The effect of humoral immunity on the composition of the oral microbiota is less intensively investigated than hygiene and diet, in part due to a lack of simple and robust systems for investigating interactions between salivary immunoglobulins and oral bacteria. Here we report the application of an ex situ method to investigate the specificity of salivary immunoglobulins for salivary bacteria. Saliva collected from six volunteers was separated into immunoglobulin and microbial fractions, and the microbial fractions were then directly exposed to salivary immunoglobulins of "self" and "non-self" origin. Antibody-selected bacteria were separated from their congeners using a magnetic bead system, selective for IgA or IgG isotypes. The positively selected fractions were then characterized using gel-based eubacterial-specific DNA profiling. The eubacterial profiles of positively selected fractions diverged significantly from profiles of whole salivary consortia based on volunteer (P≤ 0.001%) and immunoglobulin origin (P≤ 0.001%), but not immunoglobulin isotype (P = 0.2). DNA profiles of separated microbial fractions were significantly (p≤ 0.05) less diverse than whole salivary consortia and included oral and environmental bacteria. Consortia selected using self immunoglobulins were generally less diverse than those selected with immunoglobulins of non-self origin. Magnetic bead separation facilitated the testing of interactions between salivary antibodies and oral bacteria, showing that these interactions are specific and may reflect differences in recognition by self and non-self immunoglobulins. Further development of this system could improve understanding of the relationship between the oral microbiota and the host immune system and of mechanisms underlying the compositional stability of the oral microbiota.

Prognosis of HPV negative head and neck squamous cell carcinoma (HNSCC) patients remains poor despite surgical and medical advances and inadequacy of predictive and prognostic biomarkers in this type of cancer highlights one of the challenges to successful therapy. Statins, widely used for the treatment of hyperlipidaemia, have been shown to possess anti-tumour effects which were partly attributed to their ability to interfere with metabolic pathways essential in the survival of cancer cells. Here, we have investigated the effect of statins on the metabolic modulation of HNSCC cancers with a vision to predict a personalised anticancer therapy. Although, treatment of tumour-bearing mice with simvastatin did not affect tumour growth, pre-treatment for 2 weeks prior to tumour injection, inhibited tumour growth resulting in strongly increased survival. This was associated with increased expression of the monocarboxylate transporter 1 (MCT1) and a significant reduction in tumour lactate content, suggesting a possible reliance of these tumours on oxidative phosphorylation for survival. Since MCT1 is responsible for the uptake of mitochondrial fuels into the cells, we reasoned that inhibiting it would be beneficial. Interestingly, combination of simvastatin with AZD3965 (MCT1 inhibitor) led to further tumour growth delay as compared to monotherapies, without signs of toxicity. In clinical biopsies, prediagnostic statin therapy was associated with a significantly higher MCT1 expression and was not of prognostic value following conventional chemo-radiotherapy. These findings provide a rationale to investigate the clinical effectiveness of MCT1 inhibition in patients with HNSCC who have been taking lipophilic statins prior to diagnosis.

N-Methyl carbamoylimidazole is a safe and practical alternative to methyl isocyanate for carbamoylation reactions. We have developed a new chemical route for its synthesis from methyl iodide and applied this to the synthesis of N-[11C]methyl carbamoylimidazole as a new [11C]synthon to radiolabel biomolecules for PET imaging research. N-[11C]methyl carbamoylimidazole was prepared from [11C]methyl iodide in 70-74% radiochemical yield (decay corrected) and can be used in situ for further reaction without purification. The reactivity of N-[11C]methyl carbamoylimidazole was demonstrated in a series of [11C]carbamoylation reactions.

Diabetic foot wounds are commonly colonised by taxonomically diverse microbial communities and may additionally be infected with specific pathogens. Since biofilms are demonstrably less susceptible to antimicrobial agents than are planktonic bacteria, and may be present in chronic wounds, there is increasing interest in their aetiological role. In the current investigation, the presence of structured microbial assemblages in chronic diabetic foot wounds is demonstrated using several visualization methods. Debridement samples, collected from the foot wounds of diabetic patients, were histologically sectioned and examined using bright-field, fluorescence, and environmental scanning electron microscopy and assessed by quantitative differential viable counting. All samples (n = 26) harboured bioburdens in excess of 5 log₁₀ CFU/g. Microcolonies were identified in 4/4 samples by all three microscopy methods, although bright-field and fluorescence microscopy were more effective at highlighting putative biofilm morphology than ESEM. Results in this pilot study indicate that bacterial microcolonies and putative biofilm matrix can be visualized in chronic wounds using fluorescence microscopy and ESEM, but also using the simple Gram stain.

NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.

