Human adenovirus species D type 37 (HAdV-D37) is an important etiologic agent of epidemic keratoconjunctivitis. Annotation of the whole genome revealed an open reading frame (ORF) in the E3 transcription unit predicted to encode a 31.6kDa protein. This ORF, also known as CR1-γ, is predicted to be an integral membrane protein containing N-terminal signal sequence, luminal, transmembrane, and cytoplasmic domains. HAdV-D19 (C), another viral pathogen causing epidemic keratoconjunctivitis, contains an ORF 100% identical to its HAdV-D37 homologue but only 66% identical to other HAdV-D homologues. Kinetics of RNA expression and confirmation of splicing to the adenovirus tripartite leader sequence suggest a role for the protein product of CR1-γ in the late stages of the viral replication cycle. Confocal microscopy is consistent with expression in the cytoplasm. Sequence analysis reveals a hypervariable luminal domain and a conserved cytoplasmic domain. The luminal domain is predicted to contain multiple N-glycosylation sites. The cytoplasmic domain contains a putative protein kinase C phosphorylation site and potential YXXϕ and dileucine (LL) motifs suggesting a potential role in modification of host proteins.

A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

Mammalian genomes can contain thousands of enhancers but only a subset are actively driving gene expression in a given cellular context. Integrated genomic datasets can be harnessed to predict active enhancers. One challenge in integration of large genomic datasets is the increasing heterogeneity: continuous, binary and discrete features may all be relevant. Coupled with the typically small numbers of training examples, semi-supervised approaches for heterogeneous data are needed; however, current enhancer prediction methods are not designed to handle heterogeneous data in the semi-supervised paradigm.

Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.

G-protein coupled receptors (GPCRs) control multiple physiological states by transducing a multitude of extracellular stimuli into the cell via coupling to intra-cellular heterotrimeric G-proteins. Deciphering which G-proteins couple to each of the hundreds of GPCRs present in a typical eukaryotic organism is therefore critical to understand signalling. Here, we present PRECOG (precog.russelllab.org): a web-server for predicting GPCR coupling, which allows users to: (i) predict coupling probabilities for GPCRs to individual G-proteins instead of subfamilies; (ii) visually inspect the protein sequence and structural features that are responsible for a particular coupling; (iii) suggest mutations to rationally design artificial GPCRs with new coupling properties based on predetermined coupling features.

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their in silico molecular docking into segments of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. This is believed to be the target of the erythromycin A derivative, azithromycin, which has mild anti-malarial activity. The erythromycin B derivatives were evaluated on the multi-drug (chloroquine, pyrimethamine, and sulfadoxine)-resistant strain K1 of P. falciparum for asexual growth inhibition on asynchronous culture. The erythromycin B derivatives were identified as active in vitro inhibitors of asexual growth of P. falciparum with low micro-molar IC50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC50 of 68.6 µM, d-erythromycin B 86.8 µM, and erythromycin B 9-oxime 146.0 µM on the multi-drug-resistant K1 of P. falciparum. Based on the molecular docking, it seems that a small number of favourable interactions or the presence of unfavourable interactions of investigated derivatives of erythromycin B with in silico constructed segment from the exit tunnel from the apicoplast of P. falciparum is the reason for their weak in vitro anti-malarial activities.

Exudate production is a natural part of the wound healing process, however levels of exudate need to be appropriately managed to maintain a moist wound environment which supports healing. An overly-exuding wound creates an environment favourable to bacterial growth. In recent years, a significant increase in commercially available superabsorbent dressings have become available which claim to absorb and retain excess exudate and its components. However, the effectiveness of these dressings in sequestering and retaining bacteria and host-derived proteins has not been compared. We have therefore investigated several superabsorbent dressings for their ability to absorb and retain bacteria (Staphylococcus aureus and Pseudomonas aeruginosa), their impact on bacterial viability, and their ability to sequester matrix metalloproteinases (MMP)-2 and 9 over 7 days. Whilst all dressings could sequester bacteria, some dressings internalised bacteria more effectively. There was considerable variation in bacterial viability within the dressings' core, as well as differences in bacterial retention. Some dressings effectively internalised and retained bacteria over time, whereas other dressings retained significantly less. These differences were reflected visually using scanning electron microscopy. Most dressings fully sequestered MMP-2 and 9. These data illustrate differences in the ability of superabsorbent dressings to absorb and retain exudate and its components.

