Probing the quality control mechanism of theEscherichia colitwin-arginine translocase with folding variants of ade novo-designed heme protein

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin arginine translocase (Tat). The Tat machinery exports folded and cofactor containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N-terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system’s quality control mechanism

Gastrokine-1, an anti-amyloidogenic protein secreted by the stomach, regulates diet-induced obesity

Obesity and its sequelae have a major impact on human health. The stomach contributes to obesity in ways that extend beyond its role in digestion, including through effects on the microbiome. Gastrokine-1 (GKN1) is an anti-amyloidogenic protein abundantly and specifically secreted into the stomach lumen. We examined whether GKN1 plays a role in the development of obesity and regulation of the gut microbiome. Gkn1−/− mice were resistant to diet-induced obesity and hepatic steatosis (high fat diet (HFD) fat mass (g) = 10.4 ± 3.0 (WT) versus 2.9 ± 2.3 (Gkn1−/−) p < 0.005; HFD liver mass (g) = 1.3 ± 0.11 (WT) versus 1.1 ± 0.07 (Gkn1−/−) p < 0.05). Gkn1−/− mice also exhibited increased expression of the lipid-regulating hormone ANGPTL4 in the small bowel. The microbiome of Gkn1−/− mice exhibited reduced populations of microbes implicated in obesity, namely Firmicutes of the class Erysipelotrichia. Altered metabolism consistent with use of fat as an energy source was evident in Gkn1−/− mice during the sleep period. GKN1 may contribute to the effects of the stomach on the microbiome and obesity. Inhibition of GKN1 may be a means to prevent obesity

Torture and the UK’s “war on asylum”: medical power and the culture of disbelief

When the now ‘iconic’ images of shackled, humiliated and dehumanised detainees in the Abu Ghraib prison complex in Iraq were broadcast globally, in the mid-2000s, the relationship between medical power and torture in the “war on terror” was also thrust sharply into focus. Graphic images of coalition troops photographing and posing in front of hooded, naked prisoners forced into a “human pyramid”, and of people made to wear animal collars, indicated a regime in which degradation had a defining role. The photograph of a soldier gloating over the corpse of a man who had died as a result of torture was just one picture of a network of interrogation camps in which detention by coalition forces could be fatal. Yet if there were any expectations that the presence of medical personnel may have checked this violence, these were shattered by the fact that clinicians – in some cases at least – were integral to its practice. «It is now beyond doubt that Armed Forces physicians, psychologists, and medics were active and passive partners in the systematic neglect and abuse of war on terror prisoners», wrote Steven Miles in 2009 (Miles 2009, X). And as he continued, this involved providing interrogators «with medical information to use in setting the nature and degree of physical and psychological abuse during interrogations». It involved monitoring «interrogations to devise ways to break prisoners down or to keep them alive». It involved pathologists holding back death certificates and autopsy reports in order to minimise the number of fatalities or cover up torture-related deaths as deaths by natural causes (Ibid). Procedures including «cramped confinement, dietary manipulation, sleep deprivation, and waterboarding» were among the practices that were «at times (…) legally sanctioned due to medical supervision» in the context of the “war on terror”, according to Hoffman (2011, 1535). He continued to suggest that doctors are not just important to «modern torture methods», they are «irreplaceable». In this context, the “war on terror” is no aberration. As the revolutionary psychoanalyst and philosopher Frantz Fanon documented in 1959, for example, certain medical practitioners had an integral role in the military occupation of Algeria, and «There are, for instance, psychiatrists … known to numerous prisoners», he suggested, «who have given electric shock treatments to the accused and have questioned them during the waking phase, which is characterized by a certain confusion, a relaxation of resistance, a disappearance of the person's defences.» (Fanon 1959/1965, 138). Indeed, in his analysis of the Algerian revolution, he discussed how resistance to and struggles over the meanings of medical power were integral to the revolution itself. However, while the role of medical power in the practice of torture has been subjected to sustained critique in the context of the “war on terror”, what follows examines the relationship between medical power and torture in the context of what has been depicted – metaphorically – as another (although to some extents related) “war”: the “war” on asylum. According to the UNHCR (2017, 3), between 5 and 35 per cent of those asylum seekers who have been granted refugee status have survived torture. And focusing on the UK as a case study, this chapter examines the institutional and legal structures prohibiting torture and inhuman and degrading treatment, particularly as they apply to those subject to immigration control in this context. But further, it also examines the ideological and political conditions within which claims by those seeking asylum that they have been subjected to torture prior to arrival can be (and have been) ignored, downplayed and denied. It examines how medical expertise has frequently been undermined in the asylum process when this expertise is utilised to add weight to asylum seekers’ claims to have experienced torture. It examines how there have been attempts to narrow the definition of torture in ways which exclude people from the protections to which torture survivors are entitled. But it also explores the ways in which segments of the medical profession have been complicit in riding roughshod over existing safeguards to prevent further harm to those who have experienced torture, thus potentially compounding its effects. In particular, it examines claims that in certain contexts clinicians have administered dangerous “care” in order to ensure the removal of people from the UK, despite them claiming that they – or their family members – face serious harm and persecution on arrival as a result of this. In a historical discussion of medical involvement in torture, Giovanni Maio (2001, 1609) has noted that from its earliest incarnations one of the features of torture has been its use as an «oppressive instrument used in the preservation of power». Furthermore, whilst methods of torture have certainly «developed», and continue to do so, he argues, this «function» of torture is «especially relevant today». This chapter argues that the (mis)treatment of those in the UK who say they have been tortured, preserves and is bound up with a particular manifestation of state power: the aims, rationale and dictates of immigration control. Its claims are perhaps much more mundane than the forms of direct medical complicity in torture alluded to above. But they are nonetheless important. For it is argued that the acts of omission and commission documented in this chapter expose the tensions between the rights of certain “categories” of migrants to be afforded adequate clinical care on the one hand, and the goals and aims of immigration control itself on the other. This poses profound questions about the functions of clinical care and the ethical duties, responsibilities and obligations of clinicians, it is suggested. But as this chapter also crucially explores, this is a form of power that many within the medical profession have historically challenged, and continue to do so