There is a high incidence of abnormal sphenoid sinus changes in patients with pituitary apoplexy (PA). Their pathophysiology is currently unexplored and may reflect an inflammatory or infective process. In this preliminary study, we characterised the microbiota of sphenoid sinus mucosa in patients with PA and compared findings to a control group of surgically treated non-functioning pituitary adenomas (NFPAs).

Staphylococcus aureus can develop a small colony variant (SCV) phenotype in response to sub-lethal exposure to the biocide triclosan. In the current study, whole genome sequencing was performed and changes in virulence were investigated in five Staphylococcus aureus strains following repeated exposure to triclosan. Following exposure, 4/5 formed SCV and exhibited point mutations in the triclosan target gene fabI with 2/4 SCVs showing mutations in both fabI and fabD. The SCV phenotype was in all cases immediately reversed by nutritional supplementation with fatty acids or by repeated growth in the absence of triclosan, although fabI mutations persisted in 3/4 reverted SCVs. Virulence, determined using keratinocyte invasion and Galleria mellonella pathogenicity assays was significantly (p < 0.05) attenuated in 3/4 SCVs and in the non-SCV triclosan-adapted bacterium. Proteomic analysis revealed elevated FabI in 2/3 SCV and down-regulation in a protein associated with virulence in 1/3 SCV. In summary, attenuated keratinocyte invasion and larval virulence in triclosan-induced SCVs was associated with decreases in growth rate and virulence factor expression. Mutation occurred in fabI, which encodes the main triclosan target in all SCVs and the phenotype was reversed by fatty acid supplementation, demonstrating an association between fatty acid metabolism and triclosan-induced SCV.

High expression of Ki67, a proliferation marker, is associated with reduced endometrial cancer-specific survival. Pre-surgical metformin reduces tumour Ki-67 expression in some women with endometrial cancer. Metformin's anti-cancer activity may relate to effects on cellular energy metabolism. Since tumour hypoxia and glucose availability are major cellular redox determinants, we evaluated their role in endometrial cancer response to metformin.

The endemic strain of Leishmania donovani in Sri Lanka causes cutaneous leishmaniasis (CL) rather than more common visceral form. We have visualized biofilms and profiled the microbiome of lesions and unaffected skin in thirty-nine CL patients. Twenty-four lesions (61.5%) were biofilm-positive according to fluorescence in situ hybridization. Biopsies of biofilm-positive lesions were dominated by Pseudomonas, class Bacilli and Enterobacteriaceae and distinguished by significantly lower community evenness. Higher relative abundance of a class Bacilli OTU was detected in wound swabs versus contralateral skin. Wound swabs and biopsies had significantly distinct microbiome profiles and lower diversity compared to unaffected skin. Greater abundances of potentially pathogenic organisms were observed in wet ulcers, lesions with high parasite loads and large wounds. In summary, more than half of L. donovani associated CL wounds harboured biofilms and the wounds exhibited a distinct, less diverse, microbiome than unaffected skin.

(NRH):quinone oxidoreductase 2 (NQO2) is associated with various processes involved in cancer initiation and progression probably via the production of ROS during quinone metabolism. Thus, there is a need to develop inhibitors of NQO2 that are active in vitro and in vivo. As part of a strategy to achieve this we have used the 4-aminoquinoline backbone as a starting point and synthesized 21 novel analogues. The syntheses utilised p-anisidine with Meldrum's acid and trimethyl orthoacetate or trimethyl orthobenzoate to give the 4-hydrazin-quinoline scaffold, which was derivatised with aldehydes or acid chlorides to give hydrazone or hydrazide analogues, respectively. The hydrazones were the most potent inhibitors of NQO2 in cell free systems, some with low nano-molar IC50 values. Structure-activity analysis highlighted the importance of a small substituent at the 2-position of the 4-aminoquinoline ring, to reduce steric hindrance and improve engagement of the scaffold within the NQO2 active site. Cytotoxicity and NQO2-inhibitory activity in vitro was evaluated using ovarian cancer SKOV-3 and TOV-112 cells (expressing high and low levels of NQO2, respectively). Generally, the hydrazones were more toxic than hydrazide analogues and further, toxicity is unrelated to cellular NQO2 activity. Pharmacological inhibition of NQO2 in cells was measured using the toxicity of CB1954 as a surrogate end-point. Both the hydrazone and hydrazide derivatives are functionally active as inhibitors of NQO2 in the cells, but at different inhibitory potency levels. In particular, 4-((2-(6-methoxy-2-methylquinolin-4-yl)hydrazono)methyl)phenol has the greatest potency of any compound yet evaluated (53 nM), which is 50-fold lower than its toxicity IC50. This compound and some of its analogues could serve as useful pharmacological probes to determine the functional role of NQO2 in cancer development and response to therapy.

