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This study aims to investigate if the total amount of DNA methylation in peripheral monocytes changes due to activation and differentiation to in vitro monocyte-derived macrophages. Research of this nature is ongoing at Malmö University, with particular focus on aluminum salts as vaccine adjuvants, and their effects on macrophages. Gene expression and methylation changes in the macrophages of the immune system is scarcely researched, as well as how their methylation is influenced by vaccine adjuvants. New knowledge about the amount of methylation in these cells before stimulation with aluminum salts may be a part of future vaccine research. Monocytes from peripheral blood were isolated and stimulated with cytokines for differentiation and polarization before DNA extraction and methylation analysis. Findings in this study shows a decreasing amount of DNA methylation when monocytes differentiate into inactive M0 macrophages, but unchanged amount of methylation in further polarization of the cells to pro-inflammatory M1 and anti-inflammatory M2 macrophages. By analyzing more donors and including sequencing methods, the involvement and impact on specific genes may be identified and further investigated.

The escalating global prevalence of type-2 diabetes (T2D) and obesity necessitates the development of novel oral medications. Agonism at G-protein coupled receptor-119 (GPR119) has been recognized for modulation of metabolic homeostasis in T2D, obesity, and fatty liver disease. However, off-target effects have impeded the advancement of synthetic GPR119 agonist drug candidates. Non-systemic, gut-restricted GPR119 agonism is suggested as an alternative strategy that may locally stimulate intestinal enteroendocrine cells (EEC) for incretin secretion, without the need for systemic drug availability, consequently alleviating conventional class-related side effects. Herein, we report the preclinical acute safety, efficacy, and pharmacokinetics (PK) of novel GPR119 agonist compounds ps297 and ps318 that potentially target gut EEC for incretin secretion. In a proof-of-efficacy study, both compounds demonstrated glucagon-like peptide-1 (GLP-1) secretion capability during glucose and mixed-meal tolerance tests in healthy mice. Furthermore, co-administration of sitagliptin with investigational compounds in diabetic db/db mice resulted in synergism, with GLP-1 concentrations rising by three-fold. Both ps297 and ps318 exhibited low gut permeability assessed in the in-vitro Caco-2 cell model. A single oral dose PK study conducted on healthy mice demonstrated poor systemic bioavailability of both agents. PK measures (mean ± SD) for compound ps297 (Cmax 23 ± 19 ng/mL, Tmax range 0.5 – 1 h, AUC0–24 h 19.6 ± 21 h*ng/mL) and ps318 (Cmax 75 ± 22 ng/mL, Tmax range 0.25 – 0.5 h, AUC0–24 h 35 ± 23 h*ng/mL) suggest poor oral absorption. Additionally, examinations of drug excretion patterns in mice revealed that around 25 % (ps297) and 4 % (ps318) of the drugs were excreted through faeces as an unchanged form, while negligible drug concentrations (

Abstract Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers. Infection experiments with mice transplanted with patient fecal material reveal that these antibiotic-induced microbiota perturbations impair pulmonary defense against MDR Klebsiella pneumoniae. This is dependent on inflammatory monocytes (IMs), whose fatty acid receptor (FFAR)2/3-controlled and phagolysosome-dependent antibacterial activity is compromized in mice transplanted with antibiotic-associated patient microbiota. Collectively, we characterize how clinically relevant antibiotics affect antimicrobial defense in the context of human microbiota, and reveal a critical impairment of IM´s antimicrobial activity. Our study provides additional arguments for the rational use of antibiotics and offers mechanistic insights for the development of novel prophylactic strategies to protect high-risk patients from HAP.

A checklist of 2265 spider species and subspecies, 495 genera and 71 families is provided. Data were extracted from the South African National Survey of Arachnida database and over 200 taxonomic revisions, ending December 2022. Global distributions, endemicity and conservation assessment using IUCN Criteria are provided for each species. A total of 1325 spp. are endemic to South Africa (58.5%), 126 spp. (5.6%) are of special concern and 693 spp. (30.6%) are Data Deficient (DD), while 15 species were described without exact locality data. Most species (1444 spp., 63.8%) are widely distributed with no known threats and are of Least Concern. A total of 1316 spp. (57.6%) are known from both sexes and 23 spp. (1.0%) were described from juveniles. Salticidae is the most species-rich family (354 spp.), followed by Gnaphosidae (195 spp.), Thomisidae (143 spp.) and Araneidae (100 spp.) and ten families are represented by a single species.

