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BackgroundTo compare the diagnostic performance of microbiological culture and 16S/18S rRNA gene polymerase chain reaction (PCR)-Sanger sequencing for infectious keratitis (IK) and to analyse the effect of clinical disease severity on test performance and inter-test concordance.MethodsThis was a three-arm, diagnostic cross-sectional study. We included all eligible patients who presented with presumed bacterial/fungal keratitis to the Queen's Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous culture (either direct or indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. The bacterial/fungal genus and species identified on culture were confirmed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Relevant clinical data were also collected to analyze for any potential clinico-microbiological correlation. Main outcome measures included the diagnostic yield, test accuracy (including sensitivity and specificity), and inter-test agreement [including percent agreement and Cohen's kappa (k)].ResultsA total of 81 patients (86 episodes of IK) were included in this study. All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p = 0.13). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4–95.3%), followed by indirect culture (85.4%; 95% CI, 71.6–93.5%) and PCR (73.5%; 95% CI, 59.0–84.6%), with 100% specificity noted in all tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement = 81.8–86.2%, Cohen's k = 0.67–0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with greater infection severity.ConclusionsThis study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower).

Purpose: We aimed to determine whether Descemet's membrane (DM) scrolling occurs primarily along the vertical or horizontal axis and establish whether oval trephination along the axis of least scrolling can reduce the grade of the scroll. Methods: The longest limbus-to-limbus axis on 28 sclerocorneal discs was taken as the horizontal axis. The horizontal (n = 7) or (right angles to it) vertical (n = 6) axis was marked on DM before peeling it off. The direction and grade of scrolling was observed. Narrow strips (3–4 mm wide) were then cut along the two axes (n = 4 each) and the scrolling pattern was observed. Ellipses (7 × 9 mm) of DM were punched along the two axes (n = 6 each) and the scrolls graded. Immunofluorescent staining for elastin on horizontal and vertical tissue sections from three DM samples was performed. The intensity and thickness of elastin staining were measured. Results: Twenty-four (85.72%) DM samples showed scrolling along the horizontal axis, none showed scrolling along the vertical axis, and four (14.28%) samples showed a spiral scroll, regardless of which axis was marked (grade 3.7 and 3.6). Vertically oval discs showed significantly reduced scrolling (grade 1.2) compared to horizontally oval discs (grade 3.5). Narrow strips of DM showed a similar scrolling pattern. Immunohistology showed no difference in any of the parameters examined along the two axes or from the center to the periphery. Conclusion: DM scrolls primarily along the horizontal axis. Vertically oval DM samples show minimal scrolling, which can be an advantage in DMEK. Differential scrolling is not determined by the distribution of elastin.

This paper presents a new approach to estimate the probability of line-of-sight (LoS) for unmanned aerial vehicle (UAV) communications. We provide lower and upper bounds on the probability of LoS in terms of what we call the first-building-LoS probability. We provide a statistical model for the ground distance to the first building along the line from the user to the UAV. Based on this we provide a general formula for the first-building-LoS probability for urban environments where the building heights follow a Rayleigh distribution. We show that the first-building-LoS probability is a good estimate for the probability of LoS. Our closed-form formulas estimate the probability of LoS significantly more accurately than the existing approaches. We also obtain closed-form estimates of Area Line-of-Sight Probabilities for a scenario in which the UAV provides coverage to a circular region on the ground.

Abstract Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5–25.0 μg/ml (5.2–10.4 μM)] and S. epidermidis [MIC = 12.5 μg/ml (5.2 μM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 μg/ml (10.4–20.8 μM)]. CaD23 (at 25 μg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 μg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

Sight is arguably the most important sense in human. Being constantly exposed to the environmental stress, irritants and pathogens, the ocular surface – a specialized functional and anatomical unit composed of tear film, conjunctival and corneal epithelium, lacrimal glands, meibomian glands, and nasolacrimal drainage apparatus – serves as a crucial front-line defense of the eye. Host defense peptides (HDPs), also known as antimicrobial peptides, are evolutionarily conserved molecular components of innate immunity that are found in all classes of life. Since the first discovery of lysozyme in 1922, a wide range of HDPs have been identified at the ocular surface. In addition to their antimicrobial activity, HDPs are increasingly recognized for their wide array of biological functions, including anti-biofilm, immunomodulation, wound healing, and anti-cancer properties. In this review, we provide an updated review on: (1) spectrum and expression of HDPs at the ocular surface; (2) participation of HDPs in ocular surface diseases/conditions such as infectious keratitis, conjunctivitis, dry eye disease, keratoconus, allergic eye disease, rosacea keratitis, and post-ocular surgery; (3) HDPs that are currently in the development pipeline for treatment of ocular diseases and infections; and (4) future potential of HDP-based clinical pharmacotherapy for ocular diseases.

