Small molecule inhibitors for cancer immunotherapy and associated biomarkers - the current status.

Following the success of cancer immunotherapy using large molecules against immune checkpoint inhibitors, the concept of using small molecules to interfere with intracellular negative regulators of anti-tumor immune responses has emerged in recent years. The main targets for small molecule drugs currently include enzymes of negative feedback loops in signaling pathways of immune cells and proteins that promote immunosuppressive signals within the tumor microenvironment. In the adaptive immune system, negative regulators of T cell receptor signaling (MAP4K1, DGKα/ζ, CBL-B, PTPN2, PTPN22, SHP1), co-receptor signaling (CBL-B) and cytokine signaling (PTPN2) have been preclinically validated as promising targets and initial clinical trials with small molecule inhibitors are underway. To enhance innate anti-tumor immune responses, inhibitory immunomodulation of cGAS/STING has been in the focus, and inhibitors of ENPP1 and TREX1 have reached the clinic. In addition, immunosuppressive signals via adenosine can be counteracted by CD39 and CD73 inhibition, while suppression via intratumoral immunosuppressive prostaglandin E can be targeted by EP2/EP4 antagonists. Here, we present the status of the most promising small molecule drug candidates for cancer immunotherapy, all residing relatively early in development, and the potential of relevant biomarkers.

CD9 co-operation with syndecan-1 is required for a major staphylococcal adhesion pathway.

Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5ß1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of ß1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased ß1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or ß1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and ß1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

Tetraspanin CD9-derived peptides inhibit Pseudomonas aeruginosa corneal infection and aid in wound healing of corneal epithelial cells.

Pseudomonas aeruginosa is a leading cause of corneal infection both within India and globally, often causing a loss of vision. Increasing antimicrobial resistance among the bacteria is making its treatment more difficult. Preventing initial bacterial adherence to the host membrane has been explored here to reduce infection of the cornea. Synthetic peptides derived from human tetraspanin CD9 have been shown to reduce infection in corneal cells both in vitro, ex vivo and in vivo. We found constitutive expression of CD9 in immortalized human corneal epithelial cells by flow cytometry and immunocytochemistry. The synthetic peptides derived from CD9 significantly reduced bacterial adherence to cultured corneal epithelial cells and ex vivo human cadaveric corneas as determined by colony forming units. The peptides also significantly reduced bacterial burden in a murine model of Pseudomonas keratitis and lowered the cellular infiltration in the corneal stroma. Additionally, the peptides aided corneal wound healing in uninfected C57BL/6 mice compared to control mice. These potential therapeutics had no effect on cell viability or proliferation of corneal epithelial cells and have the potential to be developed as an alternative therapeutic intervention.

Variable disruption of epithelial monolayers by Neisseria meningitidis carriage isolates of the hypervirulent MenW cc11 and MenY cc23 lineages.

Colonization of mucosal tissues by Neisseria meningitidis requires adhesion mediated by the type IV pilus and multiple outer-membrane proteins. Penetration of the mucosa and invasion of epithelial cells are thought to contribute to host persistence and invasive disease. Using Calu-3 cell monolayers grown at an air-liquid interface, we examined adhesion, invasion and monolayer disruption by carriage isolates of two clonal complexes of N. meningitidis. Carriage isolates of both the serogroup Y cc23 and the hypervirulent serogroup W cc11 lineages exhibited high levels of cellular adhesion, and a variable disruption phenotype across independent isolates. Inactivation of the gene encoding the main pilus sub-unit in multiple cc11 isolates abrogated both adhesive capacity and ability to disrupt epithelial monolayers. Contrastingly, inactivation of the phase-variable opa or nadA genes reduced adhesion and invasion, but not disruption of monolayer integrity. Adherence of tissue-disruptive meningococci correlated with loss of staining for the tight junction protein, occludin. Intriguingly, in a pilus-negative strain background, we observed compensatory ON switching of opa genes, which facilitated continued adhesion. We conclude that disruption of epithelial monolayers occurs in multiple meningococcal lineages but can vary during carriage and is intimately linked to pilus-mediated adhesion.

Quality of Life After Bariatric and Body Contouring Surgery in the Australian Public Health System.