‘Globesity’ is a foremost challenge to the healthcare system. The limited efficacy and adverse effects of available oral pharmacotherapies pose a significant obstacle in the fight against obesity. The biology of the leading incretin hormone glucagon-like-peptide-1 (GLP-1) has been highly captivated during the last decade owing to its multisystemic pleiotropic clinical outcomes beyond inherent glucoregulatory action. That fostered a pharmaceutical interest in synthetic GLP-1 analogues to tackle type-2 diabetes (T2D), obesity and related complications. Besides, mechanistic insights on metabolic surgeries allude to an incretin-based hormonal combination strategy for weight loss that emerged as a forerunner for the discovery of injectable ‘unimolecular poly-incretin-agonist’ therapies. Physiologically, intestinal enteroendocrine L-cells (EECs) are the prominent endogenous source of GLP-1 peptide. Despite comprehending the potential of various G protein-coupled receptors (GPCRs) to stimulate endogenous GLP-1 secretion, decades of translational GPCR research have failed to yield regulatory-approved endogenous GLP-1 secretagogue oral therapy. Lately, a dual/poly-GPCR agonism strategy has emerged as an alternative approach to the traditional mono-GPCR concept. This review aims to gain a comprehensive understanding by revisiting the pharmacology of a few potential GPCR-based complementary avenues that have drawn attention to the design of orally active poly-GPCR agonist therapy. The merits, challenges and recent developments that may aid future poly-GPCR drug discovery are critically discussed. Subsequently, we project the mechanism-based therapeutic potential and limitations of oral poly-GPCR agonism strategy to augment intestinal GLP-1 for weight loss. We further extend our discussion to compare the poly-GPCR agonism approach over invasive surgical and injectable GLP-1-based regimens currently in clinical practice for obesity.

Abstract Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella, E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella, E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. Video Abstract

Presbyopia occurs when the physiologically normal age-related reduction in the eyes focusing range reaches a point, when optimally corrected for distance vision, that the clarity of vision at near is insufficient to satisfy an individual’s requirements. Hence, it is more about the impact it has on an individual’s visual ability to function in their environment to maintain their lifestyle than a measured loss of focusing ability. Presbyopia has a significant impact on an individual’s quality of life and emotional state. While a range of amelioration strategies exist, they are often difficult to access in the developing world and prescribing is generally not optimal even in developed countries. This review identified the need for a standardised definition of presbyopia to be adopted. An appropriate battery of tests should be applied in evaluating presbyopic management options and the results of clinical trials should be published (even if unsuccessful) to accelerate the provision of better outcomes for presbyopes.

Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe COVID-19 in some patients and mild COVID-19 in others. Dysfunctional innate immune responses have been identified to contribute to COVID-19 severity, but the key regulators are still unknown. Here, we present an integrative single-cell multi-omics analysis of peripheral blood mononuclear cells from hospitalized and convalescent COVID-19 patients. In classical monocytes, we identified genes that were potentially regulated by differential chromatin accessibility. Then, sub-clustering and motif-enrichment analyses revealed disease condition-specific regulation by transcription factors and their targets, including an interaction between C/EBPs and a long-noncoding RNA LUCAT1, which we validated through loss-of-function experiments. Finally, we investigated genetic risk variants that exhibit allele-specific open chromatin (ASoC) in COVID-19 patients and identified a SNP rs6800484-C, which is associated with lower expression of CCR2 and may contribute to higher viral loads and higher risk of COVID-19 hospitalization. Altogether, our study highlights the diverse genetic and epigenetic regulators that contribute to COVID-19.

AimsPatients with chronic health conditions not responding to conventional treatment can access medicinal cannabis (MC) prescriptions from clinicians in Australia. We aimed to assess overall health-related quality of life (HRQL), pain, fatigue, sleep, anxiety, and depression in a large real-world sample of patients accessing prescribed medicinal cannabis. We hypothesized that all patient-reported outcomes (PROs) would improve from baseline to 3-months.MethodsThe QUEST Initiative is a large prospective multicenter study of patients with any chronic health condition newly prescribed medicinal cannabis between November 2020 and December 2021. Eligible patients were identified by 120 clinicians at medical centers across six Australian states. Consenting participants completed the EuroQol Group EQ-5D-5L health status questionnaire; European Organization for Research & Treatment of Cancer Quality of Life questionnaire (QLQ-C30); Patient-Reported Outcomes Measurement Information System (PROMIS) Short Forms in Fatigue and Sleep Disturbance, and the Depression Anxiety Stress Scale (DASS-21) before starting therapy, at 2-weeks titration, then monthly for 3-months.ResultsOf the 2762 consenting participants, 2327 completed baseline and at least one follow-up questionnaire. Ages ranged between 18-97 years (mean 51y; SD = 15.4), 62.8% were female. The most commonly treated conditions were chronic pain (n = 1598/2327; 68.7%), insomnia (n = 534/2327; 22.9%), generalized anxiety (n = 508/2327; 21.5%), and mixed anxiety and depression (n = 259/2327; 11%). Across the whole cohort both EQ-5D-5L utility scores and QLQ-C30 summary scores showed clinically meaningful improvement in HRQL from baseline to mean follow-up with d = 0.54 (95%CI:0.47 to 0.59) and d = 0.64 (95%CI:0.58 to 0.70) respectively; and clinically meaningful improvement in fatigue (d = 0.54; 95%CI:0.48 to 0.59). There was clinically meaningful reduction of pain for those with chronic pain (d = 0.65; 95%CI:0.57 to 0.72); significant improvements for those with moderate to extremely severe anxiety (X2 = 383; df = 4; pConclusionsWe observed statistically significant, clinically meaningful improvements in overall HRQL and fatigue over the first 3-months in patients with chronic health conditions accessing prescribed medical cannabis. Anxiety, depression, and pain also improved over time, particularly for those with corresponding health conditions. The study continues to follow-up patients until 12-months to determine whether improvements in PROs are maintained long-term.Trail registrationStudy registration - Australian New Zealand Clinical Trials Registry: ACTRN12621000063819. https://www.australianclinicaltrials.gov.au/anzctr/trial/ACTRN12621000063819.

