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Membrane-bound pyrophosphatases are integral membrane proteins that catalyze the hydrolysis of pyrophosphate into orthophosphate, while simultaneously facilitating the pumping of protons and/or sodium ions. Since mPPases are absent in humans but play a critical role in the life cycle of protist parasite, they represent promising therapeutic targets. We successfully expressed the Plasmodium falciparum type 1 mPPase in the baculovirus/insect cell expression system and purified the protein, yielding 0.3 mg per liter cell culture. Various detergents were tested for solubilization, with the protein remaining active under all selected detergents. n -dodecyl-β-D-maltoside combined with cholesteryl hemisuccinate provided the highest solubility (88%). Finally, the PfPPase-VP1 was assayed against a set of fourteen antimalarial drugs, along with seven Thermotoga maritima mPPase inhibitors and fourteen compounds of unknown activity against mPPases. Only three compounds, all pyrazolo[1,5- a ]pyrimidine-based TmPPase inhibitors, retained micromolar IC 50 activity against PfPPase-VP1. The expression and purification of the PfPPase-VP1 will allow to conduct structural studies as well as to develop target-based screens, two steps necessary for the development of inhibitors to combat parasite disease.

Autism (MIM 209850) is a neurodevelopmental condition with heterogeneous aetiology, characterised by restricted interests, repetitive behaviour, and atypical social interaction. Many genes have been associated with autism, several of which encode for proteins involved in synaptic processes, but mutations in the X-linked patched-domain containing 1 (PTCHD1) gene (MIM:300828; Xp22.11) account for up to 1 % of cases of autism [1,2]. PTCHD1 is expressed throughout the brain and other tissues, and its expression profile in the brain changes as development proceeds [3]. The PTCHD1 protein is synaptically localised in neurons, and mutations disrupt its expression, stability, glycosylation, and localisation [3]. While the exact functional role of PTCHD1 remains elusive, several findings implicate an essential role in developmental processes, the development of autism and intellectual disability [2]. PTCHD1 is a member of the Patched-domain containing protein family, which also includes the Hedgehog (Hh) pathway receptor Patched 1 PTCH1) and Niemann-Pick disease type C1 (NPC1). These proteins are structurally homologous and contain 12-13 transmembrane helices TM), a sterol sensing domain (SSD) composed of five TMs, a sterol sensing- like domain, and two ectodomains (ECDs). Full-length PTCHD1 is an 888-amino-acid, 12-pass transmembrane protein containing an SSD composed of TM 2-6 and a sterol sensing-like domain consisting of TM7- 12 followed by a C-terminal tail containing a PDZ-binding motif (ITTV). Additionally, two large ECDs are inserted between TM1 and 2 and TM7 and 8 (Figure 1). Cholesterol transporters are involved in fundamental cellular processes. Understanding the function of PTCHD1 could provide key information on the association of PTCHD1 with autism. We have gained insight into the unique features of PTCHD1 by combining in vitro and in silico methods, using an AlphaFold2 generated structure. Structure prediction reveals conserved cholesterol-binding elements, and a click-cholesterol assay confirms PTCHD1 binds cholesterol In our recent publication, we showed the AlphaFold predicted PTCHD1 is structurally homologous to several cholesterol transporters belonging to the Patched domain-containing family, including Dispatched 1 (DISP1), NPC1, and PTCH1 (Figure 1) [4]. While the overall sequence conservation between these proteins is low, sequence alignment revealed a higher degree of conservation in the SSD (17 %, 18 %, and 23 % identity with DISP1, NPC1 and PTCH1, respectively) than in the entire sequences, indicating a shared function in cholesterol binding/ transport. To investigate this possibility, we performed a cholesterol click-reaction assay on purified PTCHD1 elution fractions. Purified PTCHD1 was cross- linked with a PhotoClick cholesterol with UV light and then labelled by a ‘click-reaction’, followed by analysis by SDS-PAGE and imaging. The results show that PTCHD1 can bind cholesterol (Figure 2A): strong bands of ∼130 kDa and ∼100 kDa, corresponding to the approximate molecular weights of fully glycosylated and not glycosylated PTCHD1, are present in all lanes of treated PTCHD1 solubilised in varying amounts of GDN. Docking indicates PTCHD1 can bind cholesterol similarly to PTCH1 Superimposing our PTCHD1 model onto PTCH1 showed that the SSDs are nearly identical in structure [2]. To further explore the similarities in cholesterol binding between PTCHD1 and PTCH1, we performed docking analysis using AutoDock Vina. The full SSD of PTCHD1 (TM2-6 of AF- Q96NR3) or PTCH1 (TM2-6 of AF- Q13635), used as a control to validate the docking method, was defined as the docking site. We compared cholesterol binding by aligning docked cholesterols to the cryo-EM structure of the PTCH1 (6RMG) cholesterol-binding sites: site 1 is within the outer leaflet pocket, site 5 is in the inner leaflet portion of the SSD, and site 4 is on the opposite side of the SSD, adjacent to TM1 (Figure 2B). Cholesterol docked to three sites on the PTCHD1-SSD with estimated binding energies ranging from -8.7 kcal/mol to -7.2 kcal/mol. The predicted cholesterol-binding positions in PTCHD1 directly overlap with cholesterols in the PTCH1 cryo-EM structure: 25/27 cholesterol binding positions are in site 1 between TM3, TM4, and TM6. Additionally, two cholesterol binding positions are near site 5 in the putative inner leaflet portion of the SSD, adjacent to TM3 (Figure 2B). In the case of PTCH1-SSD, the estimated binding energy is between -8.4 kcal/mol and -7.3 kcal/mol. In addition, 23 docked cholesterol- binding positions overlap with binding site 1 and four near site 4 (Figure 2B). The similar binding of cholesterol is unsurprising due to the SSDs of PTCHD1 and PTCH1 sharing nearly 23 % identity. Preliminary data suggests autism-linked variants disrupt cholesterol transport To further elucidate the interaction of PTCHD1 with cholesterol, and investigate how this links to autism, we have employed a fluorescence-based in vitro cholesterol transport assay in HEK293T. Our preliminary data indicates PTCHD1 overexpression increases cholesterol export by ∼10% compared to a negative control (cells transfected with pcDNA3), whereas some autism-linked variants of PTCHD1 do not. This observation is supported by preliminary docking results, which indicate autism-linked mutations in the PTCHD1 ECDs influence how they interact with cholesterol. Structural analysis and complex prediction show PTCHD1 is unlikely to bind Shh The structurally similar protein PTCH1 uses cholesterol to modulate Smoothened activity in canonical Hh signalling. However, it is not clear if PTCHD1 shares this function [3,5]. We used AlphaFold2 to predict the complex formation of N-terminal Shh (ShhN, res 23-197) with the ectodomains (ECDs) of PTCHD1 and, as a positive control, the ECDs of PTCH1. Alignment of the predicted PTCHD1 ECD1 and ECD2 onto the full AlphaFold2 predicted structure (AF-Q96NR3) indicates the conformation of each ECD and their interaction has been predicted well (RMSD 0.961 Å). Furthermore, the predicted PTCH1:Shh structure aligns well with a complex solved by cryo-EM (RMSD 1.606Å with 6DMY) and contains 6/10 of the hydrogen bonds found in the complex indicating AlphaFold2 can predict Shh binding [4]. ShhN is predicted to bind to a β- hairpin and an α-helix on ECD2 of PTCHD1 (Figure 2C, left). However, the predicted aligned error is very high between ShhN and the ECDs for PTCHD1, suggesting ShhN is unlikely to form a complex with PTCHD1 ECDs. The low-likelihood of complex formation between PTCHD1 and ShhN is consistent with our in vitro data:a GLI luciferase assay in Ptch1-/- mouse embryonic fibroblasts showed transient expression of PTCHD1 does not reduce GLI-luciferase activity compared to the empty plasmid (pcDNA3.1+ control), whereas transfecting cells with PTCH1 inhibited GLI-luciferase activity by ∼65 %. Furthermore, a LigandTracer™ experiment showed there is no binding of Shh to PTCHD1 expressed in Sf9 cells. whereas transfecting cells with PTCH1 inhibited GLI-luciferase activity by ∼65 %. Furthermore, a LigandTracer™ experiment showed there is no binding of Shh to PTCHD1 expressed in Sf9 cells. In-depth structural analysis suggests the lack of binding is due to the absence of ECD loops, which form a negatively charged pocket and mediate binding in PTCH1 (Figure 2C, right) [4]. The inability of PTCHD1 to bind Shh and to inhibit GLI transcription in MEFs argues against the possibility of acting as a Hh receptor, despite sharing structural features with PTCH1. The absence of Hh-related phenotypes in PTCHD1- deficient mice also supports our conclusion [2]. Although our in vitro experiments and in silico structural analysis suggest PTCHD1 does not bind Shh nor inhibit Hh signalling, we cannot rule out that PTCHD1 influences the Hh pathway in a noncanonical fashion and/or in a cell type-specific manner. In conclusion, our findings support the idea that PTCHD1 is functionally unique and participates in fundamental cellular processes, different to PTCH1 and NPC1. First, we showed that PTCHD1 is not a PTCH1 functional homolog. Second, we showed that PTCHD1 binds cholesterol but not Shh and cannot inhibit canonical Hh signalling. Third, we identified key features in the PTCHD1 sequence and structure that distinguish it from other Patched-domain containing family members.

