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Leukotriene B 4 (LTB 4 ) is a potent lipid chemoattractant driving inflammatory responses during host defense, allergy, autoimmune and metabolic diseases. Gradients of LTB 4 orchestrate leukocyte recruitment and swarming to sites of tissue damage and infection. How LTB 4 gradients form and spread in live tissues to regulate these processes remains largely elusive due to the lack of suitable tools for monitoring LTB 4 levels in vivo. Here, we develop GEM-LTB 4 , a genetically encoded green fluorescent LTB 4 biosensor based on the human G-protein-coupled receptor BLT1. GEM-LTB 4 shows high sensitivity, specificity and a robust fluorescence increase in response to LTB 4 without affecting downstream signaling pathways. We use GEM-LTB 4 to measure ex vivo LTB 4 production of murine neutrophils. Transgenic expression of GEM-LTB 4 in zebrafish allows the real-time visualization of both exogenously applied and endogenously produced LTB 4 gradients. GEM-LTB 4 thus serves as a broadly applicable tool for analyzing LTB 4 dynamics in various experimental systems and model organisms., (© 2023. The Author(s).)

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission. Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The CHARMM Drude-2013 polarizable force field (FF) was developed to include the explicit treatment of induced electronic polarizability, resulting in a more accurate description of the electrostatic interactions in molecular dynamics (MD) simulations. While the Drude-2013 protein FF has shown success in improving the folding properties of α-helical peptides and to reproduce experimental observables in simulations up to 1 μs, some limitations were noted regarding the stability of β-sheet structures in simulations longer than 100 ns as well as larger deviations from crystal structures in simulations of a number of proteins compared to the additive CHARMM36 protein FF. The origin of the instability has been identified and appears to be primarily due to overestimated atomic polarizabilities and induced dipole-dipole interactions on the Cβ, Cγ, and Cδ side chain atoms. To resolve this and other issues, a number of aspects of the model were revisited, resulting in Drude-2019 protein FF. Backbone parameters were optimized targeting the conformational properties of the (Ala) 5 peptide in solution along with gas phase properties of the alanine dipeptide. Dipeptides that contain N-acetylated and N'-methylamidated termini, excluding Gly, Pro, and Ala, were used as models to optimize the atomic polarizabilities and Thole screening factors on selected Cβ, Cγ, and Cδ carbons by targeting quantum mechanical (QM) dipole moments and molecular polarizabilities. In addition, to obtain better conformational properties, side chain χ 1 and χ 2 dihedral parameters were optimized targeting QM data for the respective side chain dipeptide conformations as well as Protein Data Bank survey data based on the χ 1 , χ 2 sampling from Hamiltonian replica-exchange MD simulations of (Ala) 4 -X-(Ala) 4 in solution, where X is the amino acid of interest. Further improvements include optimizing nonbonded interactions between charged residues to reproduce QM interaction energies of the charged-protein model compounds and experimental osmotic pressures. Validation of the optimized Drude protein FF includes MD simulations of a collection of peptides and proteins including β-sheet structures, as well as transmembrane ion channels. Results showed that the updated Drude-2019 protein FF yields smaller overall root-mean-square differences of proteins as compared to the additive CHARMM36m and Drude-2013 FFs as well as similar or improved agreement with experimental NMR properties, allowing for long time scale simulation studies of proteins and more complex biomolecular systems in conjunction with the remainder of the Drude polarizable FF.

Phenotypic changes in lung fibroblasts are believed to contribute to the development of Idiopathic Pulmonary Fibrosis (IPF), a progressive and fatal lung disease. Long intergenic non-coding RNAs (lincRNAs) have been identified as novel regulators of gene expression and protein activity. In non-stimulated cells, we observed reduced proliferation and inflammation but no difference in the fibrotic response of IPF fibroblasts. These functional changes in non-stimulated cells were associated with changes in the expression of the histone marks, H3K4me1, H3K4me3 and H3K27ac indicating a possible involvement of epigenetics. Following activation with TGF-β1 and IL-1β, we demonstrated an increased fibrotic but reduced inflammatory response in IPF fibroblasts. There was no significant difference in proliferation following PDGF exposure. The lincRNAs, LINC00960 and LINC01140 were upregulated in IPF fibroblasts. Knockdown studies showed that LINC00960 and LINC01140 were positive regulators of proliferation in both control and IPF fibroblasts but had no effect upon the fibrotic response. Knockdown of LINC01140 but not LINC00960 increased the inflammatory response, which was greater in IPF compared to control fibroblasts. Overall, these studies demonstrate for the first time that lincRNAs are important regulators of proliferation and inflammation in human lung fibroblasts and that these might mediate the reduced inflammatory response observed in IPF-derived fibroblasts.