INTRODUCTION: The goals of bariatric surgery are weight loss, improved management of obesity-related diseases, and enhanced health-related quality of life (HRQoL). The aim of this study is to determine HRQoL among postoperative bariatric surgery patients. The aim of this study was to evaluate the utility of bariatric surgery and the role of body contouring surgery (BCS) when considering quality of life in low-volume centres in the Australian public health system. METHODS: This cohort study compared patients who underwent bariatric surgery between 2008 and 2018, to those awaiting surgery. An additional analysis was completed for patients who also underwent BCS. Patients completed the Short Form-36 quality of life (SF-36) survey. Linear regression was used to assess the differences in mean scores between cohorts for each of the SF-36 domains. RESULTS: A total of 131 postoperative patients were identified, with a follow up rate of 68%. The mean follow up was 5.4 y. The mean scores for all domains of the SF-36 in the postoperative group were higher than the preoperative group (P ≤ 0.0001). A significant difference in scores persisted after controlling for patients' current BMI. When considering patients who underwent BCS (n = 24), there was a further global improvement in HRQoL in physical function (P = 0.0065), role limitation to physical health (P = 0.0026), pain (P = 0.0004), energy (P = 0.0023) and general health perceptions (P = 0.0023). CONCLUSIONS: Bariatric surgery followed by BCS may improve HRQoL for the patient when compared to bariatric surgery alone. We advocate for the use of bariatric surgery followed by BCS in low-volume centres in the Australian public health system.

To wait, or too late? Modeling the effects of delayed ofatumumab treatment in relapsing-remitting multiple sclerosis.

BACKGROUND: Several disease-modifying treatments (DMTs) for relapsing-remitting multiple sclerosis (RRMS) reduce relapse rates and slow disease progression. RRMS DMTs have varying efficacy and administration routes; DMTs prescribed first may not be the most effective on relapses or disease progression. Here, we aimed to quantify the benefit of initiating ofatumumab, a high-efficacy DMT, earlier in the treatment pathway. METHODS: Aggregate data from a real-world cohort of patients with RRMS, who were eligible for dimethyl fumarate (DMF) or ofatumumab treatment within the UK National Health Service (N = 615), were used to produce a simulated patient cohort. The cohort was tracked through a discrete event simulation (DES) model, based on the Expanded Disability Status Scale (EDSS), with a lifetime time horizon. Outcomes assessed were: mean number of relapses, time to wheelchair (EDSS ≥7), and time to death. Two modeling approaches were used. The first compared outcomes between two treatment sequences (base case: ofatumumab to natalizumab versus DMF to ofatumumab). The second incorporated a time-specific delay of 1-5 years for switching from DMF to ofatumumab; the difference in outcomes as a function of increasing delay to ofatumumab are reported. RESULTS: Compared with delayed ofatumumab, fewer relapses and increased time to wheelchair were predicted for earlier ofatumumab in the treatment-sequence approach (mean relapses over the lifetime time horizon: 8.63 versus 9.00; time to wheelchair: 17.55 versus 16.60 years). Time to death was similar for both sequences. At Year 10, a numerically greater proportion of patients receiving earlier ofatumumab had mild disease (EDSS 0-3: 44.12% versus 40.06%). Greater differences, reflecting poorer outcomes, were predicted for relapses and time to wheelchair with increasing delays to ofatumumab treatment. CONCLUSIONS: The DES model provided a means by which the magnitude of benefit associated with earlier ofatumumab initiation could be quantified; fewer relapses and a prolonged time to wheelchair were predicted.

YouTube-Friend or Foe? A Closer Look at Videos on Inguinal Hernia Surgery as a Source for Patient Education.

INTRODUCTION: The Internet is an extensively used source of medical education by the public. In particular, YouTube is a valuable source of information which can be used to improve patient education. However, there is no quality assurance regime for YouTube videos pertaining to medical education. In this study, we aimed to evaluate the quality and accuracy of videos regarding inguinal hernia repair. METHODS: Two hundred videos were searched for and viewed on YouTube from the phrases: 'inguinal hernia repair,' 'patient information for inguinal hernia repair,' and 'hernia operation.' After the application of predefined exclusion criteria, 23 videos were selected and the following data were collected: number of views, duration since video was posted, and the number of likes, dislikes, and comments. The educational quality was rated using three scoring systems: Health on the Net code, Journal of the American Medical Association, and DISCERN scoring systems. All three scoring systems have been previously used to evaluate online videos; however, they have not been formally validated. RESULTS: The videos were of low quality when using the Health on the Net code, Journal of the American Medical Association, and DISCERN scoring systems. There was no association between video quality as measured by any of the scoring systems and the number of views. The number of days online was independently predictive of the number of views (P = 0.044) and explained 18% of the variance in views. Likewise, there was no significant association between video quality and video length. CONCLUSIONS: YouTube videos on inguinal hernia repair are of low quality and accuracy. However, the potential of using YouTube to educate patients cannot be ignored.

Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

Neisseria gonorrhoeae physiology and pathogenesis.

Neisseria gonorrhoeae is an obligate human pathogen that is the cause of the sexually transmitted disease gonorrhoea. Recently, there has been a surge in gonorrhoea cases that has been exacerbated by the rapid rise in gonococcal multidrug resistance to all useful antimicrobials resulting in this organism becoming a significant public health burden. Therefore, there is a clear and present need to understand the organism's biology through its physiology and pathogenesis to help develop new intervention strategies. The gonococcus initially colonises and adheres to host mucosal surfaces utilising a type IV pilus that helps with microcolony formation. Other adhesion strategies include the porin, PorB, and the phase variable outer membrane protein Opa. The gonococcus is able to subvert complement mediated killing and opsonisation by sialylation of its lipooligosaccharide and deploys a series of anti-phagocytic mechanisms. N. gonorrhoeae is a fastidious organism that is able to grow on a limited number of primary carbon sources such as glucose and lactate. The utilization of lactate by the gonococcus has been implicated in a number of pathogenicity mechanisms. The bacterium lives mainly in microaerobic environments and can grow both aerobically and anaerobically with the aid of nitrite. The gonococcus does not produce siderophores for scavenging iron but can utilize some produced by other bacteria, and it is able to successful chelate iron from host haem, transferrin and lactoferrin. The gonococcus is an incredibly versatile human pathogen; in the following chapter, we detail the intricate mechanisms used by the bacterium to invade and survive within the host.

JAK and mTOR inhibitors prevent cytokine release while retaining T cell bispecific antibody in vivo efficacy.

BACKGROUND: T cell engaging therapies, like chimeric antigen receptor T cells and T cell bispecific antibodies (TCBs), efficiently redirect T cells towards tumor cells, facilitating the formation of a cytotoxic synapse and resulting in subsequent tumor cell killing, a process that is accompanied by the release of cytokines. Despite their promising efficacy in the clinic, treatment with TCBs is associated with a risk of cytokine release syndrome (CRS). The aim of this study was to identify small molecules able to mitigate cytokine release while retaining T cell-mediated tumor killing. METHODS: By screening a library of 52 Food and Drug Administration approved kinase inhibitors for their impact on T cell proliferation and cytokine release after CD3 stimulation, we identified mTOR, JAK and Src kinases inhibitors as potential candidates to modulate TCB-mediated cytokine release at pharmacologically active doses. Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the effects of mTOR, JAK and Src kinase inhibitors combined with 2+1 T cell bispecific antibodies (TCBs) including CEA-TCB and CD19-TCB on T cell activation, proliferation and target cell killing measured by flow cytometry and cytokine release measured by Luminex. The combination of mTOR, JAK and Src kinase inhibitors together with CD19-TCB was evaluated in vivo in non-tumor bearing stem cell humanized NSG mice in terms of B cell depletion and in a lymphoma patient-derived xenograft (PDX) model in humanized NSG mice in terms of antitumor efficacy. RESULTS: The effect of Src inhibitors differed from those of mTOR and JAK inhibitors with the suppression of CD19-TCB-induced tumor cell lysis in vitro, whereas mTOR and JAK inhibitors primarily affected TCB-mediated cytokine release. Importantly, we confirmed in vivo that Src, JAK and mTOR inhibitors strongly reduced CD19-TCB-induced cytokine release. In humanized NSG mice, continuous treatment with a Src inhibitor prevented CD19-TCB-mediated B cell depletion in contrast to mTOR and JAK inhibitors, which retained CD19-TCB efficacy. Ultimately, transient treatment with Src, mTOR and JAK inhibitors minimally interfered with antitumor efficacy in a lymphoma PDX model. CONCLUSIONS: Taken together, these data support further evaluation of the use of Src, JAK and mTOR inhibitors as prophylactic treatment to prevent occurrence of CRS.

ACE2-Independent Interaction of SARS-CoV-2 Spike Protein with Human Epithelial Cells Is Inhibited by Unfractionated Heparin.