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Early Distraction for Mild to Moderate Unilateral Craniofacial Microsomia: Long-Term Follow-Up, Outcomes, and Recommendations

Background: There is controversy regarding the treatment of young patients with unilateral craniofacial microsomia and moderate dysmorphism. The relative indication for mandibular distraction in such patients poses several questions: Is it deleterious in the context of craniofacial growth and appearance? This study was designed to address these questions. Methods: A retrospective review of patients undergoing mandibular distraction by a single surgeon between 1989 and 2010 was conducted. Patients with "moderate" unilateral craniofacial microsomia (as defined by Pruzansky type I or IIa mandibles) and follow-up until craniofacial skeletal maturity were included for analysis. Patients were divided into two cohorts: satisfactory and unsatisfactory results based on photographic aesthetic evaluation by independent blinded observers at the initial presentation and at the age of skeletal maturity. Clinical variables were analyzed to detect predictors for satisfactory distraction. Results: Nineteen patients were included for analysis. The average age at distraction was 68.2 months and the average age at follow-up was 19.55 years. Thirteen patients (68.4 percent) had Pruzansky type IIA and six patients (31.6 percent) had Pruzansky type I mandibles. Twelve patients (63.2 percent) had satisfactory outcomes, whereas seven patients (36.8 percent) had unsatisfactory outcomes. Comparing the two cohorts, patients with satisfactory outcomes had distraction at an earlier age (56.4 months versus 89.8 months; p = 0.07) and a greater percentage overcorrection from craniofacial midline (41.7 percent versus 1.8 percent; p = 0.003). Conclusion: Mandibular distraction is successful in patients with mild to moderate dysmorphism, provided that there is a comprehensive clinical program emphasizing adequate mandibular bone stock, proper vector selection, planned overcorrection, and comprehensive orthodontic management. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation.

Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins