This study investigates the functional and structural impact of single nucleotide polymorphisms (SNPs) in the enterocin and associated immunity genes of Enterococcus faecium GHB21, a strain known for producing potent antimicrobial peptides. Enterocins, most of them classified as class IIa bacteriocins, exhibit strong activity against pathogens such as Listeria monocytogenes, making them promising candidates for food preservation and therapeutic interventions. Using cloning, sequencing, and bioinformatics tools, we analyzed key enterocin genes (entA, entB and entP) and their associated immunity genes (entAi and entPi). Two novel SNPs were identified that result in amino acid substitutions: G15N in pre-enterocin P (EntP), located within the leader sequence, and V36I in the EntPi immunity protein. Additionally, the V9I mutation within the conserved YGNGV motif of the mature EntP peptide and the G48S mutation in the EntAi immunity protein were analyzed. Protein Variation Effect Analyzer classified all mutations as neutral, indicating minimal disruption to protein function. DynaMut analysis revealed that V9I stabilizes EntP but slightly reduces its flexibility, potentially influencing its interaction with target bacteria. Despite these mutations, the enterocins retained critical structural features, including disulfide bonds and β-sheet arrangements, ensuring their antimicrobial efficacy. These findings underscore the structural resilience of enterocins, supporting their application in food safety and in combating multidrug-resistant pathogens.

Lipopolysaccharide (LPS) is a glycolipid found uniquely in the outer membrane of diderm bacteria, formed of 4-7 acyl chains covalently linked to an extended polysaccharide chain. While a few examples of the interaction between LPS and outer membrane proteins (OMPs) have been structurally characterised, either experimentally or computationally, the precise nature of LPS-OMP interactions and their functional consequences remains unclear. Here, we show that the addition of LPS facilitated cryoEM structure determination of FusA, a 100 kDa TonB-dependent outer membrane transporter from P. atrosepticum. A 2.8 Å structure combined with molecular dynamics of FusA with different LPS models reveals LPS binding sites with a strong LPS interaction site located adjacent to the β-seam region of the FusA β-barrel. The requirement of lipid binding for successful structure determination indicates a stabilisation of the protein, which in turn suggests a potential method for solving other, small OMPs and membrane proteins. Further, it hints at how LPS may mediate protein conformation and thus how LPS and OMPs can work in concert to maintain a structural and functional OM.

TonB-dependent uptake systems of Gram-negative bacterial pathogens constitute prominent virulence factors, allowing nutrient acquisition, primarily siderophore-bound iron, to cross the highly impermeable outer membrane (OM). Remarkably, the ferredoxin uptake system (Fus) of Pectobacteriaceae , a group of soft rot-inducing plant pathogens, imports an entire folded host protein into the periplasm and extracts its bound iron for growth. The inner membrane protein FusB, a TonB homologue, plays two roles in facilitating ferredoxin import. First, like other TonBs, it remodels the globular plug domain obstructing the lumen of the OM receptor FusA to allow ferredoxin passage. Unusually for a TonB protein, FusB then interacts directly with the FusA-bound ferredoxin substrate to facilitate its transport into the periplasm. In this work, we describe structures of FusB-ferredoxin and homodimeric FusB complexes and determine the key features of the binding interfaces formed by FusB with FusA and ferredoxin. We postulate that under resting conditions FusB exists a homodimer, stabilised by an intermolecular R241-D322 salt bridge. The homodimer dissociates when the “FusB-box” of FusA outcompetes one protomer, and FusA D53 displaces FusB D322. Upon ferredoxin binding, FusB undergoes a structural rearrangement, expanding its β-sheet from three to four strands. In agreement with the proposed sequence of events, ferredoxin binding displaces the receptor (FusA) from FusB with Arg241 forming an intramolecular salt bridge with Asp322 to stabilise the newly formed β-hairpin of FusB. We propose a mechanistic model for ferredoxin import in which FusB Arg241 acts as a molecular switch, and two distinct regions function as interaction hotspots.

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohn’s disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNFα), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

Anticoccidials are an important class of compounds for treating parasitic diseases of a wide variety of animals, especially poultry. 1-(4-Chlorobenzyl)-1H-imidazole-4,5-dicarboxamide (1) has been reported in the patent literature to possess potent anticoccidial activity. In view of the earlier published synthetic process, which relies on expensive imidazole-4,5-dicarboxylic acid (IDA) as a starting material, a potentially more cost-effective process to prepare 1 from diaminomaleonitrile (DAMN) was developed. This article describes the development details for each of the three steps involved in the new process.