Introduction: Medicinal cannabis is prescribed in Australia for patients with chronic refractory pain conditions. However, measures of safety and effectiveness of different cannabinoids are lacking. We designed an observational study to capture effectiveness, adverse events (AEs), and health-related quality of life (HRQoL) measures in patients prescribed an oral medicinal cannabis formulation at Cannabis Access Clinics through the Cannabis Access Clinics Observational study (CACOS). Objectives: We aimed to evaluate effectiveness, reported AEs, and change in patient-reported outcomes in individuals prescribed a cannabinoid oil formulation for management of chronic pain. Methods: A cross-sectional analysis was conducted on patients prescribed an oil formulation of Δ9-tetrahydrocannabinol and cannabidiol for pain symptoms of at least 3-month duration. Clinician-reported AEs were organized by system, organ, class, and frequency. Analysis of patient-reported responses to a questionnaire was conducted using published minimal clinically important differences to determine meaningful change in HRQoL over time. Results: More than half (n = 91/151, 60.3%) of the participants experienced at least one AE during the observation period (mean 133 ± 116 days). No serious AEs were reported. Patient-reported pain impact scores were significantly reduced across the cohort (p = 0.034), and pain intensity scores verged on significance (p = 0.053). The majority of patients saw meaningful improvements in sleep (49.3%) and fatigue (35.6%). Conclusion: This analysis presents real-world data collected as part of standard of care. More than one-third of patients benefited from oral medicinal cannabis, which is impactful given the refractory nature of their pain. Amelioration of the impact of pain confirms continued prescribing of this formulation and validates our observational methodology as a tool to determine the therapeutic potency of medicinal cannabinoids.

Humans have employed cannabis for multiple uses including medicine, recreation, food, and fibre. The various components such as roots, flowers, seeds, and leaves have been utilized to alleviate pain, inflammation, anxiety, and gastrointestinal disorders like nausea, vomiting, diarrhoea, and inflammatory bowel diseases (IBDs). It has occupied a significant space in ethnomedicines across cultures and religions. Despite multi-dimensional uses, the global prohibition of cannabis by the USA through the introduction of the Marijuana Tax Act in 1937 led to prejudice about the perceived risks of cannabis, overshadowing its medicinal potential. Nevertheless, the discovery of tetrahydrocannabinol (THC), the primary psychoactive compound in cannabis, and the endocannabinoid system renewed scientific interest in understanding the role of cannabis in modulating different conditions, including gastrointestinal disorders. Preparations combining cannabidiol and THC have shown promise in mitigating gut symptoms through anti-inflammatory and motility-enhancing effects. This review revisits the ethnomedicinal use of cannabis in gastrointestinal diseases and emphasizes the need for further research to determine optimal dosages, formulations, and safety profiles of cannabis-based medicines. It also underscores the future potential of cannabinoid-based therapies by leveraging the role of the expanded endocannabinoid system, an endocannabinoidome, in the modulation of gastrointestinal ailments.

Genome-wide association studies (GWAS) have identified a large number of genetic loci for coronary artery disease (CAD), with many located close to genes associated with traditional CAD risk pathways, such as lipid metabolism and inflammation. It is becoming evident with recent CAD GWAS meta-analyses that vascular pathways are also highly enriched and present an opportunity for novel therapeutics. This review examines GWAS-enriched vascular gene loci, the pathways involved and their potential role in CAD pathogenesis. The functionality of variants is explored from expression quantitative trait loci, massively parallel reporter assays and CRISPR-based gene-editing tools. We discuss how this research may lead to novel therapeutic tools to treat cardiovascular disorders.