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function.

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyse pyrophosphate and pump H + /Na + across membranes. They are crucial for the virulence of protist pathogens, making them attractive drug targets. In this study, we investigate the inhibitory effects of seven distinct bisphosphonates against Thermotoga maritima mPPase to explore their mode of action and assist in future small molecule inhibitor development. We solved two structures of mPPase bound to the inhibitors in the enzyme active sites and probed the conformational dynamics of mPPase under multiple inhibitors and functionally relevant conditions by double electron-electron resonance (DEER) spectroscopy. We found that mPPase adopts distinct conformational equilibria in solution in the presence of different inhibitors, including states consistent with asymmetric binding in the active site (closed-open), but a symmetric apo-like conformation on the periplasmic side (open-open). Combined with solid-supported membrane-based electrophysiology recordings, this revealed that during catalysis, one monomer of the dimer remains open, and Na + can only be pumped in a closed state. These results further support symmetry-breaking across the membrane, consistent with half-of-the-sites-reactivity.

The purpose of this work is to undertake an exploratory pilot study on the multiplicity of texts that strive to write or analyse the memories of the displaced individuals from the former Portuguese African colonies during the 1975–1976 forced migration to Portugal who came to be labelled retornados (returnees), rethink history and eliminate silences, to pave the way for postmemory and controlled affective ties through reparative readings and nostalgia. We undertook bibliographical research to determine the eligibility criteria and used nonprobability convenience sampling to select the texts to carry out a content analysis. An interdisciplinary approach is used to present and discuss the results. Although decades have passed since the retorno, the stigma of being a retornado still remains in the memory of Portuguese society as interest in the topic and the time distance allows for more rigorous studies. In the analysed texts, the results achieved establish that the relationship of the Portuguese people with the contemporary memory and history of the colonial era is comprised of silences and nonmemories that still have to be deconstructed to forge a positive future for the generation of postmemory.

High-resolution membrane protein structures are essential for understanding the molecular basis of diverse biological events and important in drug development. Detergents are usually used to extract these bio-macromolecules from the membranes and maintain them in a soluble and stable state in aqueous solutions for downstream characterization. However, many eukaryotic membrane proteins solubilized in conventional detergents tend to undergo structural degradation, necessitating the development of new amphiphilic agents with enhanced properties. In this study, we designed and synthesized a novel class of glucoside amphiphiles, designated tandem malonate-based glucosides (TMGs). A few TMG agents proved effective at both stabilizing a range of membrane proteins and extracting proteins from the membrane environment. These favourable characteristics, along with synthetic convenience, indicate that these agents have potential in membrane protein research.

To understand if clinicians can tell apart patients with healthcare-associated infections (HCAI) from those with community-acquired infections (CAI) and to determine the impact of HCAI in the adequacy of initial antibiotic therapy and hospital mortality. One-year prospective cohort study including all consecutive infected patients admitted to a large university tertiary care hospital. A total of 1035 patients were included in this study. There were 718 patients admitted from the community: 225 (31%) with HCAI and 493 (69%) with CAI. Total microbiologic documentation rate of infection was 68% (n = 703): 56% in CAI, 73% in HCAI and 83% in hospital-acquired infections (HAI). Antibiotic therapy was inadequate in 27% of patients with HCAI vs. 14% of patients with CAI (p<0.001). Among patients with HCAI, 47% received antibiotic therapy in accordance with international recommendations for treatment of CAI. Antibiotic therapy was inadequate in 36% of patients with HCAI whose treatment followed international recommendations for CAI vs. 19% in the group of HCAI patients whose treatment did not follow these guidelines (p = 0.014). Variables independently associated with inadequate antibiotic therapy were: decreased functional capacity (adjusted OR = 2.24), HCAI (adjusted OR = 2.09) and HAI (adjusted OR = 2.24). Variables independently associated with higher hospital mortality were: age (adjusted OR = 1.05, per year), severe sepsis (adjusted OR = 1.92), septic shock (adjusted OR = 8.13) and inadequate antibiotic therapy (adjusted OR = 1.99). HCAI was associated with an increased rate of inadequate antibiotic therapy but not with a significant increase in hospital mortality. Clinicians need to be aware of healthcare-associated infections among the group of infected patients arriving from the community since the existing guidelines regarding antibiotic therapy do not apply to this group and they will otherwise receive inadequate antibiotic therapy which will have a negative impact on hospital outcome.