Deaths resulting from drug-resistant Gram-negative bacterial infections are a growing public health concern. Pyridone methylsulfone hydroxamic acid LpxC inhibitors, such as 1, are being developed for the treatment of serious Gram-negative infections. Carboxylic acid 2 is a key intermediate in the synthesis of analogs of type 1. The current synthesis of 2 is unsuitable as a manufacturing process due to safety concerns and high cost. Two scalable and potentially lower cost processes have been developed, one based on chromatographic resolution of a novel intermediate and a second based on a classical resolution of the key intermediate 3. The advantages of these new chemical approaches are illustrated in the process details described in this letter.

Objective Risk factors associated with depression in athletes include biological sex, physical pain, history of sport-related concussion (SRC) and sport-type. However, no research has examined how these factors affect the likelihood of depression occurring. Understanding this and communicating this to sportspeople effectively could improve the attitudes they have towards concussion. Design Convenience sample: Participants completed questionnaires on the online survey platform 'Qualtrics.' Setting United Kingdom. Participants 144 participants completed a short survey that collected data on biological sex, physical pain, history of SRC and sport-type as well as the Center for Epidemiological Studies Depression Scale (CESD) which measured depressive symptoms. Outcome Measures A total score ≥16 of 60 on CESD highlights an individual may be experiencing some form of depression. This cut-off was therefore used for logistic regression to create 'depressed' and 'non-depressed' groups. Main Results Logistic regression revealed all four predictors to be significantly associated with depression. The model suggested that individuals that have sustained SRC (OR = 57), experiencing greater physical pain (OR = 1.4), being female (OR = 2.9), and participating in contact sports (OR = 71) were more likely to present depressive symptoms. Conclusions This study highlights the significant risk that SRC and physical pain have to mental health and that engaging in contact sports, where these two factors are highly prevalent increases this risk. Sportspeople should therefore be wary of contact sports, as SRC and physical pain risk is high and subsequent mental health issues are likely and communicating this to athletes is imperative.

Thiophene-2-carbonyl chloride (TCC) is a key raw material in the manufacture of active ingredients for agriculture. A new 3-step process for preparing TCC from commercial thiophene was recently developed that featured a metal-catalyzed aerobic oxidation of 2-acetylthiopehene (AcT). That process utilized pure oxygen in a flammable solvent, acetic acid, at elevated temperatures, which represented a hazardous condition by operating above the limiting oxygen concentration for the solvent and at a temperature above the flashpoint for the solvent. More recently, the same oxidation reaction has been achieved in an autoclave using diluted oxygen. Described in this Letter are details of our progress towards the identification of safe operating conditions for the aerobic oxidation of AcT.

Thiophene-2-carbonyl chloride (TCC) is the key raw material in the preparation of tioxazafen, a proprietary seed treatment that was being developed by Bayer AG as a broad-spectrum nematicide. Current manufacturing routes to TCC suffer from side product formation and significant waste generation, which lead to high manufacturing costs. A new and potentially more cost-effective process to prepare TCC from thiophene has been developed. The key step in the process utilizes homogeneous liquid-phase aerobic oxidation of 2-acetylthiophene (AcT). This article describes development details for each of the three steps involved in the new process.