Cetylpyridinium chloride (CPC) and sodium fluoride augment oral hygiene by inactivating bacteria and inhibiting enamel demineralisation, respectively. However, there are few reports in the literature documenting the antibacterial efficacy of their combined use in mouthrinses. We have used six experimental systems to compare the antibacterial effects of mouthrinses containing 0.075% CPC (test rinse, TR) or 0.075% CPC with sodium fluoride (test fluoride rinse, TFR).

NRH:quinone oxidoreductase 2 enzyme (NQO2) is a potential therapeutic target in cancer and neurodegenerative diseases, with roles in either chemoprevention or chemotherapy. Here we report the design, synthesis and evaluation of non-symmetrical furan-amidines and their analogues as novel selective NQO2 inhibitors with reduced adverse off-target effects, such as binding to DNA. A pathway for the synthesis of the non-symmetrical furan-amidines was established from the corresponding 1,4-diketones. The synthesized non-symmetrical furan-amidines and their analogues showed potent NQO2 inhibition activity with nano-molar IC50 values. The most active compounds were non-symmetrical furan-amidines with meta- and para-nitro substitution on the aromatic ring, with IC50 values of 15 nM. In contrast to the symmetric furan-amidines, which showed potent intercalation in the minor grooves of DNA, the synthesized non-symmetrical furan-amidines showed no affinity towards DNA, as demonstrated by DNA melting temperature experiments. In addition, Plasmodium parasites, which possess their own quinone oxidoreductase PfNDH2, were inhibited by the non-symmetrical furan-amidines, the most active possessing a para-fluoro substituent (IC50 9.6 nM). The high NQO2 inhibition activity and nanomolar antimalarial effect of some of these analogues suggest the lead compounds are worthy of further development and optimization as potential drugs for novel anti-cancer and antimalarial strategies.

Despite the targeted and incidental exposure of the oral cavity to probiotic bacteria, relatively little information is available concerning their bacteriological effects or ecological fate in this environment. We have investigated the effects of an oral probiotic bacterium, Lactobacillus reuteri on salivary biofilm microcosms.

The skin microbiome exists in dynamic equilibrium with the host, but when the skin is compromised, bacteria can colonize the wound and impair wound healing. Thus, the interplay between normal skin microbial interactions versus pathogenic microbial interactions in wound repair is important. Bacteria are recognized by innate host pattern recognition receptors, and we previously showed an important role for the pattern recognition receptor NOD2 in skin wound repair. NOD2 is implicated in changes in the composition of the intestinal microbiota in Crohn's disease, but its role on skin microbiota is unknown. Nod2-deficient (Nod2-/-) mice had an inherently altered skin microbiome compared with wild-type controls. Furthermore, we found that Nod2-/- skin microbiome dominated and caused impaired healing, shown in cross-fostering experiments of wild-type pups with Nod2-/- pups, which then acquired altered cutaneous bacteria and delayed healing. High-throughput sequencing and quantitative real-time PCR showed a significant compositional shift, specifically in the genus Pseudomonas in Nod2-/- mice. To confirm whether Pseudomonas species directly impair wound healing, wild-type mice were infected with Pseudomonas aeruginosa biofilms and, akin to Nod2-/- mice, were found to exhibit a significant delay in wound repair. Collectively, these studies show the importance of the microbial communities in skin wound healing outcome.

Thailand has a comparatively high prevalence of severe early childhood caries (S-ECC). S-ECC adversely affects the quality of life for children and their caregivers and represents a considerable economic burden. We have assessed the bacteriological composition of unstimulated saliva, dental plaque, and degraded dentine in a Thai cohort, including children with S-ECC and children without cavities; their siblings, and their primary caregivers. Samples were collected during a dental examination and patients were scored for plaque accumulation and their decayed, missing, and filled teeth (dmft) index. Samples were analyzed using differential bacteriological counting and gel-based eubacterial DNA profiling. Plaque Lactobacillus abundance correlated significantly with S-ECC. Whilst Lactobacillus counts were significantly higher in children with S-ECC than in their siblings and primary caregivers (five families), the opposite trend was apparent for cavity-free children. Counts of Gram-negative anaerobes were significantly lower in children with S-ECC than orally healthy children. S-ECC correlated significantly with plaque index scores, dmft, and with Lactobacillus abundance in a highly predictive manner. DNA profiles showed significant homology between families but not within non-cavity and S-ECC groups. In conclusion, salivary and plaque Lactobacillus counts were significantly associated with S-ECC in the Thai subjects. Lactobacillus counts in the children were not correlated with those of their siblings and primary caregivers. Individuals could be significantly differentiated based on family but not on caries status.

