A Conformational-Dependent Interdomain Redox Relay at the Core of Protein Disulfide Isomerase Activity.

Aims: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to holdase function, some members catalyze disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical protein disulfide isomerase A1 (PDIA1) organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a', at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER. Results: Using two fluorescent redox sensors, redox green fluorescent protein 2 (roGFP2) and HyPer (circularly permutated yellow fluorescent protein containing the regulatory domain of the H2O2-sensing protein OxyR), either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a' active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a' active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds. Innovation and Conclusions: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown.

Bioadhesive Hyaluronic Acid-Based Hydrogels for Spinal Cord Injury.

Spinal cord injuries (SCI) have devastating physical, psychological, and psychosocial consequences for patients. One challenge of nerve tissue repair is the lack of a natural extracellular matrix (ECM) that guides the regenerating axons. Hyaluronic acid (HA) is a major ECM component and plays a fundamental role in facilitating lesion healing. Herein, we developed HA-based adhesive hydrogels by modification of HA with dopamine, a mussel-inspired compound with excellent adhesive properties in an aqueous environment. The hydrogels were loaded with the anti-inflammatory drug ibuprofen and the response of neuronal cells (SH-SY5Y) was evaluated in terms of viability, morphology, and adhesion. The obtained results suggested that the developed materials can bridge lesion gaps, guide axonal growth, and simultaneously act as a vehicle for the delivery of bioactive compounds.

Biocompatible 3D-Printed Tendon/Ligament Scaffolds Based on Polylactic Acid/Graphite Nanoplatelet Composites.

Three-dimensional (3D) printing technology has become a popular tool to produce complex structures. It has great potential in the regenerative medicine field to produce customizable and reproducible scaffolds with high control of dimensions and porosity. This study was focused on the investigation of new biocompatible and biodegradable 3D-printed scaffolds with suitable mechanical properties to assist tendon and ligament regeneration. Polylactic acid (PLA) scaffolds were reinforced with 0.5 wt.% of functionalized graphite nanoplatelets decorated with silver nanoparticles ((f-EG)+Ag). The functionalization of graphene was carried out to strengthen the interface with the polymer. (f-EG)+Ag exhibited antibacterial properties against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), an important feature for the healing process and prevention of bacterial infections. The scaffolds' structure, biodegradation, and mechanical properties were assessed to confirm their suitability for tendon and ligamentregeneration. All scaffolds exhibited surface nanoroughness created during printing, which was increased by the filler presence. The wet state dynamic mechanical analysis proved that the incorporation of reinforcement led to an increase in the storage modulus, compared with neat PLA. The cytotoxicity assays using L929 fibroblasts showed that the scaffolds were biocompatible. The PLA+[(f-EG)+Ag] scaffolds were also loaded with human tendon-derived cells and showed their capability to maintain the tenogenic commitment with an increase in the gene expression of specific tendon/ligament-related markers. The results demonstrate the potential application of these new 3D-printed nanocomposite scaffolds for tendon and ligament regeneration.

Fate of microbial contamination in a South European Coastal Lagoon (Ria Formosa) under the influence of treated effluents dispersal.

AIM: Assessment of the fate of microbial contamination driven from treated wastewater disposal at a highly productive zone on a South European coastal lagoon (Ria Formosa). METHODS AND RESULTS: Microbial indicators of contamination (Total coliforms, Escherichia coli, and Enterococci) were evaluated monthly during September 2018-September 2020 at three study areas (Faro, Olhão, and Tavira) under different wastewater discharge flows and hydrodynamic conditions. Additional data on E. coli monitoring in bivalves, available from the national institution responsible for their surveillance was also considered. The maximum microbial contamination was found at Faro, the highest-load and less-flushed study area, contrasting the lowest contamination at Olhão, a lower-load and strongly flushed area. The wastewater impact decreased along the spatial dispersal gradients and during high water, particularly at Faro and Tavira study areas, due to a considerable dilution effect. Microbial contamination at Olhão increased during the summer, while at the other study areas seasonal evidence was not clear. Data also indicate that E. coli in bivalves from bivalve production zones next to the three study areas reflected the differentiated impact of the wastewater treatment plants effluents on the water quality of those areas. CONCLUSIONS: Effluent loads together with local hydrodynamics, water temperature, solar radiation, precipitation, and land runoff as well as seabirds populations and environmentally adapted faecal or renaturelized bacterial communities, contributed to microbial contamination of the study areas.

