Composition

Plasma activated water (PAW) contains a cocktail of reactive oxidative species and free radicals and has demonstrated efficacy as a sanitizer for fresh produce, however there is a need for further optimization. The antimicrobial efficacy of PAW produced by a bubble spark discharge (BSD) reactor and a dielectric barrier discharge-diffuser (DBDD) reactor operating at atmospheric conditions with air, discharge frequencies of 500, 1000 and 1500 Hz, and MilliQ and tap water, was investigated with model organisms Listeria innocua and Escherichia coli. Optimal conditions were subsequently employed for pathogenic bacteria Listeria monocytogenes, E. coli and Salmonella enterica. PAW generated with the DBDD reactor reduced more than 6-log CFU of bacteria within 1 minute of treatment. The BSD-PAW, while attaining high CFU reduction was less effective, particularly for L. innocua. Analysis of physicochemical properties revealed BSD-PAW had a greater variety of reactive species than DBDD-PAW. Scavenger assays were employed to specifically sequester reactive species, including the short-lived superoxide ({middle dot}O2-) radical that could not be directly measured in the PAW. This demonstrated a critical role of superoxide for the inactivation of both E. coli and L. innocua by DBDD-PAW, while in BSD-PAW it had a role in L. innocua inactivation only. Overall, this study demonstrates the potential of DBDD-PAW in fresh produce, where there is a need for sterilization while minimizing chemical inputs and residues and maintaining food quality. Highly effective PAW was generated using air as a processing gas and tap water, making this a feasible and cost-effective option. ImportanceThere is a growing demand for fresh food produced with minimal processing, however guaranteeing microbial safety in the absence of a thermal kill step is challenging. Plasma-activated water (PAW) is a promising novel antimicrobial but its use in high-risk applications like the sanitization of fresh produce requires further optimization. This study demonstrated the importance of reactor design in the production of reactive species in PAW with capacity to kill bacteria. Very effective PAW was generated using a dielectric barrier discharge-diffuser (DBDD) system, with antimicrobial activity attributed to the presence of superoxide radicals. The DBBD reactor used air as a processing gas and tap water, highlighting the potential of this approach as a cost-effective and green alternative to chemical treatment methods that are currently used in food decontamination.

microbiology↗

bioRxiv2020

Sotagliflozin, a dual SGLT1/2 inhibitor, improves cardiac outcomes in a mouse model of early heart failure without diabetes

AimsSelective SGLT2 inhibition reduces the risk of worsening heart failure and cardiovascular death in patients with existing heart failure, irrespective of diabetic status. We aimed to investigate the effects of dual SGLT1/2 inhibition, using sotagliflozin, on cardiac outcomes in non-diabetic and diabetic mice with cardiac pressure overload. Methods and ResultsFive-week old male C57BL/6J mice were randomized to receive a high fat diet (HFD; 60% of calories from fat) to induce diabetes or remain on normal diet (ND) for 12 weeks. Transverse aortic constriction (TAC) was then employed to induce cardiac pressure-overload (50% increase in right:left carotid pressure versus sham surgery), resulting in features representative of heart failure with preserved ejection fraction. At five weeks into the dietary protocol, mice were treated for seven weeks by oral gavage once daily with sotagliflozin (10mg/kg body weight) or vehicle (0.1% tween 80). In ND non-diabetic mice, treatment with sotagliflozin attenuated cardiac hypertrophy and histological markers of cardiac fibrosis induced by TAC. These benefits were associated with profound diuresis and glucosuria, without shifts toward whole-body fatty acid utilisation nor increased cardiac ketolysis. In HFD diabetic mice, sotagliflozin did not attenuate cardiac injury induced by TAC. HFD mice had vacuolation of proximal tubular cells, associated with less profound diuresis and glucosuria, which may have compromised drug action and subsequent cardio-protection. ConclusionWe demonstrate the utility of dual SGLT1/2 inhibition in treating heart failure risk factors in the non-diabetic state. Its efficacy in high fat-induced diabetes with proximal tubular damage requires further study.

pharmacology and toxicology↗

bioRxiv2019

Integrated analysis of different non-coding features across the Sox2 locus implicates a diencephalic enhancer in adult brain expression.