Abstract. The endocannabinoid system (ECS) is an essential endogenous signaling system that may be involved in the pathophysiology of chronic widespread pain (CWP) and fibromyalgia syndrome (FMS). Further research is required to understand the role of ECS in the development and maintenance of CWP and FMS. We provided the first systematic review and meta-analysis exploring the clinical relevance of ECS alterations in patients with CWP and FMS by comparing plasma and interstitial levels of endocannabinoids and N-acylethanolamines in patients and healthy controls. A systematic search was conducted to identify studies that measured plasma and/or interstitial levels of endocannabinoids and N-acylethanolamines in patients with CWP or FMS and healthy controls. A total of 8 studies were included for qualitative review, and 7 studies were included for meta-analysis. The findings identified increased plasma levels of oleoylethanolamide and stearoylethanolamide in patients with FMS compared with those in controls (P = 0.005 and P < 0.0001, respectively) and increased plasma levels of palmitoylethanolamide and interstitial levels of stearoylethanolamide in patients with CWP compared with those in controls (P = 0.05 and P = 0.001, respectively). There were no significant differences in other ECS parameters. Most studies did not account for variables that may influence ECS function, including cannabis use, concomitant medication, comorbidities, physical activity, stress levels, circadian rhythm, sleep quality, and dietary factors, suggesting that future studies should explore the correlation between these variables and endocannabinoid activity. We highlight the importance of investigating endocannabinoid activity in CWP and FMS because it will underpin future translational research in the area.

Abstract Background Low and middle-income countries have growing older populations and could benefit from the use of multi-domain geriatric assessments in overcoming the challenge of providing quality health services to older persons. This paper reports on the outcomes of a study carried out in Cape Town, South Africa on the validity of the interRAI Check-Up Self-Report instrument, a multi-domain assessment instrument designed to screen older persons in primary health settings. This is the first criterion validity study of the instrument. The instrument is designed to identify specific health problems and needs, including psychosocial or cognition problems and issues related to functional decline. The interRAI Check-Up Self-Report is designed to be compatible with the clinician administered instruments in the interRAI suite of assessments, but the validity of the instrument against clinician ratings has not yet been established. We therefore sought to establish whether community health workers, rather than trained healthcare professionals could reliably administer the self-report instrument to older persons. Methods We evaluated the criterion validity of the self-report instrument through comparison to assessments completed by a clinician assessor. A total of 112 participants, aged 60 or older were recruited from 7 seniors clubs in Khayelitsha, Cape Town. Each participant was assessed by one of two previously untrained, non-healthcare personnel using the Check-Up Self-report version and again by a trained assessor using the clinician version of the interRAI Check-Up within 48 h. Our analyses focused on the degree of agreement between the self-reported and clinician-rated versions of the Check-Up based on the simple or weighted kappa values for the two types of ratings. Binary variables used simple kappas, and ordinal variables with three or more levels were examined using weighted kappas with Fleiss-Cohen weights. Results Based on Cohen’s Kappa values, we were able to establish that high levels of agreement existed between clinical assessors and lay interviewers, indicating that the instrument can be validly administered by community health workers without formal healthcare training. 13% of items had kappa values ranging between 0.10 and 0.39; 51% of items had kappa values between 0.4 and 0.69; and 36% of items had values of between 0.70 and 1.00. Conclusion Our findings indicate that there is potential for the Check-Up Self-Report instrument to be implemented in under-resourced health systems such as South Africa’s.

Abstract Background Developing countries are experiencing rapid population ageing. Many do not have the resources or formal structures available to support the health and wellbeing of people as they age. In other contexts, the use of peer support programmes have shown favourable outcomes in terms of reducing loneliness, increasing physical activity and managing chronic disease. Such programmes have not been previously developed or tested in African countries. We piloted a peer-to-peer support model among vulnerable community-dwelling adults in a developing country (South Africa) to examine the program’s effect on wellbeing and social engagement. Methods A pre-post, pilot design was used to evaluate targeted outcomes, including wellbeing, social support, social interaction, mood, loneliness and physical activity. A total of 212 persons, aged 60+ years and living independently in a low-income area in Cape Town were recruited and screened for eligibility by trained assessors. Participants were assessed using the interRAI CheckUp, WHO-5 Wellbeing index, and the MOS-SS 8 instruments before and after the 5-month intervention, during which they received regular visits and phone calls from trained peer volunteers. During visits volunteers administered a wellness screening, made referrals to health and social services; built friendships with clients; encouraged social engagement; promoted healthy living; and provided emotional and informational support. Results Volunteer visits with clients significantly increased levels of self-reported wellbeing by 58%; improved emotional and informational support by 50%; decreased reports of reduced social interaction by 91%; reduced loneliness by 70%; improved mood scores represented as anxiety, depression, lack of interest or pleasure in activities, and withdrawal from activities of interest; and increased levels of physical activity from 49 to 66%. Discussion The intervention led to demonstrable improvement in client wellbeing. Policymakers should consider integrating peer-support models into existing health programs to better address the needs of the elderly population and promote healthy ageing in resource-poor community settings. Longer-term and more rigorous studies with a control group are needed to support these findings and to investigate the potential impact of such interventions on health outcomes longitudinally.

