Standard operating procedures for SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 40,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated standard operating procedure (SOP) for high-throughput SARS-CoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.

Sleep deprivation decreases the reproductive capacity by affecting the arrival of morulas in the uterus.

A previous animal study by our group found that sleep deprivation during preimplantation was associated with decreased pregnancy maintenance. Given its impact on human society, we aimed in the current study to assess whether sleep deprivation affects blastocyst gene expression and/or the implantation process. For this, pregnant mice (gestational day 0 [GD 0]) were assigned into paradoxical sleep deprivation (SD, 72 hr; multiple platform method) and, a control (CT) group. Animals were euthanized on GD 3.5 and blood, uterus (embryos) and fallopian tube were collected. Then, 89% of CT presented blastocysts in the uterus versus 25% from SD group. Compared to CT, SD presented lighter relative uterus weight, increased plasma concentrations of corticosterone and testosterone, decreased concentrations of progesterone and luteinizing hormone, but no statistical differences in plasma concentrations of 17ß-estradiol and follicle stimulating hormone. There were no differences in uterus and blastocyst gene expression related to embryo implantation and development, and no alteration in blastocysts global DNA methylation. Considering this, the decreased pregnancy maintenance after sleep deprivation seems not to be associated with implantation losses or developmental problems related to the blastocysts. It is likely that complications in morula development and/or its movement through the fallopian tubes affect the pregnancy rate, since only 25% of SD females presented a blastocyst on the GD 3.5. In fact, three out of four females without blastocysts in the uterus presented morula in the fallopian tubes due to a phase delay. Additionally, we suggest that the observed hormonal changes may play a role in this outcome.

II Brazilian consensus statement on endoscopic ultrasonography.

BACKGROUND AND OBJECTIVES: At the time of its introduction in the early 80s, endoscopic ultrasonography (EUS) was indicated for diagnostic purposes. Recently, EUS has been employed to assist or to be the main platform of complex therapeutic interventions. METHODS: From a series of relevant new topics in the literature and based on the need to complement the I Brazilian consensus on EUS, twenty experienced endosonographers identified and reviewed the pertinent literature in databases. The quality of evidence, strength of recommendations, and level of consensus were graded and voted on. RESULTS: Consensus was reached for eight relevant topics: treatment of gastric varices, staging of nonsmall cell lung cancer, biliary drainage, tissue sampling of subepithelial lesions (SELs), treatment of pancreatic fluid collections, tissue sampling of pancreatic solid lesions, celiac neurolysis, and evaluation of the incidental pancreatic cysts. CONCLUSIONS: There is a high level of evidence for staging of nonsmall cell lung cancer; biopsy of SELs as the safest method; unilateral and bilateral injection techniques are equivalent for EUS-guided celiac neurolysis, and in patients with visible ganglia, celiac ganglia neurolysis appears to lead to better results. There is a moderate level of evidence for: yield of tissue sampling of pancreatic solid lesions is not influenced by the needle shape, gauge, or employed aspiration technique; EUS-guided and percutaneous biliary drainage present similar clinical success and adverse event rates; plastic and metallic stents are equivalent in the EUS-guided treatment of pancreatic pseudocyst. There is a low level of evidence in the routine use of EUS-guided treatment of gastric varices.

MCH levels in the CSF, brain preproMCH and MCHR1 gene expression during paradoxical sleep deprivation, sleep rebound and chronic sleep restriction.

Neurons that utilize melanin-concentrating hormone (MCH) as neuromodulator are located in the lateral hypothalamus and incerto-hypothalamic area. These neurons project throughout the central nervous system and play a role in sleep regulation. With the hypothesis that the MCHergic system function would be modified by the time of the day as well as by disruptions of the sleep-wake cycle, we quantified in rats the concentration of MCH in the cerebrospinal fluid (CSF), the expression of the MCH precursor (Pmch) gene in the hypothalamus, and the expression of the MCH receptor 1 (Mchr1) gene in the frontal cortex and hippocampus. These analyses were performed during paradoxical sleep deprivation (by a modified multiple platform technique), paradoxical sleep rebound and chronic sleep restriction, both at the end of the active (dark) phase (lights were turned on at Zeitgeber time zero, ZT0) and during the inactive (light) phase (ZT8). We observed that in control condition (waking and sleep ad libitum), Mchr1 gene expression was larger at ZT8 (when sleep predominates) than at ZT0, both in frontal cortex and hippocampus. In addition, compared to control, disturbances of the sleep-wake cycle produced the following effects: paradoxical sleep deprivation for 96 and 120 h reduced the expression of Mchr1 gene in frontal cortex at ZT0. Sleep rebound that followed 96 h of paradoxical sleep deprivation increased the MCH concentration in the CSF also at ZT0. Twenty-one days of sleep restriction produced a significant increment in MCH CSF levels at ZT8. Finally, sleep disruptions unveiled day/night differences in MCH CSF levels and in Pmch gene expression that were not observed in control (undisturbed) conditions. In conclusion, the time of the day and sleep disruptions produced subtle modifications in the physiology of the MCHergic system.