A set of meta-substituted 3-arylisoquinolinones have been identified that show substantial cytotoxicity in breast, liver, lung and colon cancer cell lines; these are up to 700-fold more active than the corresponding para analogues. These compounds were initially proposed as inhibitors of N-ribosyl dihydronicotinamide (NRH): quinone oxidoreductase 2 (NQO2) but were found to be inactive against the enzyme. Instead, COMPARE analysis suggested that 6-fluoro-3-(meta-fluorophenyl)isoquinolin-1(2H)-one (4) could mimic colchicine and interact with microtubules, a recognized target for cancer therapy. Subsequent docking, molecular dynamics simulations, and free energy analysis further suggested that compound 4 bound well into the colchicine-binding pocket of tubulin. Indeed, 4 suppressed tubulin polymerization, caused G2/M cell cycle arrest, and induced apoptosis. Also, 4 inhibited the formation of endothelial cell capillary-like tubes and further disrupted the structure of preestablished tubes; the effects were not observed with para analogue 5. In accordance with this, the computed free energy of binding of 5 to tubulin was lower in magnitude than that for 4 and appeared to arise in part from the inability of the para substituent to occupy a tubulin subpocket, which is possible in the meta orientation. In conclusion, the antiproliferative potential of the novel 3-arylisoquinolinones is markedly influenced by a subtle change in the structure (meta versus para). The meta-substituted isoquinolinone 4 is a microtubule-destabilizing agent with potential tumor-selectivity and antiangiogenic and vascular disrupting features.

The gut has the largest commensal bacterial population in the body and its composition can be impacted by host factors such as production of immunoglobulin A (IgA). Eosinophils in the gut have been implicated in the production of antibacterial factors and maintenance of IgA-secreting plasma cells. We used an eosinophil-deficient mouse (∆dblGATA-1-/- ) and littermate controls to investigate the role of eosinophils in the regulation of the microbiota, with particular emphasis on mucus-resident species in the small and large intestine. We found no differences in IgA production or IgA-expressing plasma cells between naive littermates in the small or large intestine. However, denaturing gel gradient electrophoresis revealed differences in the bacterial communities of the mucus and stools between wild-type mice and ∆dblGATA-1-/- mice, with the greatest separation between the mucus microbial communities. Mucus-resident bacteria in ∆dblGATA-1-/- mice had reduced diversity in the mucus compared with the stools. A quantitative PCR panel of selected bacteria showed that the most significant differences in the microbiota were between mucus-resident bacteria and those in stool, such as the abundance of Clostridiales and Bacteroides. Our data implicate eosinophils in the regulation of the microbiota, especially the bacteria most hyperlocal to the gut barrier. Although we see differences between host genotypes in the overall microbial communities, further work is required to establish specifically which bacteria are different between these groups. Most importantly, the data revealed that the mucus and stool microbiota are discrete communities. Stool analysis alone may be insufficient to comprehensively explore and define the role of the gut microbiota in health and disease.

The use of manuka honey for the topical treatment of wounds has increased worldwide owing to its broad spectrum of activity towards bacteria in both planktonic and biofilm growth modes. Despite this, the potential consequences of bacterial exposure to manuka honey, as may occur during the treatment of chronic wounds, are not fully understood. Here, we describe changes in antimicrobial susceptibility and virulence in a panel of bacteria, including wound isolates, following repeated exposure (ten passages) to sub-inhibitory concentrations of a manuka honey based wound gel. Changes in antibiotic sensitivity above 4-fold were predominantly related to increased vancomycin sensitivity in the staphylococci. Interestingly, Staphylococcus epidermidis displayed phenotypic resistance to erythromycin following passaging, with susceptibility profiles returning to baseline in the absence of further honey exposure. Changes in susceptibility to the tested wound gel were moderate (≤ 1-fold) when compared to the respective parent strain. In sessile communities, increased biofilm eradication concentrations over 4-fold occurred in a wound isolate of Pseudomonas aeruginosa (WIBG 2.2) as evidenced by a 7-fold reduction in gentamicin sensitivity following passaging. With regards to pathogenesis, 4/8 bacteria exhibited enhanced virulence following honey wound gel exposure. In the pseudomonads and S. epidermidis, this occurred in conjunction with increased haemolysis and biofilm formation, whilst P. aeruginosa also exhibited increased pyocyanin production. Where virulence attenuation was noted in a passaged wound isolate of S. aureus (WIBG 1.6), this was concomitant to delayed coagulation and reduced haemolytic potential. Overall, passaging in the presence of a manuka honey wound gel led to changes in antimicrobial sensitivity and virulence that varied between test bacteria.