The present study characterizes Bemisia tabaci and Bemisia afer from cassava in eastern Democratic Republic of Congo (DRC). The Mitochondrial COI sequencing revealed the occurrence of six cassava B. tabaci mitotypes, which were designated into four haplogroups (SSA-ECA, SSA-CA, SSA2, and SSA-ESA) using KASP SNP genotyping. SSA-ECA (72%) was the most prevalent and occurred in the northern part of the surveyed area, in the Ituri and Nord/Sud-Kivu provinces, whilst SSA-CA (21%) was present in the south, primarily in Haut-Katanga. SSA-ECA was predominant in the areas of north-eastern DRC most severely affected by cassava brown streak disease and was also reported in the new outbreak area in Pweto territory, Haut-Katanga, in the south. Bemisia afer comprised two major clusters with 85.5% of samples in cluster one, while the rest were in cluster two, which has no reference sequence in GenBank. This study provides important information on the genetic diversity of B. tabaci and B. afer in eastern DRC. This knowledge will be used as a basis for further studies to understand and to identify the role of whitefly haplogroups, their population densities and consequences for virus epidemics and spread as well as leading to improved vector and virus management strategies.

Abstract Background There is lack of adequate training and policy support for employed care workers (CWs) employed in the South African (SA) older persons’ sector. Existing literature neglects the influence of training and policy support on CWs’ experiences in long-term care (LTC) for older adults in residential care facilities (RCFs). We investigated the ways in which CWs’ experiences are rooted in the lack of adequate training and policy support. Methods Qualitative data was collected through focus group (FG) interviews with 32 CWs employed in RCFs in the City of Cape Town. Data was also collected using semi-structured interviews with representatives of five RCFs for older adults and four training organisations providing CW training in the City of Cape Town, South Africa. Results Despite some positive caregiving experiences, CWs face role ambiguity and experience care work as a ‘career-less job’. They also face poor employment conditions, negative interpersonal relations at work, and role overload. They are not coping with the demands of LTC due to role overload, and lack of basic caregiving skills, coping skills and socio-emotional support. Their motivation to cope and provide quality care is hamstrung by their experiences of role ambiguity, poor employment conditions, negative interpersonal relations at work, and lack of career growth opportunities. Conclusions Findings suggest that CWs’ experiences derive from the policy and structural context of caregiving. Policy inadequacies and lack of structural support create conditions for adverse conditions which negatively impact on CWs motivation and ability to cope with the demands of LTC. Lack of policy implementation presents structural barriers to quality LTC in the older persons’ sector. Implementation of policies and systems for professionalising care work is long overdue.

The classic understanding of molecular disease-mechanisms is largely based on protein-centric models. During the past decade however, genetic studies have identified numerous disease-loci in the human genome that do not encode proteins. Such non-coding DNA variants increasingly gain attention in diagnostics and personalized medicine. Of particular interest are long non-coding RNA (lncRNA) genes, which generate transcripts longer than 200 nucleotides that are not translated into proteins. While most of the estimated ~20,000 lncRNAs currently remain of unknown function, a growing number of genetic studies link lncRNA gene aberrations with the development of human diseases, including diabetes, AIDS, inflammatory bowel disease, or cancer. This suggests that the protein-centric view of human diseases does not capture the full complexity of molecular patho-mechanisms, with important consequences for molecular diagnostics and therapy. This review illustrates well-documented lncRNA gene aberrations causatively linked to human diseases and discusses potential lessons for molecular disease models, diagnostics, and therapy.

ABSTRACT Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneumophila infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages. IMPORTANCE Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila.

ABSTRACT A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens. IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen’s infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.

The knowledge of cytogenetics in the harvestmen family Phalangiidae has been based on taxa from the Northern Hemisphere. We performed cytogenetic analysis on Guruia africana (Karsch, 1878) (2n=24) and four species of the genus Rhampsinitus Simon, 1879 (2n=24, 26, 34) from South Africa. Fluorescence in situ hybridization with an 18S rDNA probe was used to analyze the number and the distribution of this cluster in the family Phalangiidae for the first time. The results support the cytogenetic characteristics typical for the majority of harvestmen taxa, i.e. the predominance of small biarmed chromosomes and the absence of morphologically well-differentiated sex chromosomes as an ancestral state. We identified the number of 18S rDNA sites ranging from two in R. qachasneki Kauri, 1962 to seven in one population of R. leighi Pocock, 1903. Moreover, we found differences in the number and localization of 18S rDNA sites in R. leighi between populations from two localities and between sexes of R. capensis (Loman, 1898). The heterozygous states of the 18S rDNA sites in these species may indicate the presence of XX/XY and ZZ/ZW sex chromosomes, and the possible existence of these systems in harvestmen is discussed. The variability of the 18S rDNA sites indicates intensive chromosomal changes during the differentiation of the karyotypes, which is in contrast to the usual uniformity in chromosomal morphology known from harvestmen so far.

