ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.

Orthogonal Enzyme-Substrate Design Strategy for Discovery of Human Protein Palmitoyltransferase Substrates.

Protein palmitoylation, with more than 5000 substrates, is the most prevalent form of protein lipidation. Palmitoylated proteins participate in almost all areas of cellular physiology and have been linked to several human diseases. Twenty-three zDHHC enzymes catalyze protein palmitoylation with extensive overlap among the substrates of each zDHHC member. Currently, there is no global strategy to delineate the physiological substrates of individual zDHHC enzymes without perturbing the natural cellular pool. Here, we outline a general approach to accomplish this on the basis of synthetic orthogonal substrates that are only compatible with engineered zDHHC enzymes. We demonstrate the utility of this strategy by validating known substrates and use it to identify novel substrates of two human zDHHC enzymes. Finally, we employ this method to discover and explore conserved palmitoylation in a family of host restriction factors against pathogenic viruses, including SARS-CoV-2.

Exploring facilitators and barriers associated with oral care for inpatients with dysphagia post-stroke.

OBJECTIVE: To explore the attitudes, facilitators and barriers in providing oral care for inpatients with dysphagia post-stroke as perceived by healthcare professionals. BACKGROUND: Dysphagia is a common complication of stroke and is associated with a higher incidence of aspiration pneumonia, malnutrition and dehydration. In the acute phase of stroke recovery, a dental professional is not usually part of the multidisciplinary team caring for the patient and oral care is the responsibility of the healthcare professionals in the stroke unit. There is a lack of high-quality evidence to demonstrate the most effective method of providing oral care for patients with dysphagia post-stroke. MATERIALS AND METHODS: This was a single-site study conducted with healthcare professionals working in the Stroke Unit of Cork University Hospital in Ireland, using focus groups and a qualitative thematic analysis approach. RESULTS: A total of three focus groups were conducted in the Stroke Unit with 17 healthcare professionals. The focus groups included representation from all healthcare professional groups providing direct clinical care to patients on the Stroke Unit including geriatric medicine, dietetics, speech and language therapy, healthcare assistance, nursing, occupational therapy and physiotherapy. A qualitative thematic analysis was carried out and seven overarching themes emerged from the data. Three themes related to facilitators to providing oral care for this patient group: (i) a focus on oral care in both policy and practice, (ii) expanding professional roles in the provision of oral care, (iii) perceived importance of oral care in recovery and rehabilitation. Four themes related to barriers to the provision of oral care for this patient group: (i) lack of confidence and concerns related to the perceived risk for patients with dysphagia, (ii) unique challenges of patient and stroke-related factors, (iii) lack of resources and time and (iv) perceived importance of oral care in recovery and its relative importance with competing demands. CONCLUSION: Members of the stroke multidisciplinary team believe that they all have a part to play in the delivery of oral care for patients with dysphagia post-stroke. Opportunities exist for the development of multidisciplinary interventions to improve the oral cavity assessment and oral care provided in the Stroke Unit.

Chopped! Newfound GSDMD cleavage facilitates tolerance to food allergens.

In a recent article, He et al. report that, in response to dietary protein antigens, mouse intestinal epithelial cells (IECs) accumulate a newfound 13-kDa N terminus of gasdermin D (GSDMD-N13), cleaved by caspase-3/7. Unlike the pyroptotic 30-kDa fragment, GSDMD-N13 translocates to the nucleus, inducing CIITA and major histocompatibility complex class II (MHCII) expression to promote type 1 regulatory T (T1r) cell development, thus revealing its role in balancing immunity and food tolerance.

ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Here we report the surprising finding that GSDMD cleavage is not sufficient for its pore formation. Instead, GSDMD is lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation, suggesting that palmitoylation licenses GSDMD activation. Treatment by the palmitoylation inhibitor 2-bromopalmitate and alanine mutation of Cys191 abrogate GSDMD membrane localization, cytokine secretion, and cell death, without affecting GSDMD cleavage. Because palmitoylation is formed by a reversible thioester bond sensitive to free thiols, we tested if GSDMD palmitoylation is regulated by cellular redox state. Lipopolysaccharide (LPS) mildly and LPS plus the NLRP3 inflammasome activator nigericin markedly elevate reactive oxygen species (ROS) and GSDMD palmitoylation, suggesting that these two processes are coupled. Manipulation of cellular ROS by its activators and quenchers augment and abolish, respectively, GSDMD palmitoylation, GSDMD pore formation and cell death. We discover that zDHHC5 and zDHHC9 are the major palmitoyl transferases that mediate GSDMD palmitoylation, and when cleaved, recombinant and partly palmitoylated GSDMD is 10-fold more active in pore formation than bacterially expressed, unpalmitoylated GSDMD, evidenced by liposome leakage assay. Finally, other GSDM family members are also palmitoylated, suggesting that ROS stress and palmitoylation may be a general switch for the activation of this pore-forming family. One-Sentence Summary: GSDMD palmitoylation is induced by ROS and required for pore formation.

S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection, in vitro reconstitution and role in viral infectivity.

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of ß-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.

Attending community-based lung cancer screening influences smoking behaviour in deprived populations.

OBJECTIVES: The impact of lung cancer screening on smoking is unclear, especially in deprived populations who are underrepresented in screening trials. The aim of this observational cohort study was to investigate whether a community-based lung cancer screening programme influenced smoking behaviour and smoking attitude in socio-economically deprived populations. MATERIAL AND METHODS: Ever-smokers, age 55-74, registered at participating General Practices were invited to a community-based Lung Health Check (LHC). This included an assessment of respiratory symptoms, lung cancer risk (PLCOm2012), spirometry and signposting to stop smoking services. Those at high risk (PLCOM2012≥1.51%) were offered annual low-dose CT screening over two rounds. Self-reported smoking status and behaviour were recorded at the LHC and again 12 months later, when attitudes to smoking were also assessed. RESULTS: 919 participants (51% women) were included in the analysis (77% of attendees); median deprivation rank in the lowest decile for England. At baseline 50.3% were current smokers. One-year quit rate was 10.2%, quitting was associated with increased baseline symptoms (adjOR 2.62, 95% CI 1.07-6.41; p = 0.035) but not demographics or screening results. 55% attributed quitting to the LHC. In current smokers, 44% reported the LHC had made them consider stopping, 29% it made them try to stop and 25% made them smoke less whilst only 1.7% and 0.7% said it made them worry less about smoking or think it acceptable to smoke. CONCLUSIONS: Our data suggest a community-based lung cancer screening programme in deprived areas positively impacts smoking behaviour, with no evidence of a 'licence to smoke' in those screened.

Developing a disease management program for the improvement of heart failure outcomes: the do's and the don'ts.

INTRODUCTION: Heart failure is a highly prevalent condition affecting approximately 2% of people worldwide. Heart failure disease management programs (DMP) have shown a reduction in mortality and reduced hospitalization and are an established part of clinical guidelines; however, their presence is not widespread. Focusing on the application of proven therapies, patient education, diagnosis with work up of cause and easy access for clinical deterioration should be fundamental to the structure of the DMP. Multidisciplinary team care with early and timely recognition of potentially critical patients is essential, along with the inclusion of patients diagnosed in hospital as well as the community. Areas covered: The fundamental structure of a DMP along with the current gaps in evidence is outlined. Current challenges with the heart failure condition along with the current best evidence are covered. Articles were searched using MEDLINE containing the keywords; Chronic Heart Failure, Disease Management Program. We have also provided clinical opinion. Expert opinion: A multidisciplinary approach to disease management programs is essential to providing adequate care to patients. DMPs are an established part of current guidelines and should be a benchmark of treatment. Future resources should be focused on identifying patients at risk and early prevention.

Relapse after treatment withdrawal of antiepileptic drugs for Juvenile Absence Epilepsy and Juvenile Myoclonic Epilepsy.