A putative role for homocysteine in the pathophysiology of acute bacterial meningitis in children.

BACKGROUND: Acute bacterial meningitis frequently causes cortical and hippocampal neuron loss leading to permanent neurological sequelae. Neuron death in acute bacterial meningitis involves the excessive activation of NMDA receptors and p53-mediated apoptosis, and the latter is triggered by the depletion of NAD + and ATP cellular stores by the DNA repair enzyme poly(ADP-ribose) polymerase. This enzyme is activated during acute bacterial meningitis in response to DNA damage induced, on its turn, by reactive oxygen and nitrogen species. An excess of homocysteine can also induce this cascade of events in hippocampal neurons. The present work aimed at investigating the possible involvement of homocysteine in the pathophysiology of meningitis by comparing its concentrations in cerebrospinal fluid (CSF) samples from children with viral or acute bacterial meningitis, and control individuals. METHODS: Homocysteine and cysteine concentrations were assessed by high-performance liquid chromatography in CSF samples from nine patients with acute bacterial meningitis, 13 patients with viral meningitis and 18 controls (median age: 4 years-old; range: <1 to 13) collected by lumbar puncture at admission at the Children's Hospital Joao Paulo II - FHEMIG, from January 2010 to November 2011. RESULTS: We found that homocysteine accumulates up to neurotoxic levels within the central nervous system of patients with acute bacterial meningitis, but not in those with viral meningitis or control individuals. No correlation was found between homocysteine and cysteine concentrations and the cerebrospinal fluid standard cytochemical parameters. CONCLUSIONS: Our results suggest that HCY is produced intrathecally in response to acute bacterial meningitis and accumulates within the central nervous system reaching potentially neurotoxic levels. This is the first work to propose a role for HCY in the pathophysiology of brain damage associated with acute bacterial meningitis.

Endoscopic management and prevention of migrated esophageal stents.

The use of self-expandable metallic stents has increased recently to palliate inoperable esophageal neoplasia and also in the management of benign strictures. Migration is one of the most common complications after stent placement and the endoscopist should be able to recognize and manage this situation. Several techniques for managing migrated stents have been described, as well as new techniques for preventing stent migration. Most stents have a "lasso" at the upper flange which facilitates stent repositioning or removal. An overtube, endoloop and large polypectomy snare may be useful for the retrieval of stents migrated into the stomach. External fixation of the stent with Shim's technique is efficient in preventing stent migration. Suturing the stent to the esophageal wall, new stent designs with larger flanges and double-layered stents are promising techniques to prevent stent migration but they warrant validation in a larger cohort of patients.

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior.

BACKGROUND: Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity. METHODS: MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses. RESULTS: After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months. CONCLUSIONS: These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.

Optimal cutoff points for body mass index, waist circumference and HOMA-IR to identify a cluster of cardiometabolic abnormalities in normal glucose-tolerant Brazilian children and adolescents / Valores de corte ótimos do índice de massa corporal, circunferência abdominal e HOMA-IR para a identificação de um conjunto de anormalidades cardiometabólicas em crianças e adolescentes brasileiros com tolerância normal à glicose

OBJECTIVE: The aim of this study was to establish the best cutoff values for waist circumference (WC), body mass index (BMI) and HOMA-IR (HR) to identify a cluster (> 3) of cardiovascular risk factors (CVRF) in normal glucose-tolerant (NGT) Brazilian children and adolescents. SUBJECTS AND METHODS: Cross-sectional study of 319 individuals (aged 10 to 19y) from a southern Brazilian city. Gender-specific receiver-operating characteristics (ROC) curves were constructed to assess cutoffs values of BMI (kg/m², WC (cm), and HR. RESULTS: The areas under the ROC curves to detect a cluster of CVRF were 0.92, 0.93 and 0.68 (females), and 0.93, 0.93 and 0.89 (males), for WC, BMI and HR, respectively. The cutoff values were 83.0 and 80.5 cm (WC), 22.7 and 20.4 kg/m2 (BMI), and 1.65 and 1.95 (HR), for females and males, respectively, to detect the cluster of CVRF. CONCLUSION: These values of BMI, WC-) and (HR) detected a high proportion of NGTt Brazilian children and adolescents with a cluster of CVRF.