Background/Aim: Host defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and the secondary structure and underlying mechanism of action of CaD23.Methods: Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard and time-kill assays. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FIC1.0 and ≤4), or antagonistic (FIC>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes. Circular dichroism (CD) spectroscopy was also performed to examine the secondary structures of CaD23.Results: CaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI = 0.60–0.69) and MRSA43300 (FICI = 0.56–0.60) but an indifferent effect against P. aeruginosa (FIC = 1.03–1.15). CaD23 (at 25 μg/ml; 2xMIC) completely killed S. aureus within 30 min. When used at sub-MIC concentration (3.1 μg/ml; 0.25xMIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23. The secondary structures of CaD23 observed in MD simulations were validated by CD spectroscopy.Conclusion: CaD23 is a novel alpha-helical, membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulations serves as a powerful tool in revealing the peptide secondary structure, dissecting the mechanism of action, and guiding the design and optimisation of HDPs.

Objectives The largest proportion of general practitioner (GP) magnetic resonance imaging (MRI) is musculoskeletal (MSK), with consistent annual growth. With limited supporting evidence and potential harms from early imaging overuse, we evaluated practice to improve pathways and patient safety.Methods Cohort evaluation of routinely collected diagnostic and general practice data across a UK metropolitan primary care population. We reviewed patient characteristics, results and healthcare utilisation.Results Of 306 MSK-MRIs requested by 107 clinicians across 29 practices, only 4.9% (95% CI ±2.4%) appeared clearly indicated and only 16.0% (95% CI ±4.1%) received appropriate prior therapy. 37.0% (95% CI ±5.5%) documented patient imaging request. Most had chronic symptoms and half had psychosocial flags. Mental health was addressed in only 11.8% (95% CI ±6.3%) of chronic sufferers with psychiatric illness, suggesting a solely pathoanatomical approach to MSK care. Only 7.8% (95% CI ±3.0%) of all patients were appropriately managed without additional referral. 1.3% (95% CI ±1.3%) of scans revealed diagnoses leading to change in treatment (therapeutic yield). Most imaged patients received pathoanatomical explanations to their symptoms, often based on expected age or activity-related changes. Only 16.7% (95% CI ±4.2%) of results appeared correctly interpreted by GPs, with spurious overperception of surgical targets in 65.4% (95% CI ±5.3%) who suffered ‘low-value’ (ineffective, harmful or wasteful) post-MRI referral cascades due to misdiagnosis and overdiagnosis. Typically, 20%–30% of GP specialist referrals convert to a procedure, whereas MRI-triggered referrals showed near-zero conversion rate. Imaged patients experienced considerable delay to appropriate care. Cascade costs exceeded direct-MRI costs and GP-MSK-MRI potentially more than doubles expenditure compared with physiotherapist-led assessment services, for little-to-no added therapeutic yield, unjustifiable by cost–consequence or cost–utility analysis.Conclusion Unfettered GP-MSK-MRI use has reached unaccceptable indication creep and disutility. Considerable avoidable harm occurs through ubiquitous misinterpretation and salient low-value referral cascades for two-thirds of imaged patients, for almost no change in treatment. Any marginally earlier procedural intervention for a tiny fraction of patients is eclipsed by negative consequences for the vast majority. Only 1–2 patients need to be scanned for one to suffer mismanagement. Direct-access imaging is neither clinically, nor cost-effective and deimplementation could be considered in this setting. GP-MSK-MRI fuels unnecessary healthcare utilisation, generating nocebic patient beliefs and expectations, whilst appropriate care is delayed and a high burden of psychosocial barriers to recovery appear neglected.

One of the greatest challenges facing the medical community today is the ever-increasing trajectory of antimicrobial resistance (AMR), which is being compounded by the decrease in our antimicrobial armamentarium. From their initial discovery to the current day, antibiotics have seen an exponential increase in their usage, from medical to agricultural use. Benefits aside, this has led to an exponential increase in AMR, with the fear that over 10 million lives are predicted to be lost by 2050, according to the World Health Organisation (WHO). As such, medical researchers are turning their focus to discovering novel alternatives to antimicrobials, one being Host Defence Peptides (HDPs). These small cationic peptides have shown great efficacy in being used as an antimicrobial therapy for currently resistant microbial variants. With the sudden emergence of the SARS-CoV-2 variant and the subsequent global pandemic, the great versatility and potential use of HDPs as an alternative to conventional antibiotics in treating as well as preventing the spread of COVID-19 has been reviewed. Thus, to allow the reader to have a full understanding of the multifaceted therapeutic use of HDPs, this literature review shall cover the association between COVID-19 and AMR whilst discussing and evaluating the use of HDPs as an answer to antimicrobial resistance (AMR).

The golden era of antibiotics, heralded by the discovery of penicillin, has long been challenged by the emergence of antimicrobial resistance (AMR). Host defense peptides (HDPs), previously known as antimicrobial peptides, are emerging as a group of promising antimicrobial candidates for combatting AMR due to their rapid and unique antimicrobial action. Decades of research have advanced our understanding of the relationship between the physicochemical properties of HDPs and their underlying antimicrobial and non-antimicrobial functions, including immunomodulatory, anti-biofilm, and wound healing properties. However, the mission of translating novel HDP-derived molecules from bench to bedside has yet to be fully accomplished, primarily attributed to their intricate structure-activity relationship, toxicity, instability in host and microbial environment, lack of correlation between in vitro and in vivo efficacies, and dwindling interest from large pharmaceutical companies. Based on our previous experience and the expanding knowledge gleaned from the literature, this review aims to summarize the novel strategies that have been employed to enhance the antimicrobial efficacy, proteolytic stability, and cell selectivity, which are all crucial factors for bench-to-bedside translation of HDP-based treatment. Strategies such as residues substitution with natural and/or unnatural amino acids, hybridization, L-to-D heterochiral isomerization, C- and N-terminal modification, cyclization, incorporation with nanoparticles, and “smart design” using artificial intelligence technology, will be discussed. We also provide an overview of HDP-based treatment that are currently in the development pipeline.

