NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Relationship between different sources of non-protein nitrogen and supplementation times on performance and metabolism of grazing Nellore cattle during the dry season.

Two experiments were conducted to evaluate the effect of supplementation with two sources of non-protein nitrogen at different feeding times on the performance, ingestive behavior, and rumen metabolism of growing Nellore bulls during the dry season. Exp. 1: One hundred and twenty Nellore bulls, weighing 206 ± 39 kg of initial body weight (BW) and 12 months of age, were divided into 20 paddocks, and they were used in randomized block design in a 2 × 2 factorial arrangement to evaluate performance and ingestive behavior. Exp. 2: 12 rumen cannulated animals with 509 ± 59 BW, divided into 4 paddocks, were used in a triple Latin square 4 × 4 in a 2 × 2 factorial arrangement to evaluate metabolism. The factors were 2 non-protein nitrogen sources (urea or slow-release urea) and 2 feeding times (07:00 or 13:00 at 4 g/kg BW of supplement). There was no influence of non-protein sources, supplementation time, or their interaction on the grazing time or the trough time during daytime, nighttime, or total (P ≥ 0.16). There were no interactions or factor effects on ADG (P ≥ 0.45) or final body weight (P ≥ 0.39). There was an interaction between supplementation time and collection time (P < 0.01) on ruminal pH. Animals supplemented in the morning had greater total SCFA at 18 h after supplementation (P = 0.03). The supplementation time and the non-protein nitrogen sources did not alter the ingestive behavior or animal performance of young Nellore cattle.

Impact of social isolation on the level of physical activity in young Brazilian athletes caused by COVID-19.

BACKGROUND: Social detachment to prevent the spread of the COVID-19 pandemics in the year 2020 can significantly contribute to the physical inactivity of citizens worldwide. The study aimed to analyze the level of physical activity by identifying the training methods used during the social isolation resulting from the COVID-19 pandemic. METHODS: Sixty-eight Brazilian athletes (both sexes, 14.7±1.68 years) answered and adapted the International Physical Activity Questionnaire (I-PAQ) through an online platform. Participants were asked to report their level of physical activity before and during the period of social distance. RESULTS: According to our results, 67.7% of the interviewed athletes said they were able to adapt their sports training to the isolation environment under the guidance of a distance physical education professional. Only 4.38% of the sample was not training under such supervision and, therefore, inactive. Among the activities performed in the isolation environment, calisthenics was the primary practice (effect size: ƒ2=0.50, P<0.0001) and the sport practiced at home was the secondary practice (effect size: ƒ2=0.27, P=0.004). During the pandemic, the training hours of athletes reduced significantly from ~3h to ~1h per day (effect size: 1.74, P<0.0001), as well as the perceived intensity decreased from "high" to "moderate" (effect size: 1.38, P<0.0001). The weekly training frequency decreased from ~6 to 7 days to ~3 to 5 days (effect size: 0.40, P=0.03). CONCLUSIONS: Despite the social distance and the reduced pace of training, the young Brazilian athletes analyzed managed to remain physically active during the COVID-19 pandemic.

Testicular pathology in fatal COVID-19: A descriptive autopsy study.

BACKGROUND: Multi-organ damage is a common feature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, going beyond the initially observed severe pneumonia. Evidence that the testis is also compromised is growing. OBJECTIVE: To describe the pathological findings in testes from fatal cases of COVID-19, including the detection of viral particles and antigens, and inflammatory cell subsets. MATERIALS AND METHODS: Postmortem testicular samples were obtained by percutaneous puncture from 11 deceased men and examined by reverse-transcription polymerase chain reaction (RT-PCR) for RNA detection and by light and electron microscopy (EM) for SARS-CoV-2. Immunohistochemistry (IHC) for the SARS-CoV-2 N-protein and lymphocytic and histiocytic markers was also performed. RESULTS: Eight patients had mild interstitial orchitis, composed mainly of CD68+ and TCD8+ cells. Fibrin thrombi were detected in five cases. All cases presented congestion, interstitial edema, thickening of the tubular basal membrane, decreased Leydig and Sertoli cells with reduced spermatogenesis, and strong expression of vascular cell adhesion molecule (VCAM) in vessels. IHC detected SARS-Cov-2 antigen in Leydig cells, Sertoli cells, spermatogonia, and fibroblasts in all cases. EM detected viral particles in the cytoplasm of fibroblasts, endothelium, Sertoli and Leydig cells, spermatids, and epithelial cells of the rete testis in four cases, while RT-PCR detected SARS-CoV-2 RNA in three cases. DISCUSSION AND CONCLUSION: The COVID-19-associated testicular lesion revealed a combination of orchitis, vascular changes, basal membrane thickening, Leydig and Sertoli cell scarcity, and reduced spermatogenesis associated with SARS-CoV-2 local infection that may impair hormonal function and fertility in men.