There is accumulating evidence to indicate that long non-coding RNAs (lncRNAs) are important regulators of the inflammatory response. In this report, we have employed next generation sequencing to identify 14 lncRNAs that are differentially expressed in human lung fibroblasts following the induction of inflammation using interleukin-1β (IL-1β). Knockdown of the two most highly expressed lncRNAs, IL7AS, and MIR3142HG, showed that IL7AS negatively regulated IL-6 release whilst MIR3142HG was a positive regulator of IL-8 and CCL2 release. Parallel studies in fibroblasts derived from patients with idiopathic pulmonary fibrosis showed similar increases in IL7AS levels, that also negatively regulate IL-6 release. In contrast, IL-1β-induced MIR3142HG expression, and its metabolism to miR-146a, was reduced by 4- and 9-fold in IPF fibroblasts, respectively. This correlated with a reduced expression of inflammatory mediators whilst MIR3142HG knockdown showed no effect upon IL-8 and CCL2 release. Pharmacological studies showed that IL-1β-induced IL7AS and MIR3142HG production and release of IL-6, IL-8, and CCL2 in both control and IPF fibroblasts were mediated via an NF-κB-mediated pathway. In summary, we have cataloged those lncRNAs that are differentially expressed following IL-1β-activation of human lung fibroblasts, shown that IL7AS and MIR3142HG regulate the inflammatory response and demonstrated that the reduced inflammatory response in IPF fibroblast is correlated with attenuated expression of MIR3142HG/miR-146a.

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLR•RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLR•RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLR•RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLR•RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KR•RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLR•RAMP2.

Despite increasing evidence to indicate that long non-coding RNAs (lncRNAs) are novel regulators of immunity, there has been no systematic attempt to identify and characterize the lncRNAs whose expression is changed following the induction of the innate immune response. To address this issue, we have employed next-generation sequencing data to determine the changes in the lncRNA profile in four human (monocytes, macrophages, epithelium, and chondrocytes) and four mouse cell types (RAW 264.7 macrophages, bone marrow-derived macrophages, peritoneal macrophages, and splenic dendritic cells) following exposure to the pro-inflammatory mediators, lipopolysaccharides (LPS), or interleukin-1β. We show differential expression of 204 human and 210 mouse lncRNAs, with positional analysis demonstrating correlation with immune-related genes. These lncRNAs are predominantly cell-type specific, composed of large regions of repeat sequences, and show poor evolutionary conservation. Comparison within the human and mouse sequences showed less than 1% sequence conservation, although we identified multiple conserved motifs. Of the 204 human lncRNAs, 21 overlapped with syntenic mouse lncRNAs, of which five were differentially expressed in both species. Among these syntenic lncRNA was IL7-AS (antisense), which was induced in multiple cell types and shown to regulate the production of the pro-inflammatory mediator interleukin-6 in both human and mouse cells. In summary, we have identified and characterized those lncRNAs that are differentially expressed following activation of the human and mouse innate immune responses and believe that these catalogs will provide the foundation for the future analysis of the role of lncRNAs in immune and inflammatory responses.

Myositis is characterised by muscle inflammation and weakness. Although generally thought to be driven by a systemic autoimmune response, increasing evidence suggests that intrinsic changes in the muscle might also contribute to the pathogenesis. Long non-coding RNAs (lncRNAs) are a family of novel genes that regulate gene transcription and translation. To determine the potential role of lncRNAs, we employed next generation sequencing to examine the transcriptome in muscle biopsies obtained from two histologically distinct patient populations, inclusion body myositis (IBM) and anti-Jo-1-associated myositis (Jo-1). 1287 mRNAs and 1068 mRNAs were differentially expressed in the muscle from Jo-1 and IBM patients, respectively. Pathway analysis showed the top canonical pathway in both Jo-1 and IBM was oxidative phosphorylation and mitochondrial dysfunction. We identified 731 known and 325 novel lncRNAs in the muscles biopsies. Comparison with controls showed 55 and 46 lncRNAs were differentially expressed in IBM and Jo-1 myositis, respectively. Of these, 16 lncRNAs were differentially expressed in both IBM and Jo-1 myositis and included upregulated H19, lncMyoD and MALAT1. Given that these are known to regulate muscle proliferation and differentiation, we speculate that changes in lncRNAs might contribute to the phenotypic changes in Jo-1 and IBM myositis.