Rates of random, spontaneous mutation can vary plastically, dependent upon the environment. Such plasticity affects evolutionary trajectories and may be adaptive. We recently identified an inverse plastic association between mutation rate and population density at 1 locus in 1 species of bacterium. It is unknown how widespread this association is, whether it varies among organisms, and what molecular mechanisms of mutagenesis or repair are required for this mutation-rate plasticity. Here, we address all 3 questions. We identify a strong negative association between mutation rate and population density across 70 years of published literature, comprising hundreds of mutation rates estimated using phenotypic markers of mutation (fluctuation tests) from all domains of life and viruses. We test this relationship experimentally, determining that there is indeed density-associated mutation-rate plasticity (DAMP) at multiple loci in both eukaryotes and bacteria, with up to 23-fold lower mutation rates at higher population densities. We find that the degree of plasticity varies, even among closely related organisms. Nonetheless, in each domain tested, DAMP requires proteins scavenging the mutagenic oxidised nucleotide 8-oxo-dGTP. This implies that phenotypic markers give a more precise view of mutation rate than previously believed: having accounted for other known factors affecting mutation rate, controlling for population density can reduce variation in mutation-rate estimates by 93%. Widespread DAMP, which we manipulate genetically in disparate organisms, also provides a novel trait to use in the fight against the evolution of antimicrobial resistance. Such a prevalent environmental association and conserved mechanism suggest that mutation has varied plastically with population density since the early origins of life.

Women with a prior history of polycystic ovary syndrome (PCOS) have an increased risk of endometrial cancer (EC).

Although many antibiotics are active against Gram-positive bacteria, fewer also show activity against Gram-negative bacteria. Here, we present a combination of in silico (electron ion-interaction potential, molecular docking, ADMET), NMR, and microbiological investigations of selected macrolides (14-membered, 15-membered, and 16-membered), aiming to discover the pattern of design for macrolides active against Gram-negative bacteria. Although the conformational studies of 14-membered and 15-membered macrolides are abundant in the literature, 16-membered macrolides, and their most prominent representative tylosin A, have received relatively little research attention. We therefore report the complete 1H and 13C NMR assignment of tylosin A in deuterated chloroform, as well as its 3D solution structure determined through molecular modelling (conformational search) and 2D ROESY NMR. Additionally, due to the degradation of tylosin A in deuterated chloroform, other species were also detected in 1D and 2D NMR spectra. We additionally studied the anti-bacterial activity of tylosin A and B against selected Gram-positive and Gram-negative bacteria.

Microbial growth within a wound often manifests as biofilms, which can prevent healing and is difficult to eradicate. Novel silver dressings claim to combat wound infection, but anti-biofilm efficacy and effects on healing independent of infection are often unclear. Using in vitro and in vivo S. aureus and P. aeruginosa biofilm models, we report the efficacy of a dressing which produces Ag1+ ions; an Ag1+ dressing containing ethylenediaminetetraacetic acid and benzethonium chloride (Ag1+/EDTA/BC), and a dressing containing silver oxynitrate (Ag Oxysalts) which produces Ag1+, Ag2+ and Ag3+ ions, against wound biofilms, and their effects on healing. Ag1+ dressings had minimal effect on in vitro and murine (C57BL/6j) wound biofilms. In contrast, Ag Oxysalts and Ag1+/EDTA/BC dressings significantly reduced viable bacteria within in vitro biofilms and demonstrated a visible reduction in bacteria and EPS components within murine wound biofilms. The dressings had different effects on the healing of biofilm-infected and uninfected wounds, with Ag Oxysalts dressings having a greater beneficial effect on re-epithelialisation, wound size and inflammation than the control treatment and the other silver dressings. The different physicochemical properties of the silver dressings result in varied effects on wound biofilms and healing which should be considered when selecting dressings to treat biofilm-infected wounds.

The use of non-nutritive sweeteners (NNSs) as an alternative to caloric sugars has increased in recent years. Stevia is an NNS that has demonstrated beneficial effects on appetite and energy intake. However, the impact on the gut microbiota is not well understood. Therefore, we investigated how regular consumption of stevia, for up to 12 weeks, impacts the human gut microbiota. Healthy subjects with a normal body mass index participated in our study; the stevia group (n = 14) was asked to consume five drops of stevia twice daily, compared to control participants (n = 13). Faecal samples collected before and after treatment were analysed by 16S rRNA gene sequencing. Stevia did not cause significant changes in the alpha or beta diversity when compared to the control groups. When the relative abundances of taxa were investigated, no clear differences were detected. Conversely, a random forest analysis correctly associated the gut microbiome with the control and stevia groups with an average of 75% accuracy, suggesting that there are intrinsic patterns that could discriminate between control and stevia use. However, large-scale changes in the gut microbiota were not apparent in this study, and, therefore, our data suggest that stevia does not significantly impact the gut microbiota.