Causes of death in former asbestos-cement workers in the state of São Paulo, Brazil.

BACKGROUND: In low- and middle-income countries, such as Brazil, studies on the causes of death in asbestos-exposed workers are scarce. METHODS: A cohort study was performed involving 988 males who had worked in the asbestos-cement industry in the state of São Paulo, with a total of 12,217 person-years of observation between 1995 and 2016. The standardized mortality ratio (SMR) stratified by age was calculated as the ratio between the observed rate and the expected rate in the state of São Paulo. RESULTS: Increased SMRs were observed for overall mortality (SMR 1.1, 95% confidence interval [CI], 0.98-1.23) and mortality due to pleural malignant neoplasms (MN) (SMR, 69.4; 95% CI, 22.55-162.1), asbestosis (SMR, 975.7; 95% CI, 396.4-2031), peritoneal MN (SMR, 5.0; 95% CI, 0.13-27.78), laryngeal MN (SMR, 1.4; 95% CI, 0.30-4.20), and pulmonary MN (SMR, 1.5; 95% CI, 0.82-2.64). CONCLUSION: The present study highlights the damage caused by asbestos exposure and reinforces the existing evidence of a causal association between exposure and increased mortality due to pleural MN, pulmonary MN, and asbestosis.

Effects of a blend of live yeast and organic minerals or monensin on performance of dairy cows during the hot season.

The objective of this study was to evaluate the effects of feed additives on intake and digestibility of nutrients, milk yield and composition, feeding behavior, and physiological parameters of dairy cows during the hot season. Forty Holstein cows were assigned to a randomized block design experiment with a 2 × 2 factorial treatment arrangement to evaluate (1) control diet without inclusion of additives; (2) monensin (MON), 20 mg/kg diet dry matter sodium monensin (Rumensin; Elanco); (3) Milk Sacc+ (MS+), inclusion of 40 g/cow per d of Milk Sacc+ (a blend of live yeast and organic minerals, Alltech); and (4) combination of MON and MS+. The average temperature-humidity index throughout the experimental period was 73 ± 2.84 (standard deviation). The experiment lasted 11 wk, including 2 preliminary weeks for covariate adjustments. Cows fed MS+ increased dry matter intake (% body weight), milk yield, 3.5% fat-corrected milk, and solids yield, and cows fed MON had greater milk urea nitrogen content in comparison with counterparts. Feeding MS+ increased the intake of feed particles with size between 8 and 19 mm and decreased the intake of particles shorter than 4 mm compared with other treatments. Rumination time (min/d) and chewing time (min/kg of neutral detergent fiber) were lower for cows fed MS+. Physiologic parameters (i.e., heart and respiratory rates, and body temperature) were not affected by the treatments. Overall, the use of monensin did not differ from control, and Milk Sacc+ improved performance of cows.

Systemic delivery of antagomirs during blood-brain barrier disruption is disease-modifying in experimental epilepsy.