Coronaviruses such as SARS-CoV-2, which is responsible for COVID-19, depend on virus spike protein binding to host cell receptors to cause infection. The SARS-CoV-2 spike protein binds primarily to ACE2 on target cells and is then processed by membrane proteases, including TMPRSS2, leading to viral internalisation or fusion with the plasma membrane. It has been suggested, however, that receptors other than ACE2 may be involved in virus binding. We have investigated the interactions of recombinant versions of the spike protein with human epithelial cell lines that express low/very low levels of ACE2 and TMPRSS2 in a proxy assay for interaction with host cells. A tagged form of the spike protein containing the S1 and S2 regions bound in a temperature-dependent manner to all cell lines, whereas the S1 region alone and the receptor-binding domain (RBD) interacted only weakly. Spike protein associated with cells independently of ACE2 and TMPRSS2, while RBD required the presence of high levels of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we tested the effects of various heparins on ACE2-independent spike protein interaction with cells. Unfractionated heparin inhibited spike protein interaction with an IC50 value of <0.05 U/mL, whereas two low-molecular-weight heparins were less effective. A mutant form of the spike protein, lacking the arginine-rich putative furin cleavage site, interacted only weakly with cells and had a lower affinity for unfractionated and low-molecular-weight heparin than the wild-type spike protein. This suggests that the furin cleavage site might also be a heparin-binding site and potentially important for interactions with host cells. The glycosaminoglycans heparan sulphate and dermatan sulphate, but not chondroitin sulphate, also inhibited the binding of spike protein, indicating that it might bind to one or both of these glycosaminoglycans on the surface of target cells.

Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture.

Tendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell subpopulations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation methods preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior. Statement of clinical significance: The development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.

Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data.

We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

Contrast effects in backward evaluative conditioning: Exploring effects of affective relief/disappointment versus instructional information.

Past studies of backward evaluative conditioning (EC) have found an assimilation effect, in that neutral conditional stimuli (conditional stimulus [CS]) were found to acquire the valence of co-occurring unconditional stimuli (US). Recent studies employing a concurrent forward and backward conditioning paradigm with instructions suggesting a contrastive relation between the US and the backward CS have resulted in contrast effects, in that backward CSs acquired valence opposite to the US. The current research investigated whether these effects were in fact due to the instructions highlighting the contrastive relation between the US and CS, or whether affective relief/disappointment experienced at US offset could account for this result. Consistent with the hypothesized role of instructions, backward CS contrast effects occurred only when instructions highlighted the valence of the US and attributed control of that US to the CSs. In contrast to the affective relief/disappointment hypothesis, no backward CS contrast effects were found without such instructions. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Cryptic prophages within a Streptococcus pyogenes genotype emm4 lineage.

The major human pathogen Streptococcus pyogenes shares an intimate evolutionary history with mobile genetic elements, which in many cases carry genes encoding bacterial virulence factors. During recent whole-genome sequencing of a longitudinal sample of S. pyogenes isolates in England, we identified a lineage within emm4 that clustered with the reference genome MEW427. Like MEW427, this lineage was characterized by substantial gene loss within all three prophage regions, compared to MGAS10750 and isolates outside of the MEW427-like lineage. Gene loss primarily affected lysogeny, replicative and regulatory modules, and to a lesser and more variable extent, structural genes. Importantly, prophage-encoded superantigen and DNase genes were retained in all isolates. In isolates where the prophage elements were complete, like MGAS10750, they could be induced experimentally, but not in MEW427-like isolates with degraded prophages. We also found gene loss within the chromosomal island SpyCIM4 of MEW427-like isolates, although surprisingly, the SpyCIM4 element could not be experimentally induced in either MGAS10750-like or MEW427-like isolates. This did not, however, appear to abolish expression of the mismatch repair operon, within which this element resides. The inclusion of further emm4 genomes in our analyses ratified our observations and revealed an international emm4 lineage characterized by prophage degradation. Intriguingly, the USA population of emm4 S. pyogenes appeared to constitute predominantly MEW427-like isolates, whereas the UK population comprised both MEW427-like and MGAS10750-like isolates. The degraded and cryptic nature of these elements may have important phenotypic and fitness ramifications for emm4 S. pyogenes, and the geographical distribution of this lineage raises interesting questions on the population dynamics of the genotype.

Rapid Transmission of a Hyper-Virulent Meningococcal Clone Due to High Effective Contact Numbers and Super Spreaders.

