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.

Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5' to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism.IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota.

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).

In November of 2007 a human adenovirus (HAdV) was isolated from a bronchoalveolar lavage (BAL) sample recovered from a biopsy of an AIDS patient who presented with fever, cough, tachycardia, and expiratory wheezes. To better understand the isolated virus, the genome was sequenced and analyzed using bioinformatic and phylogenomic analysis. The results suggest that this novel virus, which is provisionally named HAdV-D59, may have been created from multiple recombination events. Specifically, the penton, hexon, and fiber genes have high nucleotide identity to HAdV-D19C, HAdV-D25, and HAdV-D56, respectively. Serological results demonstrated that HAdV-D59 has a neutralization profile that is similar yet not identical to that of HAdV-D25. Furthermore, we observed a two-fold difference between the ability of HAdV-D15 and HAdV-D25 to be neutralized by reciprocal antiserum indicating that the two hexon proteins may be more similar in epitopic conformation than previously assumed. In contrast, hexon loops 1 and 2 of HAdV-D15 and HAdV-D25 share 79.13 and 92.56 percent nucleotide identity, respectively. These data suggest that serology and genomics do not always correlate.

Increased levels of serum antibody titers against human adenovirus 36 (HAdV-D36) are associated with human obesity and experimental obesity in laboratory animals. While HAdV-D36 has been studied as an infectious agent implicated in obesity for over a decade, the complete genome sequence and its analysis have yet to be reported. A detailed analysis of the genome sequence of HAdV-D36 may be important to understand its role in obesity. Genomic and bioinformatic comparisons with other HAdVs identified differences that suggested unique functions. Global pairwise genome alignment with all sequenced human adenovirus D (HAdV-D) genomes revealed areas of nonconserved sequences in the hexon, E3 CR1 beta, E3 CR1 gamma, and fiber genes. Phylogenetic analysis of all HAdV-D36 proteins confirmed that this virus belongs to species Human adenovirus D. This genomic analysis of HAdV-D36 provides an important tool for comprehending the role that this unique adenovirus may play in human obesity. Low amino acid sequence identity in the E3 CR1 beta and CR1 gamma genes may suggest distinctive roles for these proteins. Furthermore, the predicted molecular models of the HAdV-D36 fiber protein seem to implicate a unique tissue tropism for HAdV-D36.

Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFkappaB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFkappaB in adenoviral infection of the cornea.

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.

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a method for microglia depletion, with the assumption that it does not affect peripheral immune cells. Here, we show that CSF1R inhibition by PLX5622 indeed affects the myeloid and lymphoid compartments, causes long-term changes in bone marrow-derived macrophages by suppressing interleukin 1β, CD68, and phagocytosis but not CD208, following exposure to endotoxin, and also reduces the population of resident and interstitial macrophages of peritoneum, lung, and liver but not spleen. Thus, small-molecule CSF1R inhibition is not restricted to microglia, causing strong effects on circulating and tissue macrophages that perdure long after cessation of the treatment. Given that peripheral monocytes repopulate the central nervous system after CSF1R inhibition, these changes have practical implications for relevant experimental data.

To describe a novel microporous drug delivery system (DDS) for sustained anti- vascular endothelial growth factor (VEGF) delivery to the eye and to evaluate its efficacy in a corneal injury model.

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.

Studies suggest diurnal patterns of occurrence of some eye conditions. Leveraging new information sources such as web-based search data to learn more about such patterns could improve the understanding of patients' eye-related conditions and well-being, better inform timing of clinical and remote eye care, and improve precision when targeting web-based public health campaigns toward underserved populations.

Gelatin based adhesives have been used in the last decades in different biomedical applications due to the excellent biocompatibility, easy processability, transparency, non-toxicity, and reasonable mechanical properties to mimic the extracellular matrix (ECM). Gelatin adhesives can be easily tuned to gain different viscoelastic and mechanical properties that facilitate its ocular application. We herein grafted glycidyl methacrylate on the gelatin backbone with a simple chemical modification of the precursor, utilizing epoxide ring-opening reactions and visible light-crosslinking. This chemical modification allows the obtaining of an elastic protein-based hydrogel (GELGYM) with excellent biomimetic properties, approaching those of the native tissue. GELGYM can be modulated to be stretched up to 4 times its initial length and withstand high tensile stresses up to 1.95 MPa with compressive strains as high as 80% compared to Gelatin-methacryloyl (GeIMA), the most studied derivative of gelatin used as a bioadhesive. GELGYM is also highly biocompatible and supports cellular adhesion, proliferation, and migration in both 2 and 3-dimensional cell-cultures. These characteristics along with its super adhesion to biological tissues such as cornea, aorta, heart, muscle, kidney, liver, and spleen suggest widespread applications of this hydrogel in many biomedical areas such as transplantation, tissue adhesive, wound dressing, bioprinting, and drug and cell delivery.

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.

Post-keratoplasty infectious keratitis (PKIK) represents a unique clinical entity that often poses significant diagnostic and therapeutic challenges. It carries a high risk of serious complications such as graft rejection and failure, and less commonly endophthalmitis. Topical corticosteroids are often required to reduce the risk of graft rejection but their use in PKIK may act as a double-edged sword, particularly in fungal infection. The increased uptake in lamellar keratoplasty in the recent years has also led to complications such as graft-host interface infectious keratitis (IIK), which is particularly difficult to manage. The reported incidence of PKIK differs considerably across different countries, with a higher incidence observed in developing countries (9.2-11.9%) than developed countries (0.02-7.9%). Common risk factors for PKIK include the use of topical corticosteroids, suture-related problems, ocular surface diseases and previous corneal infection. PKIK after penetrating keratoplasty or (deep) anterior lamellar keratoplasty is most commonly caused by ocular surface commensals, particularly Gramme-positive bacteria, whereas PKIK after endothelial keratoplasty is usually caused by Candida spp. Empirical broad-spectrum antimicrobial treatment is the mainstay of treatment for both PKIK, though surgical interventions are required in medically refractory cases (during the acute phase) and those affected by visually significant scarring (during the late phase). In this paper, we aim to provide a comprehensive overview on PKIK, encompassing the epidemiology, risk factors, causes, management and outcomes, and to propose a treatment algorithm for systematically managing this challenging condition.

(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.

Three-dimensional (3D) spectral domain optical coherence tomography (OCT) volume scans of the optic nerve head (ONH) and the peripapillary area are useful in the management of glaucoma in patients with a type I or II Boston Keratoprosthesis (KPro).