Oligonucleotide therapies offer precision treatments for a variety of neurological diseases, including epilepsy, but their deployment is hampered by the blood-brain barrier (BBB). Previous studies showed that intracerebroventricular injection of an antisense oligonucleotide (antagomir) targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in animal models of epilepsy. In this study, we used assays of serum protein and tracer extravasation to determine that BBB disruption occurring after status epilepticus in mice was sufficient to permit passage of systemically injected Ant-134 into the brain parenchyma. Intraperitoneal and intravenous injection of Ant-134 reached the hippocampus and blocked seizure-induced upregulation of miR-134. A single intraperitoneal injection of Ant-134 at 2 h after status epilepticus in mice resulted in potent suppression of spontaneous recurrent seizures, reaching a 99.5% reduction during recordings at 3 months. The duration of spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. In vivo knockdown of LIM kinase-1 (Limk-1) increased seizure frequency in Ant-134-treated mice, implicating de-repression of Limk-1 in the antagomir mechanism. These studies indicate that systemic delivery of Ant-134 reaches the brain and produces long-lasting seizure-suppressive effects after systemic injection in mice when timed with BBB disruption and may be a clinically viable approach for this and other disease-modifying microRNA therapies.

The influence of biological maturation and neuromuscular performance on peak bone mineral density in children.

BACKGROUND: The growth and development cycle of the adult is characterized by substantial changes in physical size and maturity, and is controlled by the simultaneous influence of genetic components and the environment. Changes in the development of bones, muscles and adipose tissue are among the most important aspects of adolescent physical development. The aim of this research was to examine the correlation between bone mineral density (BMD) and neuromuscular performance in children at different stages of maturation. METHODS: The study included 31 participants of both sexes (11.7±1.89 years). Body composition was assessed through bone densitometry using an energy duo X-ray source. The neuromuscular performance of the upper limbs was analyzed by the medicine ball hand grip and throwing tests. The lower limbs by the vertical counter-motion jump test, and skeletal maturation was analyzed by bone age. Maturation was used as an effect control between the association of BMD and neuromuscular performance. RESULTS: The results demonstrated significant associations between the performance of upper (P<0.0001) and lower (P=0.01) limbs and BMD. On inter-group comparison for only the accelerated maturational stage, male participants had greater BMD than female participants (P=0.001; effect size 3.13). Regarding the maturational stages, male participants in the accelerated maturational stage demonstrated higher BMD than those in the normal and delayed maturational stages (P=0.04; effect size 3.10), whereas female participants did not demonstrate significant differences in BMD accumulation. CONCLUSIONS: BMD has a strong correlation with the neuromuscular performance of upper and lower limbs of children of both sexes.

Effectiveness of the 40-Minute Handmade Manikin Program to Teach Hands-on Cardiopulmonary Resuscitation at School Communities.

Bystander training in cardiopulmonary resuscitation (CPR) is crucial to improve the victims' survival and quality of life after sudden cardiac arrest. This observational study aimed to determine the success rate of 2 different programs of CPR training for children, adolescents, and adults in school communities. We assessed the development and acquisition of the following CPR skills: checking local safety, assessing victim's responsiveness, calling for help, assessing victim's breathing, and performing chest compression (hands and straight arms placement on the chest, compression velocity, depth, and chest release) using a 40-minute program with handmade manikins or the 120-minute program using intermediate-fidelity manikins. There were 1,630 learners (mean age 16 years, 38% male) in the 40-minute program, and 347 learners (mean age 27 years, 32% male) in the 120-minute program. The lowest successful pass rate of learners that developed CPR skills was 89.4% in the 40-minute program and 84.5% in the 120-minute program. The chances of success increased with age in the same program (compression rate and depth). The success rate also increased with the more extended and intermediate-cost program at the same age (assessing victim's responsiveness, calling for help, and assessing the victim's respiration). In conclusion, a 40-minute and cheaper (low-cost handmade manikin) CPR program was adequate to develop and acquire the overall CPR skills for ≥89% at school communities, independently of gender. However, some individual CPR skills can be further improved with increasing age and using the longer and intermediate-cost program.

Total Synthesis of (+)-Raputindole A: An Iridium-Catalyzed Cyclization Approach.