PURPOSE: Conventional teaching is that juvenile myoclonic epilepsy (JME) and juvenile absence epilepsy (JAE) require lifelong antiepileptic drug (AED) treatment. We therefore wanted to determine how many patients attending our epilepsy service with JAE or JME went into 2 year remission, and then relapsed, both off and on AEDs. METHOD: This was a retrospective case-notes review. Patients with JAE and JME were systematically ascertained from clinic lists and databases at one teaching hospital. Data was extracted systematically. Simple descriptive statistics were used. RESULTS: JAE: 14/36 (39%) were seizure free on AEDs for at least 2 years. Of the 6 (43%) attempting AED withdrawal, all (100%) relapsed, compared with only 25% of those who did not withdraw AEDs. Only 2/5 who relapsed and restarted AEDs regained remission. JME: 32/145 (22%) were seizure free on AEDs for at least 2 years. Of the 10 (31%) attempting AED withdrawal, 8 (80%) relapsed, compared with only 36% of those who did not withdraw AEDs. Only 2/8 who relapsed and restarted AEDs regained remission. CONCLUSION: Remission rates for JAE and JME was lower than expected. Higher proportions of seizure free patients underwent physician-supervised withdrawal than anticipated. Relapse rates off AEDs were similar for JAE and JME, and at least twice as high as for those remaining on AEDs, and a further remission was not invariable on restarting AEDs. Our experience, comparing relapse in those withdrawing to those staying on AEDs will help in discussions with patients keen to try AED withdrawal.

An unexpected rash with gastroenteritis: erythema multiforme associated with campylobacter infection.

We present the first documented case of erythema multiforme following campylobacter gastroenteritis.

Effects of oral contrast on dose in abdominopelvic computed tomography with pure iterative reconstruction.

AIM: To assess the effect of neutral (NC) and positive (PC) oral contrast use on patient dose in low-dose abdominal computed tomography (CT). METHODS: Low-dose clinically indicated CTs were performed on 79 Crohn's patients (35 = PC, 1 L 2% gastrografin; 44 = NC, 1.5 L polyethylene glycol). Scanner settings for both acquisitions were identical apart from 25 s difference in intravenous contrast timing. Body mass index (BMI), scan-ranges, dose-length product and size-specific dose estimated were recorded. Data was reconstructed with pure model-based iterative reconstruction. Image quality was objectively and subjectively analysed. Data analysis was performed with Statistical Package for Social Scientists. RESULTS: Higher doses were seen in neutral contrast CTs (107.60 ± 78.7 mGy.cm, 2.47 ± 1.21 mGy vs 85.65 ± 58.2 mGy.cm, 2.18 ± 0.96 mGy). The difference was significant in 2 of 4 BMI groups and in those that had both NC and PC investigations. Image-quality assessment yielded 6952 datapoints. NC image quality was significantly superior (P < 0.001) (objective noise, objective signal to noise ratio, subjective spatial resolution, subjective contrast resolution, diagnostic acceptability) at all levels. NC bowel distension was significantly (P < 0.001) superior. CONCLUSION: The use of polyethylene glycol as a neutral OC agent leads to higher radiation doses than standard positive contrast studies, in low dose abdominal CT imaging. This is possibly related to the osmotic effect of the agent resulting in larger intraluminal fluid volumes and resultant increased overall beam attenuation.

Effects of centrally acting angiotensin converting enzyme inhibitors on functional decline in patients with Alzheimer's disease.

BACKGROUND: Centrally acting angiotensin converting enzyme inhibitors (CACE-Is) are associated with reduced rates of cognitive decline in patients with dementia. CACE-Is may also improve exercise tolerance in functionally impaired older adults with normal cognition, suggesting that CACE-Is may positively influence activities of daily living (ADL) in dementia. OBJECTIVE: To compare rates of decline in patients with mild to moderate Alzheimer's disease (AD) receiving CACE-Is to those not currently treated with CACE-Is (NoCACE-I), included in the Doxycycline and Rifampicin for Alzheimer's Disease study (n = 406). METHODS: Patients were included if baseline and end-point (twelve months apart) scores were available for measures including the Standardized Alzheimer's Disease Assessment Scale - Cognitive Subscale; Quick Mild Cognitive Impairment screen; Clinical Dementia Rating Scale (CDR-SB), and Lawton-Brody ADL Scale. RESULTS: There was a significant, 25% difference (median one-point) in the 12-month rate of decline in ADL scores in patients taking CACE-Is (n = 91), compared to the NoCACE-I group (n = 274), p = 0.024. This remained significant after adjusting for age, gender, education, and blood pressure, p = 0.034. When individual CACE-Is were compared to the NoCACE-I group, a significant reduction in the rate of decline in ADLs (median one versus four points), were only observed for perindopril, p = 0.01. The CDR-SB was also reduced (median one-point) for the perindopril compared to the NoCACE-I group, p = 0.04. CONCLUSION: This observational study suggests that CACE-Is, and potentially perindopril in particular, are associated with a reduced rate of functional decline in patients with AD, without an association with mood or behavior. This suggests that CACE-Is may slow disease progression in AD.