Numerous applications of liquid crystals rely on control of molecular orientation at an interface. However, little is known about the precise molecular structure of such interfaces. In this work, synchrotron X-ray reflectivity measurements, accompanied by large-scale atomistic molecular dynamics simulations, are used for the first time to reconstruct the air-liquid crystal interface of a nematic material, namely, 4-pentyl-4'-cyanobiphenyl (5CB). The results are compared to those for 4-octyl-4'-cyanobiphenyl (8CB) which, in addition to adopting isotropic and nematic states, can also form a smectic phase. Our findings indicate that the air interface imprints a highly ordered structure into the material; such a local structure then propagates well into the bulk of the liquid crystal, particularly for nematic and smectic phases.

The human genome is widely transcribed outside of protein-coding genes, producing thousands of noncoding RNAs from different subfamilies including enhancer RNAs. Functional studies to determine the role of individual genes are challenging with noncoding RNAs appearing to be more difficult to knockdown than mRNAs. One factor that may have hindered progress is that the majority of noncoding RNAs are thought to be located within the nucleus, where the efficiency of traditional RNA interference techniques is debatable. Here we present an alternative RNA interference technique utilizing Locked Nucleic Acids, which is able to efficiently knockdown noncoding RNAs irrespective of intracellular location.

Objective: To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA., Methods: OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non-OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase-polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex., Results: RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin-1β (IL-1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50-associated cyclooxygenase 2-extragenic RNA (PACER) and 2 novel chondrocyte inflammation-associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non-OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL-1-stimulated secretion of proinflammatory cytokines., Conclusion: The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation-driven cartilage degeneration in OA., (© 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Recent studies have indicated that non-coding RNAs transcribed from enhancer regions are important regulators of enhancer function and gene expression. In this report, we have characterised the expression of six enhancer RNAs (eRNAs) induced in human monocytic THP1 cells following activation of the innate immune response by lipopolysaccharide (LPS). Specifically, we have demonstrated that LPS-induced expression of individual eRNAs is mediated through divergent intracellular signalling pathways that includes NF-κB and the mitogen activated protein kinases, extracellular regulated kinase-1/2 and p38., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

G protein-coupled receptors (GPCRs) are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types. Here, we review some of the main interacting proteins of GPCRs. A greater understanding of the mechanisms regulating their interactions may lead to the discovery of new drug targets for therapy.

Closed surgical mitral valvotomy is the procedure of choice in most patients with symptomatic pliable mitral stenosis in developing countries. The procedure is efficacious and safe. Mitral valvotomy performed with a balloon has shown similar good results, with infrequent complications in selected subjects. Because there is a paucity of studies comparing the two techniques, this study was undertaken to compare the results of percutaneous balloon mitral valvuloplasty with those of closed commissurotomy as determined by catheterization studies. Forty-five patients with tight pliable mitral stenosis were randomly assigned to one of two groups: 23 patients had balloon valvuloplasty by the single catheter technique (group I) and 22 underwent closed surgical valvotomy (group II). The two groups were similar with regard to clinical and hemodynamic findings before intervention. Mitral valve area increased from 0.8 +/- 0.3 to 2.1 +/- 0.7 cm2 in group I (p less than 0.001) and from 0.7 +/- 0.2 to 1.3 +/- 0.3 cm2 in group II (p less than 0.001). Pulmonary artery pressure and pulmonary vascular resistance decreased in both groups, but these changes did not reach statistical significance in group II. Treadmill exercise time increased from 3.8 +/- 2.3 to 7.3 +/- 2.6 min in group I (p less than 0.001) and from 4 +/- 2.6 to 5.6 +/- 2.6 min in group II (p less than 0.001). There were no deaths. One patient in each group developed moderate (3+) mitral regurgitation. A small interatrial shunt (less than 1.5:1) was detected in three patients in group I immediately after the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Only 1 case of lymphomatoid granulomatosis has previously been reported from South Africa. Experience with 4 such adult patients (2 blacks and 2 whites) is described. These patients were followed up for 15-48 months and none developed evidence of a lymphoma during this period. Fever, weight loss, cough and breathlessness were prominent symptoms in all patients. One patient, a black woman, with a diffuse interstitial pattern of lung involvement, had digital clubbing--a rare accompaniment that resolved after therapy. Dilated congestive cardiomyopathy was found in association with pulmonary nodules in a black male patient. All 4 patients were treated with cytotoxic regimens. The 2 patients treated with oral cyclophosphamide and prednisolone responded favourably. The possible explanation for paucity of reports of lymphomatoid granulomatosis from South Africa could be under-reporting, underdiagnosis or a true geographic/ethnic variation in the incidence of this condition.