This work describes the total synthesis of raputindole A (1) through a convergent approach that features (1) an iridium-catalyzed cyclization to assemble the tricyclic core of the northern part, (2) enzymatic resolution to secure the preparation of an enantiomerically pure benzylic alcohol intermediate, and (3) the installation of the isobutenyl side chain via methallylation of the corresponding benzylic carbocation and coupling of the northern and southern parts via the Heck reaction. (+)-Raputindole A (1) was prepared in 10 steps (longest linear sequence) in 3.3% overall yield.

Genetic deletion of microRNA-22 blunts the inflammatory transcriptional response to status epilepticus and exacerbates epilepsy in mice.

MicroRNAs perform important roles in the post-transcriptional regulation of gene expression. Sequencing as well as functional studies using antisense oligonucleotides indicate important roles for microRNAs during the development of epilepsy through targeting transcripts involved in neuronal structure, gliosis and inflammation. MicroRNA-22 (miR-22) has been reported to protect against the development of epileptogenic brain networks through suppression of neuroinflammatory signalling. Here, we used mice with a genetic deletion of miR-22 to extend these insights. Mice lacking miR-22 displayed normal behaviour and brain structure and developed similar status epilepticus after intraamygdala kainic acid compared to wildtype animals. Continuous EEG monitoring after status epilepticus revealed, however, an accelerated and exacerbated epilepsy phenotype whereby spontaneous seizures began sooner, occurred more frequently and were of longer duration in miR-22-deficient mice. RNA sequencing analysis of the hippocampus during the period of epileptogenesis revealed a specific suppression of inflammatory signalling in the hippocampus of miR-22-deficient mice. Taken together, these findings indicate a role for miR-22 in establishing early inflammatory responses to status epilepticus. Inflammatory signalling may serve anti-epileptogenic functions and cautions the timing of anti-inflammatory interventions for the treatment of status epilepticus.

Pulmonary and systemic involvement in COVID-19 patients assessed with ultrasound-guided minimally invasive autopsy.

AIMS: Brazil ranks high in the number of coronavirus disease 19 (COVID-19) cases and the COVID-19 mortality rate. In this context, autopsies are important to confirm the disease, determine associated conditions, and study the pathophysiology of this novel disease. The aim of this study was to assess the systemic involvement of COVID-19. In order to follow biosafety recommendations, we used ultrasound-guided minimally invasive autopsy (MIA-US), and we present the results of 10 initial autopsies. METHODS AND RESULTS: We used MIA-US for tissue sampling of the lungs, liver, heart, kidneys, spleen, brain, skin, skeletal muscle and testis for histology, and reverse transcription polymerase chain reaction to detect severe acute respiratory syndrome coronavirus 2 RNA. All patients showed exudative/proliferative diffuse alveolar damage. There were intense pleomorphic cytopathic effects on the respiratory epithelium, including airway and alveolar cells. Fibrinous thrombi in alveolar arterioles were present in eight patients, and all patients showed a high density of alveolar megakaryocytes. Small thrombi were less frequently observed in the glomeruli, spleen, heart, dermis, testis, and liver sinusoids. The main systemic findings were associated with comorbidities, age, and sepsis, in addition to possible tissue damage due to the viral infection, such as myositis, dermatitis, myocarditis, and orchitis. CONCLUSIONS: MIA-US is safe and effective for the study of severe COVID-19. Our findings show that COVID-19 is a systemic disease causing major events in the lungs and with involvement of various organs and tissues. Pulmonary changes result from severe epithelial injury and microthrombotic vascular phenomena. These findings indicate that both epithelial and vascular injury should be addressed in therapeutic approaches.

Sequential Michael Addition/Electrophilic Alkynylation: Synthesis of α-Alkynyl-ß-Substituted Ketones and Chromanones.

We describe the synthesis of α-alkynyl-ß-substituted cyclic ketones and analogue chromanones via one-pot Michael addition/hypervalent iodine-based α-alkynylation. Cu(I)-catalyzed Michael addition using either alkyl-aluminum or Grignard reagents, followed by diastereoselective electrophilic alkynylation of the resulting enolate by 1-ethynyl-1λ3,2-benziodoxol-3(1H)-one (EBX) resulted in the α-alkynyl-ß-substituted cyclic ketones or chromanones within 34-89% yield (16 examples). The reaction was successfully upscaled to the 5 mmol scale, and further functionalization of a model alkynylated ketone was demonstrated.