Human Stem Cells for Cardiac Disease Modeling and Preclinical and Clinical Applications-Are We on the Road to Success?

Cardiovascular diseases (CVDs) are pointed out by the World Health Organization (WHO) as the leading cause of death, contributing to a significant and growing global health and economic burden. Despite advancements in clinical approaches, there is a critical need for innovative cardiovascular treatments to improve patient outcomes. Therapies based on adult stem cells (ASCs) and embryonic stem cells (ESCs) have emerged as promising strategies to regenerate damaged cardiac tissue and restore cardiac function. Moreover, the generation of human induced pluripotent stem cells (iPSCs) from somatic cells has opened new avenues for disease modeling, drug discovery, and regenerative medicine applications, with fewer ethical concerns than those associated with ESCs. Herein, we provide a state-of-the-art review on the application of human pluripotent stem cells in CVD research and clinics. We describe the types and sources of stem cells that have been tested in preclinical and clinical trials for the treatment of CVDs as well as the applications of pluripotent stem-cell-derived in vitro systems to mimic disease phenotypes. How human stem-cell-based in vitro systems can overcome the limitations of current toxicological studies is also discussed. Finally, the current state of clinical trials involving stem-cell-based approaches to treat CVDs are presented, and the strengths and weaknesses are critically discussed to assess whether researchers and clinicians are getting closer to success.

Adhesive and self-healing materials for central nervous system repair.

The central nervous system (CNS) has a limited ability to regenerate after a traumatic injury or a disease due to the low capacity of the neurons to re-grow and the inhibitory environment formed in situ. Current therapies include the use of drugs and rehabilitation, which do not fully restore the CNS functions and only delay the pathology progression. Tissue engineering offers a simple and versatile solution for this problem through the use of bioconstructs that promote nerve tissue repair by bridging cavity spaces. In this approach, the choice of biomaterial is crucial. Herein, we present recent advances in the design and development of adhesive and self-healing materials that support CNS healing. The adhesive materials have the advantage of promoting recovery without the use of needles or sewing, while the self-healing materials have the capacity to restore the tissue integrity without the need for external intervention. These materials can be used alone or in combination with cells and/or bioactive agents to control the inflammation, formation of free radicals, and proteases activity. We discuss the advantages and drawbacks of different systems. The remaining challenges that can bring these materials to clinical reality are also briefly presented.

Transient neonatal hypocalcaemia caused by maternal hyperparathyroidism.

Hypocalcaemia in neonates can range from asymptomatic to a potentially life-threatening condition. We present a case of a 36 weeks gestational age boy, admitted to our neonatal intensive care unit for jitteriness, mild hypotonia and breastfeeding difficulties. By the ninth day of life, he presented with late-onset hypocalcaemia, hypomagnesaemia, low 25-OH-vitamin D and inappropriately normal parathyroid hormone. Further investigation revealed maternal hypercalcaemia with high parathyroid hormone. Maternal asymptomatic hyperparathyroidism was diagnosed and admitted as the cause of neonatal hypocalcaemia. There was a clinical improvement and calcium levels stabilisation after treatment with calcium gluconate and vitamin D3 This case highlights the importance of careful evaluation of neonatal late-onset hypocalcaemia in uncovering asymptomatic maternal hyperparathyroidism.

Sweet's Syndrome: A Case Report of a Rare Extraintestinal Manifestation of Ulcerative Colitis.

Sweet's syndrome is a neutrophilic dermatosis of unknown etiology and a rare extraintestinal manifestation of ulcerative colitis. Classically, it is more common in women with active inflammatory bowel disease (IBD). This syndrome typically presents in patients with acute-onset painful tender erythematous skin lesions and is usually accompanied by fever, arthralgia, and elevated inflammatory markers. Histological examination is characterized by diffuse dense dermal neutrophilic infiltrate with leukocytoclasia, without vasculitis. The treatment goals are to reduce morbidity and complications, and the most effective therapy is systemic corticosteroids. Early recognition of this syndrome is essential to improve our diagnostic and therapeutic abilities. We report a case of a 59-year-old female with ulcerative colitis, which presented with manifestations of Sweet's syndrome.

Adhesive and biodegradable membranes made of sustainable catechol-functionalized marine collagen and chitosan.