The occurrence of a bleeding placenta praevia in a young woman, with a viable fetus and an acutely obstructed Lillehei-Kaster valve, provided serious difficulties in priorities and decision making. This has prompted an analysis of the problems which others may encounter.

One hundred and eighty-one patients with carcinoma of the upper thoracic oesophagus were intubated perorally using a Procter-Livingstone tube. The mortality was 16-6 per cent but, in the patients who survived, the palliation achieved, as judged by improved swallowing, was considered satisfactory. Factors influencing the success of intubation are also considered.

The life of a young woman was threatened by a large, recurrent, invasive desmoid tumour which penetrated the sternum. The combined thoracic and plastic surgical management is described.

This paper reviews our experience with the Lillehei-Kaster pivoting disc prosthesis in 155 patients with aortic and mitral valve disease. We employed 189 valves during the period 1971 to 1974. The early surgical mortality rates for isolated mitral, isolated aortic, and combined mitral and aortic valve replacements were 9 per cent, 14 per cent, and 3 per cent, respectively. Postoperatively, there was no evidence of significant hemolysis, and the gradients across the prostheses were satisfactory. Clinical evaluation of prosthetic function was made difficult by the infrequency of an opening click and the common occurrence of mid-diastolic murmurs even with minimal gradients. The most disturbing complication, which has led to our abandoning use of this valve, was thrombosis, which occurred in at least 10 per cent of the mitral and 5 per cent of the aortic valves. The cause is thought to be late prosthetic disproportion as the heart shrinks in size.

Malignant esophago-respiratory fistula is an incurable condition calsing severe distress to those patients suffering from it. Sixty two successive patients with esophago-respiratory fistulae, secondary to squamous cell carcinomas of the esophagus, were intubated for palliation. The first 14 were intubated by a traction technique using the Celestin tube, and the remaining 48 were intubated by the Procter-Livingstone tube inserted by a pulsion method. Celestin intubation had a mortality of 64.3% but 75% of those intubated by the Procter-Livingstone tube were discharged from hospital, swallowing satisfactorily and relieved of their respiratory distress. In our experience the insertion of the Proctor-Livingstone tube by a pulsion technique, can be performed in a few minutes, with little morbidity and allows the patient to be discharged from hospital within 3-4 days of the procedure. It is a successful and acceptable method of treating malignant esophago-respiratory fistulae.

Isolated mitral valve surgery was carried out in 182 children under the age of 16 years. Closed valvulotomy was performed in 72, annuloplasty or reconstruction in 10 and valve replacement in 100 of the children. Preoperative atrial fibrillation implied severe disease. Acute rheumatic carditis was not a contraindication to operation. Two patients died after closed mitral commissurotomy and the results were unsatisfactory in nine children with valve calcification and in 12 with a small jet of incompetence. Restenosis occurred in 10 patients during a review period of five years. The long-term results of annuloplasty were disappointing, and this procedure is not recommended for children with mitral incompetence. Ten of the 100 patients who underwent mitral valve replacement died in hospital and there were two late deaths. Seventy-seven patients are completely well and 11 have grade II disability. Severe mitral valve disease in children is associated with high morbidity and mortality, unless normal hemodynamics are restored. Since the results of operation are so satisfactory, we believe that the natural history of this condition is most favorably altered by surgical treatment.

Thirty-five children under 16 years of age were prepared for pericardiectomy on account of constrictive pericarditis; three died in the immediate preparatory period. Pericardiectomy was undertaken in 32; in 28 the cause was tuberculous. The purpose of this report is to draw attention to the high incidence of constrictive pericarditis in African children in Natal.

Although the removal of hair as a pre-operative preparation of the skin is desirable, it need only be removed over an area limited to a planned incision, and then only in hirsute patients. The use of a depilatory cream as an alternative to a razor would seem to have the advantages of increasing patient comfort; of avoiding injuries inflicted by even skilful shaving; and perhaps of diminishing the incidence of wound infection, without significant risk of reaction to the depilatory. Routine extensive pre-operative hair removal in all patients is unnecessary.