Effects of centrally acting ACE inhibitors on the rate of cognitive decline in dementia.

OBJECTIVES: There is growing evidence that antihypertensive agents, particularly centrally acting ACE inhibitors (CACE-Is), which cross the blood-brain barrier, are associated with a reduced rate of cognitive decline. Given this, we compared the rates of cognitive decline in clinic patients with dementia receiving CACE-Is (CACE-I) with those not currently treated with CACE-Is (NoCACE-I), and with those who started CACE-Is, during their first 6 months of treatment (NewCACE-I). DESIGN: Observational case-control study. SETTING: 2 university hospital memory clinics. PARTICIPANTS: 817 patients diagnosed with Alzheimer's disease, vascular or mixed dementia. Of these, 361 with valid cognitive scores were included for analysis, 85 CACE-I and 276 NoCACE-I. MEASUREMENTS: Patients were included if the baseline and end-point (standardised at 6 months apart) Standardised Mini-Mental State Examination (SMMSE) or Quick Mild Cognitive Impairment (Qmci) scores were available. Patients with comorbid depression or other dementia subtypes were excluded. The average 6-month rates of change in scores were compared between CACE-I, NoCACE-I and NewCACE-I patients. RESULTS: When the rate of decline was compared between groups, there was a significant difference in the median, 6-month rate of decline in Qmci scores between CACE-I (1.8 points) and NoCACE-I (2.1 points) patients (p=0.049), with similar, non-significant changes in SMMSE. Median SMMSE scores improved by 1.2 points in the first 6 months of CACE treatment (NewCACE-I), compared to a 0.8 point decline for the CACE-I (p=0.003) group and a 1 point decline for the NoCACE-I (p=0.001) group over the same period. Multivariate analysis, controlling for baseline characteristics, showed significant differences in the rates of decline, in SMMSE, between the three groups, p=0.002. CONCLUSIONS: Cognitive scores may improve in the first 6 months after CACE-I treatment and use of CACE-Is is associated with a reduced rate of cognitive decline in patients with dementia.

Comparison of the quick mild cognitive impairment (Qmci) screen and the SMMSE in screening for mild cognitive impairment.

INTRODUCTION: differentiating mild cognitive impairment (MCI) from normal cognition (NC) is difficult. The AB Cognitive Screen (ABCS) 135, sensitive in differentiating MCI from dementia, was modified to improve sensitivity and specificity, producing the quick mild cognitive impairment (Qmci) screen. OBJECTIVE: this study compared the sensitivity and specificity of the Qmci with the Standardised MMSE and ABCS 135, to differentiate NC, MCI and dementia. METHODS: weightings and subtests of the ABCS 135 were changed and a new section 'logical memory' added, creating the Qmci. From four memory clinics in Ontario, Canada, 335 subjects (154 with MCI, 181 with dementia) were recruited and underwent comprehensive assessment. Caregivers, attending with the subjects, without cognitive symptoms, were recruited as controls (n = 630). RESULTS: the Qmci was more sensitive than the SMMSE and ABCS 135, in differentiating MCI from NC, with an area under the curve (AUC) of 0.86 compared with 0.67 and 0.83, respectively, and in differentiating MCI from mild dementia, AUC of 0.92 versus 0.91 and 0.91. The ability of the Qmci to identify MCI was better for those over 75 years. CONCLUSION: the Qmci is more sensitive than the SMMSE in differentiating MCI and NC, making it a useful test, for MCI in clinical practice, especially for older adults.

Effect of silane reagent functionality for fluorinated alkyl and phenyl silica bonded stationary phases prepared in supercritical carbon dioxide.