BACKGROUND:Although no causative microbe has been yet identified or successfully targeted in the treatment of inflammatory bowel disease (IBD), the role of Escherichia coli in the pathogenesis of Crohn's disease has attracted considerable interest. AIM:In this review, we present a literature overview of the interactions between diet and E. coli and other Proteobacteria in the aetiology, outcomes and management of IBD and suggest future research directions. METHODS:An extensive literature search was performed to identify in vitro studies and research in animal models that explored mechanisms by which dietary components can interact with E. coli or Proteobacteria to initiate or propagate gut inflammation. We also explored the effect diet and dietary therapies have on the levels of E. coli or Proteobacteria in patients with IBD. RESULTS:Preclinical data suggest that the Western diet and its components influence the abundance, colonisation and phenotypic behaviour of E. coli in the gut, which may in turn initiate or contribute to gut inflammation. In contrast, the Mediterranean diet and specific dietary fibres may abrogate these effects and protect from inflammation. There are limited data from clinical trials, mostly from patients with Crohn's disease during treatment with exclusive enteral nutrition, with findings often challenging observations from preclinical research. Data from patients with ulcerative colitis are sparse. CONCLUSIONS:Preclinical and some clinical trial data suggest that E. coli and other Proteobacteria interact with certain dietary components to promote gut inflammation. Well-designed clinical trials are required before dietary recommendations for disease management can be made.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow-spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energize their transport into cells and catalyze their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Student athletes are a unique group that balances sporting and academic prowess with a reduction in social support when moving away from home. Thus, many studies indicate that student athletes may experience higher depressive symptoms compared to the general public, with sex, pain, and injury status reported to influence this. Therefore, this study aimed to identify whether sex, injury or pain predicts elevated depressive symptoms among student athletes and whether there are interactions between these factors. Depression scores were measured using the Center for Epidemiological Studies Depression Scale (CESD). Of 130 participants, 43% displayed depressive symptoms recorded as scoring >= 16 on the CESD, which is much higher than what is seen in the general public. A multiple linear regression revealed that sex (p = .022) and physical pain (p = .015) explained almost 9% of the variance in depressive symptoms (p = .022). Women were found to be more susceptible to experiencing depressive symptoms and there was a significant interaction between sex and pain (p = .002). Injury was not found to be a significant predictor (p = .566). Therefore, it is important to be aware of physical pain levels in women student athletes to protect their mental health regardless of whether they are injured or not. Improving our knowledge in this area can improve the support given towards student athletes that are experiencing physical pain. This may provide sports coaches with a greater insight into the well-being of their athletes and lead to improved athlete support.

Risk factors associated with depression in athletes include biological sex, physical pain, and history of sport-related concussion (SRC). Due to the well-documented benefits of sport and physical activity on mental health, athletes and non-athletes were recruited to assess any differences. Beyond this, athletes were also grouped by sport-type (contact/non-contact sports) due to the increased prevalence of pain and SRC in contact sports. To our knowledge, there has been no research on how these factors influence the likelihood of depression. In the current study, 144 participants completed a short survey on the above factors and the Center for Epidemiological Studies Depression Scale. Sixty-two of these reported a history of concussion. Logistic regression revealed all the above predictors to be significantly associated with the depression scale. Individuals that had previously sustained SRC, were experiencing greater physical pain and females were more likely to display poor mental health. However, we provide further evidence for the benefits of engaging in sport and physical activity as those that took part in sport were less likely to report depression. Therefore, this study provides a simple risk metric whereby sportspeople can make a better informed choice of their sporting participation, making their own cost/reward judgement.

Much of the present literature suggests that sport-related concussion (SRC) can lead to poor mental health, impaired cognition, and reduced quality of life. However, physical pain has also been reported to have the same influence on these three outcomes, and while SRC and pain often accompany each other, it is surprising that little research assesses the two simultaneously. Therefore, the present study aimed to further investigate the relationship the two factors have on the three outcomes. Depression was measured using the Center for Epidemiological Studies, anxiety was assessed using the State-Trait Anxiety Inventory while the SF-12 recorded quality of life. A trail-making test assessed cognitive flexibility of participants. Data analysis of 84 participants (Concussed 45 vs. 39 Non-concussed) revealed that (i) SRC history was related to reduced accuracy in the cognitive flexibility task but had no bearing on mental health symptoms and quality of life scores, and (ii) physical pain was not related to cognitive flexibility but was responsible for poorer mental health and lower quality of life. This study, like no other to our knowledge, highlights the different influence that SRC and pain have on mental health, cognition, and quality of life. With the knowledge that concussion appears to be more responsible for impaired cognition whereas physical pain is associated with poorer mental health and reduced quality of life, we are better placed to predict the outcome of these events and support athletes that have sustained SRC, are experiencing physical pain, or both.

The α7 nicotinic acetylcholine receptor is a calcium permeable, ligand-gated ion channel that modulates synaptic transmission in the hippocampus, thalamus, and cerebral cortex. Previously disclosed work described PNU-120596 that acts as a powerful positive allosteric modulator of the α7 nicotinic acetylcholine receptor. The initial structure–activity relationships around PNU-120596 were gleaned from screening a large thiazole library. Independent systematic examination of the aryl and heteroaryl groups resulted in compounds with enhanced potency and improved physico-chemical properties culminating in the identification of 16 (PHA-758454). In the presence of acetylcholine, 16 enhanced evoked currents in rat hippocampal neurons. In a rat model of impaired sensory gating, treatment with 16 led to a reversal of the gating deficit in a dose-dependent manner. These results demonstrate that aryl heteroaryl ureas, like compound 16, may be useful tools for continued exploration of the unique biology of the α7 nicotinic acetylcholine receptor.