We describe bioadhesive membranes developed from marine renewable biomaterials, namely chitosan and collagen extracted from fish skins. Collagen was functionalized with catechol groups (Coll-Cat) to provide the membranes with superior adhesive properties in a wet environment and blended with chitosan to improve the mechanical properties. The blended membranes were compared to chitosan and chitosan blended with unmodified collagen in terms of surface morphology, wettability, weight loss, water uptake, mechanical and adhesive properties. The metabolic activity, the viability and the morphology of L929 fibroblastic cells seeded on these membranes were also assessed. Our results show that the functionalization with catechol groups improves the adhesive and mechanical properties of the membranes and enhances cell attachment and proliferation. These data suggest that the developed marine origin-raw membranes present a potential towards the restoration of the structural and functional properties of damaged soft tissues.

A Fluorescent Nanosensor for Silver (Ag+) and Mercury (Hg2+) Ions Using Eu (III)-Doped Carbon Dots.

Carbon dots doped with Eu3+ ions (Eu-Cdots) were prepared by a hydrothermal treatment, using citric acid and urea as precursors and Eu (NO3)3 as a europium source. The Eu3+ ions are strongly coordinated with the carboxylate groups at the surface of the Cdots and incorporated within the nanographene network in the carbon core. Vibrational spectroscopy provides evidence of such interaction with identification of bands assigned to the stretching of the Eu-O bond. Eu3+ doped Cdots have larger diameters then undoped Cdots, but they are divided into smaller domains of sp2 carbon. The UV-vis excitation spectrum provides evidence of energy transfer from the Cdots to the Eu3+. The luminescence spectrum shows the characteristic sharp peaks of Eu3+ in the red part of the visible spectrum and a broad emission of Cdots centered at 450 nm. The luminescence of the Cdots is strongly quenched by Hg2+ and Ag+, but not by other cations. The quenching mechanism differs significantly depending on the nature of the ion. Both the blue emission of Cdots and the red emission of Eu3+ are quenched in the presence of Hg2+ while only the emission of the Cdots is affected by the presence of Ag+. A ratiometric sensor can be built using the ratio of luminescence intensities of the Cdots to the Eu3+ peaks.

Understanding the bioaccumulation of pharmaceutical active compounds by clams Ruditapes decussatus exposed to a UWWTP discharge.

Twenty-four pharmaceutical active compounds (PhACs) were evaluated in the soft tissues of clams Ruditappes decussatus exposed along a 1.5-km dispersal gradient of the treated effluent from an urban wastewater treatment plant discharging in Ria Formosa, and compared with those in the marine waters and discharged effluents. The clams were exposed for 1 month, in June-July 2016, 2017 and 2018. PhACs were quantified by high performance liquid chromatography coupled to tandem mass spectrometry after the quick, easy, cheap, effective, rugged and safe (QuEChERS) method (clams) or solid-phase extraction (water samples). The most representative PhACs in the effluents and receiving waters (regardless of the tidal dilution effect) were diclofenac, carbamazepine and caffeine (on average ≤ 2 µg/L) and only caffeine exhibited significant inter-annual differences, with higher values in 2017. In turn, the most bioaccumulated PhACs in clams were caffeine (0.54-27 ng/g wet weight, significantly higher in 2016) and acetaminophen (0.37-3.7 ng/g wet weight, significant lower in 2016). A multivariate principal component analysis showed (i) PhAC bioaccumulation primarily depended on biotic factors (clams length and weight), (ii) PhAC physicochemical properties Log Kow, pKa and water solubility interplaying with water abiotic variables were more relevant for explaining data variability in water than the physical dilution/tidal mixing, (iii) this process, reflected by the salinity gradient, had a tertiary role in data variation, responsible for spatial discrimination of marine waters. This study provides a better understanding of PhACs bioaccumulation by clams Ruditapes decussatus in real environmental conditions, under the influence of urban treated effluent dispersal in Ria Formosa coastal lagoon, a major producer of bivalves, ultimately disentangling key factors of PhAC bioaccumulation.

Rhabdomyolysis Due to Salmonellosis: A Case Report of a Rare Presentation.

