Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling.

Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the CLN3 gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3exΔ7/8 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.

Role of lipids in interorganelle communication.

Cell homeostasis and function rely on well-orchestrated communication between different organelles. This communication is ensured by signaling pathways and membrane contact sites between organelles. Many players involved in organelle crosstalk have been identified, predominantly proteins and ions. The role of lipids in interorganelle communication remains poorly understood. With the development and broader availability of methods to quantify lipids, as well as improved spatiotemporal resolution in detecting different lipid species, the contribution of lipids to organelle interactions starts to be evident. However, the specific roles of various lipid molecules in intracellular communication remain to be studied systematically. We summarize new insights in the interorganelle communication field from the perspective of organelles and discuss the roles played by lipids in these complex processes.

Pre-selection of fibroblast subsets prompts prevascularization of tissue engineered skin analogues.

The papillary and reticular dermis harbors phenotypically distinct fibroblasts, whose functions such as maintenance of skin's microvasculature are also distinct. Thus, we hypothesized that pre-selection of the subpopulations of fibroblasts would benefit the generation of skin tissue engineered (TE) constructs, promoting their prevascularization in vitro. We first isolated papillary and reticular fibroblasts using fluorescence-activated cell sorting and studied the effect of their secretome and extracellular matrix (ECM) on human dermal microvascular endothelial cell (hDMEC) organization. Subsequently, we developed a bilayered 3D polymeric structure with distinct layer-associated features to house the subpopulations of fibroblasts, to generate a skin analogue. Both papillary and reticular fibroblasts were able to stimulate capillary-like network formation in a Matrigel assay. However, the secretome of the two subpopulations was substantially different, being enriched in VEGF, IGF-1, and Angio-1 in the case of papillary fibroblasts and in HGF and FGF-2 for the reticular subset. In addition, the fibroblast subpopulations deposited varied levels of ECM proteins, more collagen I and laminin was produced by the reticular subset, but these differences did not impact hDMEC organization. Vessel-like structures with lumens were observed earlier in the 3D skin analogue prepared with the sorted fibroblasts, although ECM deposition was not affected by the cell's pre-selection. Moreover, a more differentiated epidermal layer was obtained in the skin analogue formed by the sorted fibroblasts, confirming that its whole structure was not affected. Overall, we provide evidence that pre-selection of papillary and reticular fibroblasts is relevant for promoting the in vitro prevascularization of skin TE constructs.

Antibiotic resistance genes in bacteriophages from wastewater treatment plant and hospital wastewaters.

Antibiotic resistant bacteria (ARB) are a major health risk caused particularly by anthropogenic activities. Acquisition of antibiotic resistances by bacteria is known to have happened before the discovery of antibiotics and can occur through different routes. Bacteriophages are thought to have an important contribution to the dissemination of antibiotic resistance genes (ARGs) in the environment. In this study, seven ARGs (blaTEM, blaSHV, blaCTX-M, blaCMY, mecA, vanA, and mcr-1) were investigated, in the bacteriophage fraction, in raw urban and hospital wastewaters. The genes were quantified in 58 raw wastewater samples collected at five WWTPs (n = 38) and hospitals (n = 20). All genes were detected in the phage DNA fraction, with the bla genes found in higher frequency. On the other hand, mecA and mcr-1 were the least frequently detected genes. Concentrations varied between 102 copies/L and 106 copies/L. The gene coding for the resistance to colistin (mcr-1), a last-resort antibiotic for the treatment of multidrug-resistant Gram-negative infections, was identified in raw urban and hospital wastewaters with positivity rates of 19 % and 10 %, respectively. ARGs patterns varied between hospital and raw urban wastewaters, and within hospitals and WWTP. This study suggests that phages are reservoirs of ARGs, and that ARGs (with particularly emphasis on resistance to colistin and vancomycin) in the phage fraction are already widely widespread in the environment with potential large implications for public health.

Gellan gum-based hydrogels support the recreation of the dermal papilla microenvironment.

The dermal papilla (DP), a specialized compartment within the hair follicle, regulates hair growth. However, human DP cells rapidly lose their inductivity in 2D-culture given the loss of positional and microenvironmental cues. Spheroids have been capable of recreating the 3D intercellular organization of DP cells, however, DP cell-matrix interactions are poorly represented. Considering the specific nature of the DP's extracellular matrix (ECM), we functionalized gellan gum (GG) with collagen IV-(HepIII) or fibronectin-(cRGDfC) derived peptide sequences to generate a 3D environment in which the phenotype and physiological functions of DP cells are restored. We further tuned the stiffness of the microenvironments by varying GG amount. Biomimetic peptides in stiffer hydrogels promoted the adhesion of DP cells, while each peptide and amount of polymer independently influenced the type and quantity of ECM proteins deposited. Furthermore, although peptides did not seem to have an influence, stiffer hydrogels improved the inductive capacity of DP cells after short term culture. Interestingly, independently of the peptide, these hydrogels supported the recapitulation of basic hair morphogenesis-like events when incorporated in an organotypic human skin in vitro model. Our work demonstrates that tailored GG hydrogels support the generation of a microenvironment in which both cell-ECM and cell-cell interactions positively influence DP cells towards the creation of an artificial DP.