Purpose: To investigate non-cycloplegic changes in refractive error prior to the onset of presbyopia. Methods: The Aston Longitudinal Assessment of Presbyopia (ALAP) study is a prospective 2.5 year longitudinal study, measuring objective refractive error using a binocular open-field WAM-5500 autorefractor at 6-month intervals in participants aged between 33 and 45 years. Results: From the 58 participants recruited, 51 participants (88%) completed the final visit. At baseline, 21 participants were myopic (MSE -3.25 ± 2.28 DS; baseline age 38.6 ± 3.1 years) and 30 were emmetropic (MSE −0.17 ± 0.32 DS; baseline age 39.0 ± 2.9 years). After 2.5 years, 10% of the myopic group experienced a hypermetropic shift (≥0.50 D), 5% a myopic shift (≥0.50 D) and 85% had no significant change in refraction (

A synthetic material of silicone rubber was used to construct an artificial lens capsule (ALC) in order to replicate the biomechanical behaviour of human lens capsule. The silicone rubber was characterised by monotonic and cyclic mechanical tests to reveal its hyper-elastic behaviour under uniaxial tension and simple shear as well as the rate independence. A hyper-elastic constitutive model was calibrated by the testing data and incorporated into finite element analysis (FEA). An experimental setup to simulate eye focusing (accommodation) of ALC was performed to validate the FEA model by evaluating the shape change and reaction force. The characterisation and modelling approach provided an insight into the intrinsic behaviour of materials, addressing the inflating pressure and effective stretch of ALC under the focusing process. The proposed methodology offers a virtual testing environment mimicking human capsules for the variability of dimension and stiffness, which will facilitate the verification of new ophthalmic prototype such as accommodating intraocular lenses (AIOLs).

Presbyopia is a common eye disorder among aged people which is attributed to the loss of accommodation of the crystalline lens due to the increasing stiffness. One of the potential techniques to correct presbyopia involves removing the lens substance inside the capsule and replacing it with an artificial lens. The development of such devices, e.g., accommodating intraocular lenses (AIOLs), relies on the understanding of the biomechanical behaviour of the lens capsule and the essential design verification ex vivo. To mimic the eye’s dynamic focusing ability (accommodation), an artificial lens capsule (ALC), from silicone rubber accompanied by a lens radial stretching system (LRSS) was developed. The ALC was manufactured to offer a dimension and deforming behaviour replicating the human lens capsule. The LRSS was calibrated to provide a radial stretch simulating the change of diameter of capsules during accommodating process. The biomechanical function of the ALC was addressed by studying its evolution behaviour and reaction force under multiaxial stretch from the LRSS. The study highlighted the convenience of this application by performing preliminary tests on prototypes of ophthalmic devices (e.g., AIOLs) to restore accommodation.

Significant treatment variation exists in the Netherlands between teams treating patients with cleft lip, alveolus, and/or palate, resulting in a confusing and undesirable situation for patients, parents, and practitioners. Therefore, to optimize cleft care, clinical practice guidelines (CPGs) were developed. The aim of this report is to describe CPG development, share the main recommendations, and indicate knowledge gaps regarding cleft care. Together with patients and parents, a multidisciplinary working group of representatives from all relevant disciplines assisted by two experienced epidemiologists identified the topics to be addressed in the CPGs. Searching the Medline, Embase, and Cochrane Library databases identified 5157 articles, 60 of which remained after applying inclusion and exclusion criteria. We rated the quality of the evidence from moderate to very low. The working group formulated 71 recommendations regarding genetic testing, feeding, lip and palate closure, hearing, hypernasality, bone grafting, orthodontics, psychosocial guidance, dentistry, osteotomy versus distraction, and rhinoplasty. The final CPGs were obtained after review by all stakeholders and allow cleft teams to base their treatment on current knowledge. With high-quality evidence lacking, the need for additional high-quality studies has become apparent.

While the important functions of long noncoding RNAs (lncRNAs) in nuclear organization are well documented, their orchestrating and architectural roles in the cytoplasmic environment have long been underestimated. However, recently developed fractionation and proximity labelling approaches have shown that a considerable proportion of cellular lncRNAs is exported into the cytoplasm and associates nonrandomly with proteins in the cytosol and organelles. The functions of these lncRNAs range from the control of translation and mitochondrial metabolism to the anchoring of cellular components on the cytoskeleton and regulation of protein degradation at the proteasome. In the present review, we provide an overview of the functions of lncRNAs in cytoplasmic structures and machineries und discuss their emerging roles in the coordination of the dense intracellular milieu. It is becoming apparent that further research into the functions of these lncRNAs will lead to an improved understanding of the spatiotemporal organization of cytoplasmic processes during homeostasis and disease.