Rapid transmission, a critical contributory factor in outbreaks of invasive meningococcal disease, requires naïve populations of sufficient size and intermingling. We examined genomic variability and transmission dynamics in a student population subject to an 11-fold increase in carriage of a hypervirulent Neisseria meningitidis serogroup W ST-11 clone. Phylogenetic clusters, mutation and recombination rates were derived by bioinformatic analyses of whole-genome sequencing data. Transmission dynamics were determined by combining observed carriage rates, cluster sizes and distributions with simple SIS models. Between 9 and 15 genetically-distinct clusters were detected and associated with seven residential halls. Clusters had low mutation accumulation rates and infrequent recombination events. Modeling indicated that effective contacts decreased from 10 to 2 per day between the start and mid-point of the university term. Transmission rates fluctuated between 1 and 4% while the R(t) for carriage decreased from an initial rate of 47 to 1. Decreases in transmission values correlated with a rise in vaccine-induced immunity. Observed carriage dynamics could be mimicked by populations containing 20% of super spreaders with 2.3-fold higher effective contact rates. We conclude that spread of this hypervirulent ST-11 meningococcal clone depends on the levels of effective contacts and immunity rather than genomic variability. Additionally, we propose that super-spreaders enhance meningococcal transmission and that a 70% MenACWY immunization level is sufficient to retard, but not fully prevent, meningococcal spread in close-contact populations.

Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis.

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.

Startle during backward evaluative conditioning is not modulated by instructions.

Instructions highlighting that backward conditional stimuli (CSs) stop unconditional stimuli (USs) result in their acquiring valence opposite to that of the US on explicit measures of valence. We assessed whether such instructions would influence startle blink modulation in the same way. Two groups were presented with concurrent forward and backward evaluative conditioning (CS-US-CS) using cartoon aliens as CSs, and pleasant, neutral, and unpleasant sounds as USs. Startle magnitude was measured during conditioning and valence ratings were assessed after conditioning. Participants in the "start-stop" instructions group (n = 41) were instructed to learn whether CSs started or stopped US presentations, while participants in the "observe" instructions group (n = 41) were told to pay attention to the stimuli as they would be asked questions about them after the experiment. In the start-stop instructions group backward CSs paired with positive USs were rated as less pleasant than backward CSs paired with neutral and negative USs (contrast effect), whereas ratings of backward CSs did not differ in the observe instructions group. Startle magnitude was larger during backward CSs paired with positive USs in comparison to CSs paired with neutral or negative USs in both instruction groups. Startle blink modulation was unaffected by instructions, suggesting that startle indexes the emotional state at the time of probe presentation rather than CS valence based on propositional information about the function of the CS.

UV-Induced 1,3,4-Oxadiazole Formation from 5-Substituted Tetrazoles and Carboxylic Acids in Flow.

A range of 1,3,4-oxadiazoles have been synthesized using a UV-B activated flow approach starting from carboxylic acids and 5-substituted tetrazoles. The application of UV light represents an attractive alternative to the traditional thermolytic approach and has demonstrated comparable efficiency and versatility, with a diverse substrate scope, including the incorporation of highly substituted amino acids.

Meningococcal core and accessory phasomes vary by clonal complex.

Neisseria meningitidis is a Gram-negative human commensal pathogen, with extensive phenotypic plasticity afforded by phase-variable (PV) gene expression. Phase variation is a stochastic switch in gene expression from an ON to an OFF state, mediated by localized hypermutation of simple sequence repeats (SSRs). Circulating N. meningitidis clones vary in propensity to cause disease, with some clonal complexes (ccs) classified as hypervirulent and others as carriage-associated. We examined the PV gene repertoires, or phasome, of these lineages in order to determine whether phase variation contributes to disease propensity. We analysed 3328 genomes representative of nine circulating meningococcal ccs with PhasomeIt, a tool that identifies PV genes by the presence of SSRs and homologous gene clusters. The presence, absence and functions of all identified PV gene clusters were confirmed by annotation or blast searches within the Neisseria PubMLST database. While no significant differences were detected in the number of PV genes or the core, conserved phasome content between hypervirulent and carriage lineages, individual ccs exhibited major variations in PV gene numbers. Phylogenetic clusters produced by phasome or core genome analyses were similar, indicating co-evolution of PV genes with the core genome. While conservation of PV clusters is high, with 76 % present in all meningococcal isolates, maintenance of an SSR is variable, ranging from conserved in all isolates to present only in a single cc, indicating differing evolutionary trajectories for each lineage. Diverse functional groups of PV genes were present across the meningococcal lineages; however, the majority directly or indirectly influence bacterial surface antigens and could impact on future vaccine development. Finally, we observe that meningococci have open pan phasomes, indicating ongoing evolution of PV gene content and a significant potential for adaptive changes in this clinically relevant genus.