Aim: Major challenges to islet transplantation in Type 1 diabetes include host-inflammation, which results in failure to maintain survival and functions of transplanted islets. Therefore, this study investigated the applications of encapsulating the bile acid ursodeoxycholic acid (UDCA) with transplanted islets within improved nano-gel systems for Type 1 diabetes treatment. Materials & methods: Islets were harvested from healthy mice, encapsulated using UDCA-nano gel and transplanted into the diabetic mice, while the control group was transplanted encapsulated islets without UDCA. The two groups' survival plot, blood glucose, and inflammation and bile acid profiles were analyzed. Results & conclusion: UDCA-nano gel enhanced survival, glycemia and normalized bile acids' profile, which suggests improved islets functions and potential adjunct treatment for insulin therapy.

Background / Aim: Metformin is widely used in type 2 diabetes and exhibits many positive biological effects on pancreatic b-cells and muscle cells, such as supporting insulin release by b-cells and glucose uptake by muscle cells and reducing oxidative stress, particularly due to diabetes-associated hyperglycaemia. Interestingly, for type 1 diabetes, transplantation of healthy b-cells has been proposed as a novel way to replace insulin therapy. Recently, bile acid-formulations containing transplantable b-cells showed best stability. Hence, this study aimed to explore the effects of metformin-bile acid formulations in b-cell encapsulation and on the biological activities of b-cells and muscle-cells. Methods: Two sets of biological effects were examined, using metformin-bile acid formulations, on encapsulated b-cells and on muscle cells exposed to the formulations. Results: Various encapsulated b-cell formulations' cell viability, insulin levels, cellular oxidative stress, cellular inflammatory profile and bioenergetics at the normoand hyperglycaemic states showed differing results based upon the metformin concentration and the inclusion or absence of bile acid. Similar effects were observed with muscle cells. Low ratios of metformin and bile acids showed best biological effects, suggesting a formulation dependent result. The formulations' positive effects were more profound at the hyperglycaemic state suggesting efficient cell protective effects. Conclusion: Overall, metformin had positive impacts on the cells in a concentration-dependent manner, with the addition of chenodeoxycholic acid further improving results.

Bile acids (BA)s are known surfactants and well-documented to play a major role in food digestion and absorption. Recently, potential endocrinological and formulation-stabilisation effects of BAs have been explored and their pharmacological effects on supporting cell survival and functions have gained wide interest. Hence, this study aimed to explore the hyper-glycaemic dependent dose-effect of the BA chenodeoxycholic acid (CDCA) when encapsulated with pancreatic β-cells, allowing assessment of CDCA’s impacts when encapsulated. Four different concentrations of the BA were prepared, and viable cells were encapsulated and incubated for 2 days. Multiple analyses were carried out including confocal imaging, glucose-induced cellular mitochondrial viability indices, insulin production, inflammatory biomarker analyses and cellular bioenergetics measurements. There was a significant dose-effect with different concentrations of the BA, affecting cellular viability and antioxidant activities, cell functions and insulin release, inflammatory biomarkers, and cellular-bioenergetics at different oxidative stress levels. The results demonstrate that, when encapsulated, the BA CDCA exerts positive pharmacological effects at the cellular level, and such effects are concentration dependent.

Recent studies in our laboratories have shown promising effects of bile acids in ➀ drug encapsulation for oral targeted delivery (via capsule stabilization) particularly when encapsulated with Eudragit NM30D® and ➁ viable-cell encapsulation and delivery (via supporting cell viability and biological activities, postencapsulation). Accordingly, this study aimed to investigate applications of bile acid-Eudragit NM30D® capsules in viable-cell encapsulation ready for delivery. Mouse-cloned pancreatic β-cell line was cultured and cells encapsulated using bile acid-Eudragit NM30D® capsules, and capsules' images, viability, inflammation, and bioenergetics of encapsulated cells assessed. The capsules' thermal and chemical stability assays were also assessed to ascertain an association between capsules' stability and cellular biological activities. Bile acid-Eudragit NM30D® capsules showed improved cell viability (e.g., F1 < F2 & F8; p < 0.05), insulin, inflammatory profile, and bioenergetics as well as thermal and chemical stability, compared with control. These effects were formulation-dependent and suggest, overall, that changes in ratios of bile acids to Eudragit NM30D® can change the microenvironment of the capsules and subsequent cellular biological activities.