Salmonella infection has been commonly associated with gastrointestinal symptoms, such as diarrhea, abdominal pain, and nausea. However, in some cases, patients can develop rare and life-threatening complications such as rhabdomyolysis. Here, we report a case of gastroenteritis due to Salmonella enteritidis infection complicated by rhabdomyolysis. The patient was successfully treated with fluids and antibiotics. Despite rare, the association of Salmonella infection and rhabdomyolysis may occur. Prompt diagnosis and treatment with aggressive fluid replacement and antibiotics are paramount to prevent acute kidney injury.

Biomarker Responses of the Clam Ruditapes decussatus Exposed to a Complex Mixture of Environmental Stressors under the Influence of an Urban Wastewater-Treatment Plant.

To evaluate the potential impact of an urban wastewater-treatment plant on Ria Formosa coastal lagoon, a sentinel species, the clam Ruditapes decussatus, was exposed along a gradient of the effluent's dispersal for 1 mo. Three exposure sites were selected to study the responses of 3 biomarkers: electron transport system, acetylcholinesterase, and lipid peroxidation. As complementary data, morphometric measurements, condition index, and lipid and protein content were considered together with in situ physicochemical characterization of the sites (temperature, salinity, pH, and dissolved oxygen). Electron transport system activity levels were between 35.7 and 50.5 nmol O2 /min g protein, acetylcholinesterase activity levels ranged from 2.6 to 3.8 nmol/min g protein, and lipid peroxidation ranged from 174.7 to 246.4 nmol malondialdehyde/g protein. The exposure sites shaped the response not only of biomarkers but also of "health" parameters (protein, lipids, and condition index). Lipid peroxidation was the most responsive biomarker also associated with electron transport system, especially at the closest site to the urban wastewater-treatment plant. Because of the presence of complex mixtures of contaminants in urban effluents, biomarker responses can provide valuable information in environmental assessment. However, it is vital to identify all biological and ecological factors induced by the natural life cycle of clams. Abiotic factors can mask or overlap the response of biomarkers and should be considered in a multibiomarker approach. Environ Toxicol Chem 2021;40:272-283. © 2020 SETAC.

Congenital SARS-CoV-2 Infection in a Neonate With Severe Acute Respiratory Syndrome.

Coronavirus disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is mainly transmitted through droplets, but other ways of transmission have been hypothesized. We report a case of vertical transmission of SARS-CoV-2 in a preterm born to an infected mother, confirmed by the presence of the virus in the neonatal blood, nasopharyngeal and oropharyngeal swabs collected in the first half an hour of life. The neonate presented with acute respiratory distress, similar to the findings in severely affected adults. This case highlights the importance of pregnancy, labor and neonatal period surveillance of affected mothers and their newborns.

Stability of Protein Formulations at Subzero Temperatures by Isochoric Cooling.

Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP ß-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.

Evaluation of saliva pools method for detection of congenital human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most frequent cause of congenital infection, leading to long-term sequelae especially sensorineural hearing loss (SNHL). Since 5-15 % of the asymptomatic newborns will develop late sequelae, the implementation of a universal screening would allow the identification of infected children and early intervention. The aim of this study was to validate the use of saliva pools of 10 and 20 samples for the detection of HCMV congenital infection. Four spiking samples (negative saliva matrix added with known concentration of AD169 strain culture supernatant) and a set of 12 saliva samples, collected from newborns with confirmed congenital infection in their first three weeks of life, were tested individually and after dilution in 10 and 20 pools by an "in-house" RT-PCR. Both pool methodologies, 10-pool and 20-pool samples, had 100 % sensitivity and specificity when compared with individual samples. This methodology could allow a cost reduction close to 85 % and 89 %, respectively for the 10-pool and 20-pool approach, when compared with testing each sample individually. This significant reduction may open the possibility to perform the newborn screening for HCMV in a large-scale.

Children Who Leave the Emergency Department Without Being Seen: Why Did They Leave and What Would Make Them Stay?