OBJETIVO: O objetivo deste estudo foi estabelecer os melhores valores de corte para circunferência abdominal (CA), índice de massa corpórea (IMC) e HOMA-IR (HR) para identificação da concomitância de um conjunto de fatores de risco cardiovascular (> 3) em crianças e adolescentes brasileiros com tolerância normal à glicose (TNG). SUJEITOS E MÉTODOS: Estudo transversal realizado em uma cidade do sudeste do Brasil com 319 indivíduos de 10 a 19 anos de idade. Curva ROC para cada gênero foi utilizada para estabelecimento dos valores de IMC (kg/m²), CA (cm) e HRR. RESULTADOS: As áreas sob as curvas ROC para detectar o conjunto de fatores cardiovascular foram 0,92, 0,93 e 0,68 (meninas) e 0,93, 0,93 e 0,89 (meninos) para CA, IMC e HR, respectivamente. Os valores de corte foram 83,0 e 80,5 cm (CA), 22,7 e 20,4 kg/m² (IMC) e 1,65 e 1,95 (HR), para meninas e meninos, respectivamente, para detecção do grupo de fatores de risco cardiovascular. CONCLUSÃO: Esses valores de IMC,CA e HR detectaram uma porcentagem significativa de crianças e adolescentes brasileiros com TNG e elevado risco cardiovascular.

Optimal cutoff points for body mass index, waist circumference and HOMA-IR to identify a cluster of cardiometabolic abnormalities in normal glucose-tolerant Brazilian children and adolescents.

OBJECTIVE: The aim of this study was to establish the best cutoff values for waist circumference (WC), body mass index (BMI) and HOMA-IR (HR) to identify a cluster (≥ 3) of cardiovascular risk factors (CVRF) in normal glucose-tolerant (NGT) Brazilian children and adolescents. SUBJECTS AND METHODS: Cross-sectional study of 319 individuals (aged 10 to 19y) from a southern Brazilian city. Gender-specific receiver-operating characteristics (ROC) curves were constructed to assess cutoffs values of BMI (kg/m(2), WC (cm), and HR. RESULTS: The areas under the ROC curves to detect a cluster of CVRF were 0.92, 0.93 and 0.68 (females), and 0.93, 0.93 and 0.89 (males), for WC, BMI and HR, respectively. The cutoff values were 83.0 and 80.5 cm (WC), 22.7 and 20.4 kg/m(2) (BMI), and 1.65 and 1.95 (HR), for females and males, respectively, to detect the cluster of CVRF. CONCLUSION: These values of BMI, WC-) and (HR) detected a high proportion of NGTt Brazilian children and adolescents with a cluster of CVRF.

Biochemical, biometrical and behavioral changes in male offspring of sleep-deprived mice.

Epidemiological and experimental studies suggest a high prevalence of cognitive impairment and social behavior deficits in adolescents and adults that have experienced prenatal exposure to adverse conditions. This study investigated whether sleep deprivation during the pre-implantation stage of development alters the physiological, behavioral and oxidative metabolic processes in adult male mouse offspring. One group of dams was continuously sleep-deprived using the platform technique from gestational days 0 to 3 (PSD 72). Three additional groups were sleep-deprived by gentle handling for 6h on gestational days 1 (GH 1), 2 (GH 2) or 3 (GH 3). After sleep deprivation, homocysteine, cysteine, corticosterone, estrogen and progesterone concentrations were measured from the experimental mothers and time-matched controls. The sizes and weights of the male pups were measured at various stages throughout the experiment. At PND 90, behavioral (Activity Box and Elevated Plus Maze) and biochemical parameters were assessed. The dams' plasma progesterone concentrations decreased in the PSD 72 group, and the levels of plasma estradiol increased in GH 2. Corticosterone levels were found to increase after all sleep-deprivation procedures. Homocysteine concentrations increased in the GH 2 but decreased in the PSD 72 group. The offspring of GH 1 mothers exhibited decreased superoxide dismutase activity. Exposure to sleep deprivation had a long-lasting impact on tissue weight; in particular, there was a decrease in hemilateral epididymal fat weight in mature animals from the PSD 72 group. Although some of the alterations observed in the mothers (elevated estrogen and corticosterone levels and decreased progesterone) might have played a role in the permanent alterations in the adult offspring, they were not the main cause. The homocysteine changes detected in the sleep-deprived dams may contribute to redox changes, controlling gene expression and shaping epigenetic development.

Homocysteine and cysteine concentrations are modified by recent exposure to environmental air pollution in São Paulo, Brazil.