Metal-Free Synthesis of Homopropargylic Alcohols from Aldehydes.

The synthesis of homopropargylic alcohols under metal-free and mild condition is described. This transformation is based on a one-pot procedure involving sequential α-alkynylation of acyclic aldehydes using hypervalent iodine reagents and borohydride reduction. The chemistry exhibits broad substrate scope and good scalability, providing a convenient route for the α-alkynylation of aldehydes along with the formation of a quaternary carbon center. The applicability of the method is demonstrated by the gram-scale synthesis of the key synthetic precursor of botulinum toxin inhibitors.

Potent Anti-seizure Effects of Locked Nucleic Acid Antagomirs Targeting miR-134 in Multiple Mouse and Rat Models of Epilepsy.

Current anti-epileptic drugs (AEDs) act on a limited set of neuronal targets, are ineffective in a third of patients with epilepsy, and do not show disease-modifying properties. MicroRNAs are small noncoding RNAs that regulate levels of proteins by post-transcriptional control of mRNA stability and translation. MicroRNA-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid (LNA), cholesterol-tagged antagomirs targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in mouse models of status epilepticus. Translation of these findings would benefit from evidence of efficacy in non-status epilepticus models and validation in another species. Here, we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre-treated with Ant-134 in the pentylenetetrazol model. Pre-treatment with Ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats, a toxin-free model of acquired epilepsy. Nevertheless, Ant-134 post-treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and Ant-134 delayed epileptiform activity in a rat ex vivo hippocampal slice model. The potent anticonvulsant effects of Ant-134 in multiple models may encourage pre-clinical development of this approach to epilepsy therapy.

Performance of post-mortem CT compared to autopsy in children.

INTRODUCTION: Radiological techniques such as non-enhanced post-mortem computed tomography (PMCT) play an increasingly important role in death investigations, especially in cases of non-medicolegal context of death, where the consent of the next of kin is required to perform autopsy. Such consent is often difficult to obtain for deceased children, and radiological methods may be an acceptable alternative. The aim of our study was to evaluate the performance of PMCT explorations compared to medicolegal conventional autopsies in children and its potential usefulness in non-medicolegal situations. METHODS: We retrospectively reviewed a group of 26 children aged 0-12 years who died of different causes, which were investigated by both conventional autopsy and PMCT. We compared the findings extracted from radiological and autopsy reports. All findings were grouped according to their importance with respect to cause of death and to the anatomical structure they covered: organs, vascular system, soft tissue, and skeletal system. RESULTS: A significantly larger number of findings were detected by autopsy compared to PMCT. Autopsy proved to be superior to PMCT, notably at detecting organ, soft tissue, and vascular findings, while PMCT was superior at detecting bone findings. However, no statistically significant differences were found between the methods concerning the essential findings used to define the cause of death. CONCLUSIONS: In children, PMCT was less sensitive than conventional autopsy for detecting general findings. However, most essential findings were detected by both methods. PMCT was superior to autopsy for the detection of bone lesions in children. ADVANCES IN KNOWLEDGE: Up to today, very rare literature exists concerning PMCT in children, especially in a forensic setting. This article investigates the advantages and limitations of PMCT compared to autopsy in a unique study group and discusses possibilities for future developments.

Metal-Free Asymmetric Synthesis of Indanes through Chiral Hypervalent Iodine(III)-Mediated Ring Contraction.

The iodine(III)-mediated asymmetric oxidative rearrangement of 1,2-dihydronaphthalenes was investigated to prepare optically active 1-substituted indanes. The chiral hypervalent iodine species is generated in situ from a chiral aryl iodide, prepared in 94% yield in one step. This metal-free protocol was applied to different cyclic alkenes, substituted with oxygen, with nitrogen, or at position 1 with aryl or methyl. Indanes can be isolated as an acetal or alcohol in up to 78% ee.