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.

Adenovirus E1A is the first viral protein expressed during infection. E1A controls critical aspects of downstream viral gene expression and cell cycle deregulation, and its function is thought to be highly conserved among adenoviruses. Various bioinformatics analyses of E1A from 38 human adenoviruses of species D (HAdV-D), including likelihood clade model partitioning, provided highly significant evidence of divergence of HAdV-Ds into two distinct groups for the conserved region 3 (CR3), present only in the E1A 13S isoform. This variance within E1A 13S of HAdV-Ds was not found in any other human adenovirus (HAdV) species. By protein sequence and structural analysis, the zinc finger motif of E1A CR3, previously shown as critical for transcriptional activation, showed the greatest differences. Subsequent codon usage bias analysis revealed substantial divergence in E1A 13S between the two groups of HAdV-Ds, suggesting that these two sub-groups of HAdV-D evolved under different cellular conditions. Hence, HAdV-D E1A embodies a previously unappreciated evolutionary divergence among HAdVs.

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.

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.

Transcriptional enhancers are critical for development and phenotype evolution and are often mutated in disease contexts; however, even in well-studied cell types, the sequence code conferring enhancer activity remains unknown. To examine the enhancer regulatory code for pluripotent stem cells, we identified genomic regions with conserved binding of multiple transcription factors in mouse and human embryonic stem cells (ESCs). Examination of these regions revealed that they contain on average 12.6 conserved transcription factor binding site (TFBS) sequences. Enriched TFBSs are a diverse repertoire of 70 different sequences representing the binding sequences of both known and novel ESC regulators. Using a diverse set of TFBSs from this repertoire was sufficient to construct short synthetic enhancers with activity comparable to native enhancers. Site-directed mutagenesis of conserved TFBSs in endogenous enhancers or TFBS deletion from synthetic sequences revealed a requirement for 10 or more different TFBSs. Furthermore, specific TFBSs, including the POU5F1:SOX2 comotif, are dispensable, despite cobinding the POU5F1 (also known as OCT4), SOX2, and NANOG master regulators of pluripotency. These findings reveal that a TFBS sequence diversity threshold overrides the need for optimized regulatory grammar and individual TFBSs that recruit specific master regulators.

(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.

Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression.

The ongoing endeavors to uncover the link between the prevalent errors in clinical endodontic training and undergraduate education are founded on tentative assumptions. This investigation was aimed at determining if cascade analysis can provide an understanding of the origins and causes of errors and if the sensitivity of student reports to the impact of errors on treatment outcomes can be established.In 2021, a group of investigators from the endodontics department concerned with clinical dental education launched the Study of Endodontic Errors (SEE). Sixty-six undergraduate dental students at one dental teaching hospital submitted anonymous narratives of problems they witnessed in their root canal treatment practices. The reports were examined to determine the sequence of events and the major errors. We kept track of the consequences of treatment outcomes, both as reported by students and as deduced by investigators.In 77% of the narratives, a chain of errors was recorded. The majority of the errors that took place were related to the working length or width of root canals. A substantial portion, 86%, of these errors could have been prevented through a deeper comprehension of the concepts that underlie working length and width. 75% of the errors that initiated cascades involved losing the correct working length. When asked whether the treatment outcome was compromised, students answered affirmatively in 16% of cases in which their narratives described compromised outcomes. Unacceptable outcomes necessitating re-treatment accounted for only 3% of student-reported consequences, but when investigator-inferred consequences were considered, the percentage more than doubled (7%).Cascade analysis of student error narratives is useful in understanding the triggering chain of events, but students provide insufficient information about how treatment outcomes are affected. Misconceptions about working length and width appear to play a significant role in the propagation of procedural errors.

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.

The recent emergence of highly virulent human adenoviruses (HAdVs) with new tissue tropisms underscores the need to determine their ontogeny. Here we report complete high quality genome sequences and analyses for all the previously unsequenced HAdV serotypes (n = 20) within HAdV species D. Analysis of nucleotide sequence variability for these in conjunction with another 40 HAdV prototypes, comprising all seven HAdV species, confirmed the uniquely hypervariable regions within species. The mutation rate among HAdV-Ds was low when compared to other HAdV species. Homologous recombination was identified in at least two of five examined hypervariable regions for every virus, suggesting the evolution of HAdV-Ds has been highly dependent on homologous recombination. Patterns of alternating GC and AT rich motifs correlated well with hypervariable region recombination sites across the HAdV-D genomes, suggesting foci of DNA instability lead to formulaic patterns of homologous recombination and confer agility to adenovirus evolution.

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.

The data presented in this article relates to the manuscript entitled 'Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production', published in the Journal Metabolic Engineering [1]. In the article here, we present data examining the overexpression of the lipid metabolism modifying genes SCD1 and SREBF1 in CHO cells by densitometry of western blots and by using mass spectrometry to investigate the impact on specific lipid species. We also present immunofluorescence data at the protein level upon SCD1 and SREBF1 overexpression. The growth profile data during batch culture of control CHO cells and CHO cells engineered to overexpress SCD1 and SREBF1 during batch culture are also reported. Finally, we report data on the yields of model secretory recombinant proteins produced from control, SCD1 or SREBF1 engineered cells using a transient expression systems.

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.