Abstract. Introduction:. This article reports the content validation of a Critical Appraisal Tool designed to Review the quality of Analgesia Studies (CATRAS) involving subjects incapable of self-reporting pain and provide guidance as to the strengths and weakness of findings. The CATRAS quality items encompass 3 domains: level of evidence, methodological soundness, and grading of the pain assessment tool. Objectives:. To validate a critical appraisal tool for reviewing analgesia studies involving subjects incapable of self-reporting pain. Methods:. Content validation was achieved using Delphi methodology through panel consensus. A panel of 6 experts reviewed the CATRAS in 3 rounds and quantitatively rated the relevance of the instrument and each of its quality items to their respective domains. Results:. Content validation was achieved for each item of the CATRAS and the tool as a whole. Item-level content validity index and kappa coefficient were at least greater than 0.83 and 0.81, respectively, for all items except for one item in domain 2 that was later removed. Scale-level content validity index was 97% (excellent content validity). Conclusions:. This 67-item critical appraisal tool may enable critical and quantitative assessment of the quality of individual analgesia trials involving subjects incapable of self-reporting pain for use in systematic reviews and meta-analysis studies.

One of the aims of South African National Survey of Arachnida (SANSA) is to survey protected areas to obtain species-specific information and compile inventories to determine species distribution patterns and evaluate their conservation status for Red Data assessments. The aim of this study, the first in a series of surveys of the Diamond Route Reserves, was to compile the first checklist of the spider species in the Northern Cape at the Tswalu Kalahari Reserve. Spiders were collected during three survey periods (2005−2013) using different collecting methods to sample both the ground and field layers. In total, 32 families represented by 108 genera and 136 species have been collected so far. The most species-rich families are the Salticidae (20 spp.) and Thomisidae (18 spp.), followed by the Gnaphosidae and Araneidae (11 spp. each), while nine families are represented by singletons. The free-living wandering spiders represent 97 spp., while 39 spp. are web-builders. Information on spider guilds, endemicity value and conservation status are provided. The Tswalu Kalahari Reserve protects approximately 6.1% of the total South African spider fauna, while 24.3% of the species found in the reserve are South African endemics, of which 5.9% are Northern Cape endemics. Approximately 6.0% of the species sampled are possibly new to science or represent new records for South Africa. Conservation implications: The Tswalu Kalahari Reserve falls within the Savanna Biome in the Northern Cape province. Only one spider species was previously known from the reserve; a further 135 spp. are reported for the first time, with 5.9% of the species being Northern Cape endemics and 24.3% South African endemics. Approximately 6.0% of the species may be new to science or represent new records for South Africa.

CRISPR/Cas9-based approaches have greatly facilitated targeted genomic deletions. Contrary to coding genes however, which can be functionally knocked out by frame-shift mutagenesis, non-coding RNA (ncRNA) gene knockouts have remained challenging. Here we present a universal ncRNA knockout approach guided by epigenetic hallmarks, which enables robust gene silencing even in provisionally annotated gene loci. We build on previous work reporting the presence of overlapping histone H3 lysine 4 tri-methylation (H3K4me3) and DNaseI hypersensitivity sites around the transcriptional start sites of most genes. We demonstrate that excision of this gene-proximal signature leads to loss of microRNA and lincRNA transcription and reveals ncRNA phenotypes. Exemplarily we demonstrate silencing of the constitutively transcribed MALAT1 lincRNA gene as well as of the inducible miR-146a and miR-155 genes in human monocytes. Our results validate a role of miR-146a and miR-155 in negative feedback control of the activity of inflammation master-regulator NFκB and suggest that cell-cycle control is a unique feature of miR-155. We suggest that our epigenetically guided CRISPR approach may improve existing ncRNA knockout strategies and contribute to the development of high-confidence ncRNA phenotyping applications.

Purpose: The aim of the study was to determine the effect of optimal spectral filters on reading performance following stroke. Methods: Seventeen stroke subjects, aged 43–85, were considered with an age-matched Control Group (n = 17). Subjects undertook the Wilkins Rate of Reading Test on three occasions: (i) using an optimally selected spectral filter; (ii) subjects were randomly assigned to two groups: Group 1 used an optimal filter, whereas Group 2 used a grey filter, for two-weeks. The grey filter had similar photopic reflectance to the optimal filters, intended as a surrogate for a placebo; (iii) the groups were crossed over with Group 1 using a grey filter and Group 2 given an optimal filter, for two weeks, before undertaking the task once more. An increase in reading speed of >5% was considered clinically relevant. Results: Initial use of a spectral filter in the stroke cohort, increased reading speed by ∼8%, almost halving error scores, findings not replicated in controls. Prolonged use of an optimal spectral filter increased reading speed by >9% for stroke subjects; errors more than halved. When the same subjects switched to using a grey filter, reading speed reduced by ∼4%. A second group of stroke subjects used a grey filter first; reading speed decreased by ∼3% but increased by ∼4% with an optimal filter, with error scores almost halving. Conclusions: The present study has shown that spectral filters can immediately improve reading speed and accuracy following stroke, whereas prolonged use does not increase these benefits significantly.