SOX2 is a prominent member of the SOX family of transcription factors that has many different functional roles. This pleiotropy is made possible by multiple regulatory mechanisms that direct appropriate spatial and temporal patterns of expression, and therefore action. The current study concerns the mechanisms that determine Sox2 gene expression in the adult mammalian brain, where SOX2 protein is absent in general, but is selectively and abundantly expressed in a majority of neurons within a ventral diencephalic brain structure, the suprachiasmatic nucleus (SCN). In this study, a comparative bioinformatic and biochemical analysis of different adult rat brain regions was conducted in order to identify SCN-selective (immaturity-related) regulatory mechanisms. The approach incorporated an integrated analysis of Sox2 enhancers, CTCF binding sites, and also expression of the Sox2-overlapping, long non-coding (lnc)RNA, Sox2ot. Initial experiments revealed brain region-specific Sox2ot expression (including region-specific novel transcripts), indicating a significant diversity of Sox2ot expression across the adult brain. However, the pattern and abundance of Sox2ot expression in the SCN, relative to selected control areas of the brain, did not indicate an overt relationship to Sox2 gene expression. Furthermore, although multiple individual Sox2ot exon sequences were shown to overlap annotated Sox2 gene enhancers at different sites across the Sox2 locus, again there was no indication of a SCN-specific functional correlation. Further integration with an analysis of selectively-active CTCF sites within the Sox2 locus directed attention to one site with both a prominent peak of activity in immature brain, and proximity to a functionally-characterized, ventral diencephalic, Sox2 enhancer termed U6 (upstream enhancer 6). Ex vivo analysis of the U6-associated CTCF site revealed SCN-selective CTCF binding, and these sequences were both localized within a known (brain region-selective) super-enhancer. Bioinformatic analysis of the U6 enhancer sequence revealed an abundance of consensus sites for the SCN-selective transcription factor LHX1, and over-expression of this factor enhanced the activity of cloned U6 sequence in transfected cells. However, despite this compelling evidence for a molecular mechanism that underlies adult brain expression of SOX2, further analysis of LHX1-SOX2 co-expression in the SCN confounded this view, indicating the presence of other concurrent mechanisms in the different cell populations of the SCN.

neuroscience↗

bioRxiv2018

Phenotypic variability correlates with clinical outcome in Cryptococcus isolates obtained from Botswanan HIV/AIDS patients

Pathogenic species of Cryptococcus cause hundreds of thousands of deaths annually. Considerable phenotypic variation is exhibited during infection, including increased capsule size, capsule shedding, giant cells ([≥] 15 m) and micro cells ([≤] 1 m). We examined 70 clinical isolates of Cryptococcus neoformans and Cryptococcus tetragattii from HIV/AIDS patients in Botswana to determine if the capacity to produce morphological variants was associated with clinical parameters. Isolates were cultured under conditions designed to simulate in vivo stresses. Substantial variation was seen across morphological and clinical data. Giant cells were more common in C. tetragattii, while micro cells and shed capsule occurred in C. neoformans only. Phenotypic variables fell into two groups associated with differing symptoms. The production of \"large\" phenotypes (greater cell and capsule size and giant cells) was associated with higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting these are induced in early-stage infection. \"Small\" phenotypes (micro cells and shed capsule) were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators and positively correlated with intracranial pressure indicators, suggesting they are produced later during infection and may contribute to immune suppression and promote proliferation and dissemination. These trends persisted at the species level, indicating that they were not driven by association with particular Cryptococcus species. Isolates possessing giant cells, micro cells, and shed capsule were rare, but strikingly were associated with patient death (p=0.0165). Our data indicate that pleomorphism is an important driver in Cryptococcus infection.\n\nImportanceCryptococcosis results in hundreds of thousands of deaths annually, predominantly in sub-Saharan Africa. Cryptococcus is an encapsulated yeast, and during infection cells have the capacity for substantial morphological changes, including capsule enlargement and shedding, and variations in cell shape and size. In this study we examined 70 Cryptococcus isolates causing meningitis in HIV/AIDS patients in Botswana in order to look for associations between phenotypic variation and clinical symptoms. Four variant phenotypes were seen across strains: giant cells [≥] 15 m, micro cells [≤] 1 m, shed extracellular capsule, and irregularly shaped cells. We found \"large\" and \"small\" phenotypes were associated with differing disease symptoms, indicating that their production may be important during the disease process. Overall, our study indicates that Cryptococcus strains that can switch on cell types under different situations may be more able to sustain infection and resist the host response.

microbiology↗