A Novel Plant-Based Protein Has Similar Effects Compared to Whey Protein on Body Composition, Strength, Power, and Aerobic Performance in Professional and Semi-Professional Futsal Players.

Introduction: The effects of dietary protein on body composition and physical performance seemingly depend on the essential amino acid profile of the given protein source, although controversy exists about whether animal protein sources may possess additional anabolic properties to plant-based protein sources. Purpose: To compare the effects of a novel plant-based protein matrix and whey protein supplementation on body composition, strength, power, and endurance performance of trained futsal players. Methods: Fifty male futsal players were followed during 8 weeks of supplementation, with 40 completing the study either with plant-based protein (N = 20) or whey protein (N = 20). The following measures were assessed: bone mineral content, lean body mass, and fat mass; muscle thickness of the rectus femoris; total body water; blood glucose, hematocrit, C-reactive protein, aspartate aminotransferase, alanine aminotransferase, creatine kinase, creatinine, and estimated glomerular filtration rate; salivary cortisol; maximal strength and 1-RM testing of the back squat and bench press exercises; muscle power and countermovement jump; VO2max and maximal aerobic speed. Subjects were asked to maintain regular dietary habits and record dietary intake every 4 weeks through 3-day food records. Results: No differences in any variable were observed between groups at baseline or pre- to post-intervention. Moreover, no time*group interaction was observed in any of the studied variables, and a time effect was only observed regarding fat mass reduction. Conclusions: Supplementing with either a novel plant-based protein matrix or whey protein did not affect any of the variables assessed in high-level futsal players over 8 wks. These results suggest that whey protein does not possess any unique anabolic properties over and above those of plant-based proteins when equated to an essential amino acid profile in the population studied. Furthermore, when consuming a daily protein intake >1.6 g/kg BW.day-1, additional protein supplementation does not affect body composition or performance in trained futsal players, regardless of protein type/source.

Edaravone for acute ischemic stroke - Systematic review with meta-analysis.

INTRODUCTION: Ischemic stroke is a major cause of death and disability. Despite major advances in reperfusion therapies, most patients don´t benefit from these treatments as the time window for such interventions is limited. Therefore, other treatment options are desirable. Edaravone has been demonstrated in previous studies to reduce neurologic deficits in stroke patients. OBJECTIVE: To test the hypothesis that edaravone reduces functional dependence in ischemic stroke patients. MATERIAL AND METHODS: Systematic review and meta-analysis of randomized controlled trials and observational studies comparing edaravone to placebo in adult patients with ischemic stroke. The efficacy outcomes of interest were good and excellent functional outcomes at 90 days, defined as modified Rankin Scale (mRS) scores of 0-2 and 0-1 respectively. The safety outcomes of interest were intracranial hemorrhage and mortality. RESULTS: 19 studies were included. Edaravone treatment was associated with improved chances of 90-day good (OR=1.31, 95% CI 1.06-1.67) and excellent (OR=1.26, 95% CI 1.04-1.54) functional outcomes. Mortality was also lower in edaravone treated patients (OR=0.50, 95% CI 0.45-0.56). There were no differences in terms of intracranial hemorrhage. Most studies were observational and performed in Asian populations, especially Japan. Heterogeneity was high for all outcomes but reduced when analysis was restricted to randomized trials. CONCLUSION: Edaravone is a promising treatment for ischemic stroke patients, with a more favorable time window. However, more randomized studies including patient populations outside Asia are required to confirm this hypothesis.

Community perceptions surrounding Lisbon's first mobile drug consumption room.

Portugal's first Mobile Drug Consumption Room (MDCR) has been operating for over three years in two Lisbon neighborhoods. This qualitative study first creates a baseline understanding of community perceptions concerning the acceptability of and expectations surrounding drug consumption rooms in the communities of intervention. Then, at least six months after the initial interviews, it determines how these perceptions changed and what changes participants perceive in the neighborhoods. Findings highlight widespread acceptance of the MDCR among participants. One of the participants' priorities related to the MDCR was to lessen the visibility of public consumption, a change some participants perceived in the neighborhood. While in the first round of data collection participants primarily conceptualized the MDCR as a service for People Who Use Drugs (PWUD), in the follow-up round, they reported changes in the community.

How can exposure to engineered nanomaterials influence our epigenetic code? A review of the mechanisms and molecular targets.

Evidence suggests that engineered nanomaterials (ENM) can induce epigenetic modifications. In this review, we provide an overview of the epigenetic modulation of gene expression induced by ENM used in a variety of applications: titanium dioxide (TiO2), silver (Ag), gold (Au), silica (SiO2) nanoparticles and carbon-based nanomaterials (CNM). Exposure to these ENM can trigger alterations in cell patterns of DNA methylation, post-transcriptional histone modifications and expression of non-coding RNA. Such effects are dependent on ENM dose and physicochemical properties including size, shape and surface chemistry, as well as on the cell/organism sensitivity. The genes affected are mostly involved in the regulation of the epigenetic machinery itself, as well as in apoptosis, cell cycle, DNA repair and inflammation related pathways, whose long-term alterations might lead to the onset or progression of certain pathologies. In addition, some DNA methylation patterns may be retained as a form of epigenetic memory. Prenatal exposure to ENM may impair the normal development of the offspring by transplacental effects and/or putative transmission of epimutations in imprinting genes. Thus, understanding the impact of ENM on the epigenome is of paramount importance and epigenetic evaluation must be considered when assessing the risk of ENM to human health.

An unusual case of osteoarthritis?

Forestier disease is a condition characterized by calcification and ossification of ligaments and entheses particularly affecting axial skeleton. Diagnosis is difficult and mandates a high suspicion level, but unexpensive and accessible examinations like a simple radiography might provide useful diagnostic clues in these challenging clinical scenarios and improve clinical assistance.

Barium aspiration - Accident or complication?

Aspiration of barium sulfate is possible during radiographic contrast procedures and is potentially life-threatening in severe cases. In patients with dysphagia or suspected tracheoesophageal fistula, barium compounds may be used, considering lateral projection fluoroscopy of the pharyngeal phase of swallowing, but iso-osmotic agents should also be considered.

Vascular tumor or malformation of the pediatric airway?

Distinguishing airway vascular tumors from malformations can be challenging. Failure to treat a suspected airway hemangioma with a beta-blocker and systemic corticosteroid should raise suspicion of a possible airway malformation.

Outer Nuclear Layer as the Main Predictor to Anatomic Response to Half Dose Photodynamic Therapy in Chronic Central Serous Retinopathy.

PURPOSE: To evaluate the predictors for subretinal fluid resorption in patients with chronic central serous retinopathy (cCSR) submitted to half-dose photodynamic therapy (HD-PDT). METHODS: Observational, longitudinal, and retrospective study of patients with cCSR submitted to HD-PDT in a tertiary ophthalmology department in Portugal between January 2015 and February 2018. Best-corrected visual acuity (BCVA) and SD-OCT at baseline and 12 ± 3 months after treatment were performed. The central macular thickness (CMT), outer nuclear layer (ONL) thickness, integrity of the external limiting membrane (ELM), ellipsoid (EZ) and interdigitation zone (IZ), subretinal fluid (SFR) height, and choroidal thickness (CT) were evaluated. Patients were classified into responders and nonresponders based on SRF resorption. RESULTS: Sixty-one eyes of 42 patients were included; 75.4% were classified as responders. Final BCVA was significantly better in responders (p=0.002). The baseline ONL was thicker (p < 0.01) and intact ELM (67.2% vs. 16.4%), EZ (49.2% vs. 8.2%), and IZ (31.2% vs. 1.6%) were more prevalent in responders than in nonresponders. Anatomic response was correlated with a thicker ONL (rs (59) = 0.416, p=0.001 ∗ ), intact ELM (rs (59) = 0.261, p=0.04 ∗ ), EZ (rs (59) = 0.278, p=0.03 ∗ ), and IZ (rs (59) = 0.318, p=0.01 ∗ ). Binary logistic regression showed that a thicker ONL thickness increased the chance of an anatomic response to HD-PDT. The other evaluated retinal layers did not have statistical significance in the binary regression model. CONCLUSIONS: cCSR responders to HD-PDT have a better final BCVA, a thicker baseline ONL, and an intact baseline ELM, EZ, and IZ. However, ONL was the only predictor in a logistic regression model for SRF resorption.

Unusual Pneumonia Mimic.

Disease in atypical organ locations can mimic other pathologies, hampering the right diagnosis. Such conditions may even be emergencies, like appendicitis. Subhepatic appendix is a very rare entity which may be caused by caecum dehiscence failure. The authors present the case of a 55-year-old immunocompetent man admitted to the Emergency Department with sepsis and severe hypoxaemia. Chest x-ray showed right lower lobe infiltrate, and community-acquired pneumonia was diagnosed. The patient was started on broad-spectrum antibiotics, but he continued to deteriorate and after 3 days developed abdominal complaints. Exploratory laparoscopy revealed an abscess caused by perforated subhepatic appendicitis. Subhepatic appendicitis presents a diagnostic challenge and its clinical presentation may mimic that of other entities. This case highlights an atypical presentation, where the early development of inflammatory lung injury mimicked common pneumonia. Maintenance of a high index of suspicion and knowledge of these atypical locations is crucial. LEARNING POINTS: Control of the source of infection in sepsis is vital for survival.Acute inflammatory lung injury is common in sepsis and is correlated with clinical severity.A high index of suspicion and awareness of anatomical variants of the appendix are necessary for correct diagnosis of appendicitis mimicking other conditions.

Polineuropatía amiloidótica familiar Val30Met, insuficiencia cardiaca y ascitis quilosa: una cómbinacion inesperada / Val30Met familial amyloid polyneuropathy, heart failure, and chylous ascites: an unexpected combination

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