Case summary This case report describes the clinical signs and treatment of an alfaxalone 10 times overdose in a 12-year-old cat undergoing anaesthesia for MRI. The cat was discharged from hospital following a prolonged recovery including obtunded mentation and cardiorespiratory depression for several hours following cessation of anaesthesia. The cat received supportive therapy that included supplemental oxygen via a face mask, intravenous crystalloid fluids and active rewarming. The benefits of using alfaxalone for maintenance of anaesthesia, its pharmacokinetics and previously reported lethal doses are discussed. Strategies for reducing the incidence of medication errors are presented. Relevance and novel information An unintentional overdose of alfaxalone by continuous rate infusion has not been reported previously in a cat. Treatment is supportive and directed towards maintenance of the cardiorespiratory systems. Whenever possible, smart pumps that have been designed to reduce human error should be used to help prevent medication errors associated with continuous rate infusions.

This study aimed to quantify biometric modifications of the anterior segment (AS) during accommodation and to compare them against changes in both accommodative demand and response. Thirty adults, aged 18–25 years were rendered functionally emmetropic with contact lenses. AS optical coherence tomography (AS-OCT) images were captured along the 180° meridian (Visante, Zeiss Meditec, Jena, Germany) under stimulated accommodative demands (0–4 D). Images were analysed and lens thickness (LT) was measured, applying a refractive index correction of 1.00. Accommodative responses were also measured sequentially through a Badal optical system fitted to an autorefractor (Shin Nippon NVision-K 5001, Rexxam, Japan). Data were compared with Dubbelman schematic eye calculations. Significant changes occurred in LT, anterior chamber depth (ACD), lens centroid (i.e., ACD + LT/2), and AS length (ASL = ACD + LT) with accommodation (all p < 0.01). There was no significant change in CT with accommodation (p = 0.81). Measured CT, ACD, and lens centroid values were similar to Dubbelman modelled parameters, however AS-OCT overestimated LT and ASL. As expected, the accommodative response was less than the demand. Interestingly, up until approximately 1.5 D of response (2.0 D demand), the anterior crystalline lens surface appears to be the primary correlate. Beyond this point, the posterior lens surface moves posteriorly resulting in an over-all sigmoidal trajectory. he posterior crystalline lens surface demonstrates a sigmoidal response with increasing accommodative effort.

We investigate the biological effects of radiation using adult Drosophila melanogaster as a model organism, focusing on gene expression and lifespan analysis to determine the effect of different radiation doses. Our results support a threshold effect in response to radiation: no effect on lifespan and no permanent effect on gene expression is seen at incident radiation levels below 100 J/kg. We also find that it is more appropriate to compare radiation effects in flies using the absorbed energy rather than incident radiation levels.

Purpose. To examine the influence of positional misalignments on intraocular pressure (IOP) measurement with a rebound tonometer. Methods. Using the iCare rebound tonometer, IOP readings were taken from the right eye of 36 healthy subjects at the central corneal apex (CC) and compared to IOP measures using the Goldmann applanation tonometer (GAT). Using a bespoke rig, iCare IOP readings were also taken 2 mm laterally from CC, both nasally and temporally, along with angular deviations of 5 and 10 degrees, both nasally and temporally to the visual axis. Results. Mean IOP ± SD, as measured by GAT, was 14.7±2.5 mmHg versus iCare tonometer readings of 17.4±3.6 mmHg at CC, representing an iCare IOP overestimation of 2.7±2.8 mmHg (P

Accurate estimation of biological diversity in environmental DNA samples using high-throughput amplicon pyrosequencing must account for errors generated by PCR and sequencing. We describe a novel approach to distinguish the underlying sequence diversity in environmental DNA samples from errors that uses information on the abundance distribution of similar sequences across independent samples, as well as the frequency and diversity of sequences within individual samples. We have further refined this approach into a bioinformatics pipeline, Amplicon Pyrosequence Denoising Program (APDP) that is able to process raw sequence datasets into a set of validated sequences in formats compatible with commonly used downstream analyses packages. We demonstrate, by sequencing complex environmental samples and mock communities, that APDP is effective for removing errors from deeply sequenced datasets comprising biological and technical replicates, and can efficiently denoise single-sample datasets. APDP provides more conservative diversity estimates for complex datasets than other approaches; however, for some applications this may provide a more accurate and appropriate level of resolution, and result in greater confidence that returned sequences reflect the diversity of the underlying sample.