\"Navigating colonialism through Sports in Portuguese Africa\" aims to present a brief review of literature on the importance of sports in the African colonies/provinces of the Portuguese Colonial Empire, namely regarding the diffusion and the appropriation of sports - as a tool of colonial control and repression by the coloniser and as a tool of resistance by the colonised. The research question focuses on the fact that the colonised accepted (European) sports but also used the opportunity to resist the colonial system. The selected articles reflect on the role of colonialism and its link to the effort placed on the use of sports to promote national cohesion and identity building (coloniser) or to promote resistance (colonised). The different studies prove that football/soccer was the most practised sport in the former colonies during the Portuguese occupation.

Background There is a lack of consensus regarding the definition of risk factors for healthcare-associated infection (HCAI). The purpose of this study was to identify additional risk factors for HCAI, which are not included in the current definition of HCAI, associated with infection by multidrug-resistant (MDR) pathogens, in all hospitalized infected patients from the community. Methods This 1-year prospective cohort study included all patients with infection admitted to a large, tertiary care, university hospital. Risk factors not included in the HCAI definition, and independently associated with MDR pathogen infection, namely MDR Gram-negative (MDR-GN) and ESKAPE microorganisms (vancomycin-resistant Enterococcus faecium , methicillin-resistant Staphylococcus aureus , extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella species, carbapenem-hydrolyzing Klebsiella pneumonia and MDR Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species), were identified by logistic regression among patients admitted from the community (either with community-acquired or HCAI). Results There were 1035 patients with infection, 718 from the community. Of these, 439 (61%) had microbiologic documentation; 123 were MDR (28%). Among MDR: 104 (85%) had MDR-GN and 41 (33%) had an ESKAPE infection. Independent risk factors associated with MDR and MDR-GN infection were: age (adjusted odds ratio (OR) = 1.7 and 1.5, p = 0.001 and p = 0.009, respectively), and hospitalization in the previous year (between 4 and 12 months previously) (adjusted OR = 2.0 and 1,7, p = 0.008 and p = 0.048, respectively). Infection by pathogens from the ESKAPE group was independently associated with previous antibiotic therapy (adjusted OR = 7.2, p < 0.001) and a Karnofsky index <70 (adjusted OR = 3.7, p = 0.003). Patients with infection by MDR, MDR-GN and pathogens from the ESKAPE group had significantly higher rates of inadequate antibiotic therapy than those without (46% vs 7% , 44% vs 10% , 61% vs 15%, respectively, p < 0.001). Conclusions This study suggests that the inclusion of additional risk factors in the current definition of HCAI for MDR pathogen infection, namely age >60 years, Karnofsky index <70, hospitalization in the previous year, and previous antibiotic therapy, may be clinically beneficial for early diagnosis, which may decrease the rate of inadequate antibiotic therapy among these patients.

Background The provision of Intensive Care (IC) can lead to a health care provider’s physical, psychological and emotional exhaustion, which may develop into burnout. We notice the absence of specific studies regarding this syndrome in Portuguese Intensive Care Units (ICUs). Our main objective is to study the incidence and risk factors of burnout in Portuguese ICUs. Methods A self-fulfilment questionnaire containing 3 items: (i) socio-demographic data of the study population; (ii) experiences in the workplace; (iii) Maslach Burnout Inventory (MBI) - was applied to evaluate the influence of distinct factors on the prevalence of burnout among physicians and nurses working in ICUs. Results Three hundred professionals (82 physicians and 218 nurses) from ten ICUs were included in the study, out of a total of 445 who were eligible. There was a high rate of burnout among professionals working in Portuguese ICUs, with 31% having a high level of burnout. However, when burnout levels among nurses and physicians were compared, no significant difference was found. Using multivariate analysis, we identified gender as being a risk factor, where female status increases the risk of burnout. In addition, higher levels of burnout were associated with conflicts and ethical decision making regarding withdrawing treatments. Having a temporary work contract was also identified as a risk factor. Conversely, working for another service of the same health care institution acts as a protective factor. Conclusions A high rate of burnout was identified among professionals working in Portuguese ICUs. This study highlights some new risk factors for burnout (ethical decision making, temporary work contracts), and also protective ones (maintaining activity in other settings outside the ICU) that were not previously reported. Preventive and interventive programmes to avoid and reduce burnout syndrome are of paramount importance in the future organization of ICUs and should take the above results into account.