INTRODUCTION: Children who visit emergency departments and leave without being seen represent a multifactorial problem. We aimed to compare the sociodemographic characteristics of children who left and of those who did not leave, as well as to evaluate parental reasoning, subsequent use of medical care and patient outcome. MATERIAL AND METHODS: This was a prospective case-control study of a random sample of children who left without being seen and their matched controls from an emergency department during a three-month period. We performed a phone questionnaire to obtain information concerning reasons for leaving, patient outcomes and general feedback. RESULTS: During the study period, 18 200 patients presented to the emergency department, of whom 92 (0.5%) left without being seen. Fifty-five (59.8%) completed the questionnaire and there were 82 controls. The most common reasons for leaving were 'excessive waiting time' (92.7%) and 'problem could wait' (21.8%). A significantly higher number of patients who left sought further medical care (78.2% vs 11%) but they did not experience higher levels of unfavourable outcomes. DISCUSSION: The waiting time seems to be the major factor that drives the decision to leave. The fact that parents felt safe in leaving and the low level of adverse outcomes highlights the low-acuity nature of the majority of patients who leave. CONCLUSION: Reducing the waiting times may be the logical strategic mean to decrease the rates of patients who leave without being seen. However, our data seems to indicate that the concerns surrounding clinical outcome after leaving may be partly unwarranted.

Introdução: O abandono de doentes do serviço de urgência pediátrico antes da observação médica constitui um problema multifatorial. Procurámos comparar características sociodemográficas de crianças que abandonaram a urgência e das que não abandonaram, assim como avaliar os motivos, recurso subsequente a cuidados de saúde e outcome clínico. Material e Métodos: Estudo caso-controlo prospetivo de amostra aleatória de crianças que abandonaram a urgência e de controlos pareados durante um período de três meses. Foi realizado um questionário telefónico para recolha de informação relacionada com os motivos para o abandono, outcomes clínicos e opiniões gerais. Resultados: Durante o período do estudo, 18 200 doentes recorreram ao Serviço de Urgência Pediátrica, dos quais 92 (0,5%) abandonaram. Um total de 55 casos (59,8%) e 82 controlos completaram o questionário. As razões mais comuns para o abandono foram 'tempo de espera excessivo' (92,7%) e 'problema podia esperar' (21,8%). Um número significativamente superior de doentes que abandonaram recorreu subsequentemente a cuidados de saúde (78,2% vs 11%), não tendo contudo apresentado uma incidência superior de outcomes adversos. Discussão: O tempo de espera parece ser o fator prioritário que motiva a decisão de abandonar a urgência. A segurança referida pelos pais aquando da decisão e a incidência reduzida de outcomes adversos parecem reforçar a noção de que se trata de doentes com casos clínicos de baixa gravidade. Conclusão: A redução do tempo de espera parece ser a medida estratégica lógica para diminuir as taxas de abandono. No entanto, a preocupação associada ao outcome clínico após o abandono poderá ser parcialmente injustificada.

Pseudohypoparathyroidism type 1B - a rare cause of tetany: case report.

Pseudohypoparathyroidism (PHP) is a rare group of disorders characterised by end-organ resistance to the parathyroid hormone (PTH). A 16-year-old boy presented with a 2-year history of involuntary dystonic movements involving mainly the left hand, initially after writing and later during physical exercise. Serum calcium was 1.37 mmol/L (2.20-2.69), phosphate 2.1 mmol/L (0.8-1.45) and PTH 302 ng/L (12-88). CT scan of the head demonstrated multiple subcortical and diffuse basal ganglia calcifications. Genetic analysis confirmed a methylation defect in the GNAS cluster on chromosome 20q13.32 which established the diagnosis. Treatment with calcitriol and calcium carbonate led to complete remission of symptoms. Causes of hypocalcaemia should be considered in evaluating patients with movement disorders. The diagnosis of PHP-1B is challenging but the overall prognosis is excellent.

Use of gabapentin in the treatment of chronic pain in an adolescent with sickle cell disease.

Vaso-occlusive crises are the most common manifestation of sickle cell disease (SCD) and the main cause of hospital admission in these patients. There is emerging evidence that vaso-occlusive pain has both nociceptive and neuropathic components. However, the treatment of SCD-related pain with neuropathic drugs has not yet been systematically studied, particularly in children. We describe a 14-year-old girl with SCD and multiple hospital admissions for pain management for severe acute vaso-occlusive pain episodes. The patient was evaluated by a multidisciplinary team of specialists which considered that the chronic, refractory pain she was experiencing for years was probably neuropathic in origin and it was decided to start oral gabapentin (300 mg/day). At 10 months follow-up, the patient reported remarkable improvement in her quality of life with a significant decrease in the number of hospital admissions (three admissions for acute vaso-occlusive pain episodes in 10 months versus the previously monthly recurrences).