Pseudomonas aeruginosa (PA) is the leading cause of corneal blindness worldwide. A constant increase in multi-drug resistant PA strains have heightened the challenge of effectively managing corneal infections with conventional antibiotics. Antimicrobial peptides are promising antibiotic analogs with a unique mode of action. Cathelicidin-derived shorter peptides (FK13 and FK16) have previously been shown to kill a range of pathogens in both in vitro and in vivo systems. Here, our aim was to exploit the potential of FK13 or FK16 to enhance the anti-Pseudomonas activity of vancomycin, which normally has low clinical efficacy against PA. Our results have demonstrated that FK16 is more potent than FK13 against different PA strains including a clinical isolate from a patient’s ocular surface. FK16 was shown to enhance the membrane permeability of PAO1 at sub-inhibitory concentrations. Moreover, FK16 at lower concentrations was shown to increase the antibacterial susceptibility of vancomycin against PA strains up to eightfold. The bactericidal synergism between FK16 and vancomycin was shown to be stable in the presence of physiological tear salt concentration and did not cause toxic effects on the human corneal epithelial cells and human red blood cells. Our results have revealed that sub-inhibitory concentration of FK16 could augment the antimicrobial effects of vancomycin against PA. It is anticipated that the future exploitation of the peptide design approach may enhance the effectiveness of FK16 and its application as an adjuvant to antibiotic therapy for the treatment of multi-drug resistant infections.

BACKGROUND: Leprosy, being a chronic infectious disease with profound social stigma, remains associated with high psychological mortidity. PURPOSES: To find out the pattern of psychiatric morbidity in leprosy patients and the relationship of various factors with the morbidity. METHODS: Ninty patients attending leprosy clinic were randomly chosen for the study group alongwith 40 patients suffering from acute skin problem other than leprosy as control group. The socio-demographic data were recorded in semi-structural proforma; all patients were given Goldbery Health Questioneaire (GHQ). Patients having GHQ score> 2 was assessed by Disability Assessent Questionaire (DAQ). The psychiatric diagnoses was made according to ICD-10 by W ho0 and physical deformity by W ho 0 Disability Scale. FINDINGS: The mean GHQ score of the study grant was 3.44 and that of control group was 1.62. The mean DAQ score was 45.13. Psychiatric disorder was seen in 44.4% and 7.5% of study group and control group respectively. The psychiatric illness was generalised anoxidy disorder (GAD) (27.8%). CONCLUSIONS: Leprosis highly associated with psychiatric mobidity. LIMITATIONS: The findings can not be generalised due to small sample size and clinic-based data.

PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB

BACKGROUND:During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain. METHODOLOGY/PRINCIPAL FINDINGS:To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 -/- mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit. CONCLUSIONS/SIGNIFICANCE:These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

Background: Overprescribing of potentially harmful medication in UK general practice has a complex association with socioeconomic deprivation. Aim: To assess trends in general practice prescribing of five high-risk medications and their relationship with deprivation. Design & setting: An observational study was conducted using general practice data from three English regions with varied sociodemographic factors: West Yorkshire and Harrogate (WY), Black Country and West Birmingham (BC), and Surrey and East Sussex (SE). Method: Practice-level prescribing data were obtained from 2016–2021 for five drug classes: opioids, hypnotics, gabapentinoids, non-steroidal anti-inflammatory drugs (NSAIDs), and antibacterials. Prescribing trends were demonstrated using a linear model. Results: Reduction in NSAID, opioid, hypnotic and antibacterial prescriptions, and the increase in gabapentinoid prescriptions, were significant at each financial year time period. Index of Multiple Deprivation (IMD) was positively associated with all drug classes except antibacterials, which showed a positive association when incorporating the interaction term between IMD and age. When adjusting for IMD and population, region was independently associated with prescribing rate. Compared with WY, IMD had a smaller association with prescribing in BC for NSAIDs (coefficient = −0.01578, P = 0.004) and antibacterials (coefficient = −0.02769, P = 0.007), whereas IMD had a greater association with prescribing in SE for NSAIDs (coefficient = 0.02443, P<0.001), opioids (coefficient = 0.08919, P<0.001), hypnotics (coefficient = 0.09038, P<0.001), gabapentinoids (coefficient = 0.1095, P<0.001), and antibacterials (coefficient = 0.01601, P = 0.19). Conclusion: The association of socioeconomic deprivation with overprescribing of high-risk medication in general practice varies by region and drug type. Geographical location is associated with overprescribing, independent of socioeconomic status. [ABSTRACT FROM AUTHOR]