Millions of people worldwide are affected by anthropogenic air pollution derived from the combustion of fossil fuels. In this work, we tested the effects of fetal, lactation and post-weaning ambient air pollution exposure on total homocysteine (tHcy) concentrations and on a downstream pathway element, the plasma cysteine (Cys) concentration. Two similar exposure chambers (polluted and filtered chamber) were located near an area with heavy traffic in São Paulo, Brazil, and male Swiss mice were housed there from the pre-natal period until 3 months of age. Groups during fetal, lactation and adult periods of exposure were apportioned, and tHcy and Cys plasma concentrations were assessed when the animals were 3 months old. In our study, both the tHcy and Cys concentrations were decreased in groups that spent their final stage of life in polluted chambers, suggesting recent alterations in tHcy and Cys concentrations due to air pollution exposure. The possible relationship of these data with cardiovascular dysfunction is still a matter of controversy in animals; nevertheless, epigenetic mechanisms emerge as a possible issue to consider in the investigation of the link between air pollution and Hcy measurement.

Avaliação "In Vitro" da resistência a tração de bráquetes metálicos colados sobre superfície de resina acrílica com diferentes condicionamentos / Evaluation \"In Vitro\" the resistance of metallic brackets traction bonding over acrylic artificial teeth with different primed surfaces

O objetivo desta pesquisa foi avaliar a resistência a tração de bráquetes metálicos colados sobre dentes artificiais de resina acrílica com superfícies condicionadas diferentemente. Foram confeccionados 50 corpos de prova, constituído de cílindro de PVC preenchidos com resina acrílica com dente artificial inserido no meio com a face vestibular voltada para cima. Esses corpos de prova foram divididos em cinco grupos: Grupo I – controle: superfície de resina acrílica sem qualquer tipo de tratamento; Grupo II – superfície de resina jateada com óxido de alumínio (Bioart) de 50 micrômetros; Grupo III – superfície de resina de ranhuras realizadas com ponta diamantada cilíndrica (KG Sorensen), em alta rotação com refrigeração durante 5 a 10 segundos; Grupo IV – superfície de resina tratada com ácido hidrofluorídrico a 9,6% durante 2 a 4 minutos; Grupo V – aplicação do silano sobre a superfície de resina acrílica. Após o condicionamento das superfícies, bráquetes metálicos Mini Dyna-Lock (Unitek, 3M) foram colados com resina fotopolimerizável Transbond XT (3M). Após 24 horas os corpos de prova foram submetidos ao teste de tração. Os resultados foram submetidos à análise de variância e ao Teste de Tukey, com significância de 5%. A média da resistência a tração foi de 0,6780 KgF/mm2 para o grupo III; 0,6688 KgF/mm2 para o grupo V; 0,6287 KgF/mm2 para o grupo II, 0,6137 para o grupo I e 0,4408 para o grupo IV. Os resultados mostraram que o grupo utilizando ácido hidrofluoridico a 9,6% apresentou o menor valor, porém sem nenhuma diferença estatística significante entre os outros grupos.

How variable are hydroxyproline determinations made in different samples of the same liver?

OBJECTIVES: The haphazard distribution of fibrous tissue can interfere with quantitative methods for evaluating hepatic fibrosis. Inter-sample variation may represent a crucial issue when hydroxyproline measurement is used to quantify fibrosis. A comparative study of the hydroxyproline levels in normal and fibrotic rats is herein reported. MATERIAL AND METHODS: Twelve normal and 20 Capillaria hepatica-infected Wistar rats were used. Two fragments of the liver (A and B) of each rat were taken from separate areas and hydroxyproline measurements were made. Calculated differences in hydroxyproline measurements between samples from the same liver were analyzed by BOOTSTRAP. RESULTS: Differences in normal rats varied from 0.026 to 1.85 micromol of HP/g, in ten rats, the difference was less than 0.50 micromol. In infected rats, it varied from 0.04 to 2.86 micromol HP/g. Differences higher than 0.69 micromol/g were significant for normal rats (p<0.05) and above 1.22 micromol/g (p<0.05) for fibrotic rats. CONCLUSIONS: Hydroxyproline ratio in a normal liver kept a fair degree of reproducibility. In the presence of hepatic fibrosis, the levels of hydroxyproline may vary significantly between samples from a single liver and may have limited value in quantifying the extent of fibrosis.

Capillaria hepatica in rats: focal parasitic hepatic lesions and septal fibrosis run independent courses.

Capillaria hepatica causes two main lesions in the liver of rats: multifocal chronic inflammation, directly related to the presence of disintegrating parasites and their eggs, and a process of systematized septal fibrosis. The comparative behavior of these two lesions was investigated in rats experimentally infected with 600 embryonated eggs, following either corticosteroid treatment or specific antigenic stimulation, in an attempt to understand the relationship between these two lesions, and the pathogenesis of septal fibrosis. The two treatments differently modified the morphological aspects of the focal parasitic-related lesions, but did not interfere with the presentation of diffuse septal fibrosis, although a mild decrease in the degree of fibrosis occurred in corticoid-treated animals. These findings indicate that although the two lesions are C. hepatica induced, they are under different pathogenetic control, the induction of septal fibrosis being triggered during early infection to follow an independent pathway.