This research examines the effect of silane reagent functionality for the preparation of fluorinated alkyl silica-bonded stationary phases prepared using supercritical carbon dioxide (sc-CO(2)) as a bonding medium. We present results that demonstrate that alkyl (C(8) and C(10)) and phenyl (pentafluorophenylpropyl, PFPP) silica bonded stationary phases can be prepared under sc-CO(2) conditions of 100 degrees C, 414bar and 3h, with surface coverages comparable to those obtained using organic solvent based methods. Fluorinated alkyl silica bonded phase preparation with a trichloro silane generates high ligand densities and more chemically uniform silica surface species compared to phases prepared using a monochloro or alkoxy silanes, as evidenced by thermogravimetric analysis (TGA) and (29)Si cross polarisation magic angle spinning (CP-MAS) NMR spectroscopy. In addition, the sc-trichloro prepared fluorinated C(8) bonded phases have demonstrated the ability to separate solutes on the basis of their molecular shape with the separation of the LC column shape selectivity test mixture, SRM 869a in the elution order of PhPh

Preparation of a mercaptopropyl bonded silica intermediate in supercritical carbon dioxide: synthesis, characterisation and chromatography of a quinine based chiral stationary phase.

This research examines the preparation of a mercaptopropyl bonded silica intermediate in supercritical carbon dioxide (sc-CO(2)) and the subsequent conversion in sc-CO(2) to a quinine derived chiral stationary phase (CSP). The effects of reaction temperature, pressure and time on the surface coverage of the silica intermediate were investigated when porous silica particles (Exsil-Avanti, 3microm) were reacted with 3-trimethoxymercaptopropylsilane in sc-CO(2). We present results which demonstrate that a stable mercaptopropyl bonded silica intermediate can be successfully prepared under supercritical conditions of 40 degrees C, 483bar, in a substantially reduced reaction time of 1h with superior surface coverages compared to organic solvent based methods. The further utility of this supercritical fluid technology was demonstrated by the free radical addition of a quinine derived chiral selector onto a mercaptopropyl bonded silica intermediate in sc-CO(2). This supercritical fluid generated chiral stationary phase (CSP) was utilised for the direct LC enantioseparation of a series of 3,5-dinitrobenzoyl (DNB) amino acids. Bonded silica samples were characterised using elemental analysis, diffuse reflectance infrared fourier transform (DRIFT) spectroscopy, solid state (13)C and (29)Si CP-MAS NMR spectroscopy, and thermogravimetric analysis (TGA). This supercritical fluid functionalisation approach offers an efficient and cleaner alternative to existing organic solvent based approaches for the preparation of bonded silica phases.

Supercritical fluid generated stationary phases for liquid chromatography and capillary electrochromatography.

Chromatographic silica-bonded stationary phases have been prepared using supercritical CO(2) as the reaction medium. (29)Si solid-state NMR spectra of the generated bonded silica phases show unreacted silica species Q(3) and Q(4), alongside important resonances for surface-bound ligands, T(1), T(2), and T(3). Initially, a fluorinated octyl silica (C(8)) phase was produced, by reacting (1)H,(1)H,(2)H,(2)H-perfluorooctyltriethoxysilane with silica particles (3 microm) in sc-CO(2) at 60 degrees C and 450 atm for 3 h. In-house-packed LC columns of this fluorinated sc-C(8) silica phase yielded typical reversed-phase behavior when a standard test mix of benzamide (k' = 1.03), benzophenone (k' = 8.11), and biphenyl (k' = 14.92) was eluted. When packed into fused-silica capillaries for CEC, this fluorinated sc-C(8) silica phase gave linear plots of log k' versus percentage MeOH for benzophenone and biphenyl and, in contrast to octyl or octadecyl silica phases, displayed selectivity for aromatic thioureas when chromatographed among a series of synthetic organic thiourea test solutes. Similarily, an octadecyl silica phase (sc-C(18) silica) was prepared by reaction of n-octadecyltriethoxysilane in sc-CO(2), packed at 9500 psi and examined by LC. The sc-C(18) silica LC column yielded high column efficiency (up to 141 000 N/m (fluorene)) and excellent asymmetry factors (1.06, fluorene) without resource to end-capping. Following a second silylating or end-capping step using hexamethyldisilazane in sc-CO(2), sc-end-capped sc-C(18) silica phases elute N,N-DMA before toluene and the toluidine isomers as a single peak, indicating lowered silanol activity according to the Engelhardt test. A rapid separation of the important pharmaceutical substances, ketoprofen, naproxen, fentoprofen, and ibuprofen, on an sc-end-capped sc-C(18) silica phase is also shown.