Clinical and molecular features of four Brazilian families with multiple endocrine neoplasia type 1.

Objective: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome characterized by its clinical variability and complexity in diagnosis and treatment. We performed both clinical and molecular descriptions of four families with MEN1 in a follow-up at a tertiary center in Brasília. Methods: From a preliminary review of approximately 500 medical records of patients with pituitary neuroendocrine tumor (PitNET) from the database of the Neuroendocrinology Outpatient Clinic of the University Hospital of Brasília, a total of 135 patients met the criteria of at least two affected family members. From this cohort, we have identified 34 families: only four with a phenotype of MEN1 and the other 30 families with the phenotype of familial isolated pituitary adenoma (FIPA). Eleven patients with a clinical diagnosis of MEN1 from these four families were selected. Results: Variants in MEN1 gene were identified in all families. One individual from each family underwent genetic testing using targeted high-throughput sequencing (HTS). All patients had primary hyperparathyroidism (PHPT), and the second most common manifestation was PitNET. One individual had well-differentiated liposarcoma, which has been previously reported in a single case of MEN1. Three variants previously described in the database and a novel variant in exon 2 have been found. Conclusions: The study allowed the genotypic and phenotypic characterization of families with MEN1 in a follow-up at a tertiary center in Brasília.

Metabolites of endophytic fungi isolated from leaves of Bauhinia variegata exhibit antioxidant activity and agonist activity on peroxisome proliferator-activated receptors α, ß/δ and γ.

Diabetes mellitus is a metabolic disorder that affects millions of people worldwide and is linked to oxidative stress and inflammation. Thiazolidinediones (TZD) improve insulin sensitization and glucose homeostasis mediated by the activation of peroxisome proliferator-activated receptors γ (PPARγ) in patients with type 2 diabetes. However, their use is associated with severe adverse effects such as loss of bone mass, retention of body fluids, liver and heart problems, and increased risk of bladder cancer. Partial PPARγ agonists can promote the beneficial effects of thiazolidinediones with fewer adverse effects. Endophytic fungi colonize plant tissues and have a particularly active metabolism caused by the interaction with them, which leads to the production of natural products with significant biological effects that may be like that of the colonized plant. Here, we identify seven endophytic fungi isolated from Bauhinia variegata leaves that have antioxidant activities. Also, one of the extracts presented pan-agonist activity on PPAR, and another showed activity in PPARα and PPARß/δ. A better understanding of this relationship could help to comprehend the mechanism of action of antioxidants in treating diabetes and its complications. Moreover, compounds with these capabilities to reduce oxidative stress and activate the receptor that promotes glucose homeostasis are promising candidates in treatment of diabetes.

Antioxidant activity of mycelia methanolic extracts of endophytic fungi BvFV and BvFIX isolated from leaves of Bauhinia variegata.

Endophytes are considered an essential source of natural products. Skin is the body's largest organ; its primary function is the protection of other organs, and aging is one of the most relevant problems associated with this organ. UV radiation generates reactive oxygen species (ROS), which lead to skin degeneration and consequent aging. The main endogenous antioxidants that neutralize ROS are enzymatic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase, and non-enzymatic antioxidants, such as glutathione and α-tocopherol. Nuclear receptors are involved in molecular mechanisms that control the aging process, especially peroxisome proliferator-activated receptors (PPAR), which regulate the function and expression of genes that modulate the balance between matrix metalloproteinases (MMP) activity and the expression of collagen. Some natural compounds, such as polyphenols, can activate PPAR and reduce the activation of MMP and collagen degradation. In this work, the antioxidant activity of the mycelia methanolic extracts of two endophytic fungi isolated from leaves of Bauhinia variegata, named BvFV and BvFIX, their action as PPAR agonists, and their effect on the activity of antioxidant defense system enzymes were evaluated. The mycelia methanolic extract of BvFV showed a weak agonist effect on PPARß/δ, a high capability to inhibit lipid peroxidation, increased catalase activity, and increased superoxide dismutase activity by approximately 64%. In contrast, BvFIX increased catalase activity and increased superoxide dismutase activity in a dose-dependent manner, with an increase of 49.62% ± 7.87%, 56.64% ± 12.27%, and 240.46% ± 26.11% at concentrations of 25 µg/mL, 50 µg/mL and 100 µg/mL, respectively, in human dermal fibroblasts submitted to oxidative stress. These results suggest that the metabolites of the mycelia of endophytic fungi studied are promising to act in the chemoprevention of skin aging.

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR.

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method-nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18-23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow.

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented.

Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge.

The cell-free genomic DNA (gDNA) concentration in serum ranges from 1500 to 7500 copies/mL within 2 h after phlebotomy (6⁻24 times the concentration observed in plasma). Here, we aimed to evaluate the gDNA size distribution in serum with time after coagulation and to test if crude serum can be directly used as a source of gDNA for qPCR. Next, we investigated if single nucleotide polymorphisms (SNPs) could be genotyped directly from the crude serum isolated from capillary blood using a hand-powered paper centrifuge. All tested PCR targets (65, 100, 202 and 688 base pairs) could be successfully amplified from DNA extracted from serum, irrespective of their amplicon size. The observed qPCR quantitation cycles suggested that the genomic DNA yield increased in serum with incubation at room temperature. Additionally, only 65 and 101 base pair qPCR targets could be amplified from crude serum soon after the coagulation. Incubation for 4 days at room temperature was necessary for the amplification of PCR targets of 202 base pairs. The 688 base pair qPCR target could not be amplified from serum directly. Lastly, serum was successfully separated from capillary blood using the proposed paper centrifuge and the genotypes were assigned by testing the crude serum using allele-specific qPCR, producing small amplicon sizes in complete agreement with the genotypes assigned by testing the DNA extracted from whole blood. The serum can be used directly as the template in qPCR for SNP genotyping, especially if small amplicon sizes are applied. This shortcut in the SNP genotyping process could further molecular point-of-care diagnostics due to elimination of the DNA extraction step.

Association between suspected Zika virus disease during pregnancy and giving birth to a newborn with congenital microcephaly: a matched case-control study.

OBJECTIVE: In early 2015, an outbreak of an acute exanthematous illness with dengue-like symptoms occurred in northeastern Brazil. By the end of the same year, an unexpected increase in the number of cases of microcephaly was observed in the region. The microcephaly outbreak cause was unknown and rumors pointing to various potential causes arose. Since we were unaware at the time if this scenario would attract the interest of the broader scientific community, due to the neglected regions associated and as often happens with many others health conditions related to infectious diseases in Latin America. This coupled with the fact that diagnostic testing for Zika virus was not available, prompted us to design a study that could demonstrate the correlation between the development of an exanthematous illness with Zika-like symptoms during pregnancy and the delivery of a newborn with congenital microcephaly. RESULTS: Mothers who experienced symptoms associated with the Zika virus during pregnancy had 10 times higher odds of delivering newborns with congenital microcephaly when compared with mothers who did not exhibit Zika-like symptoms. Thus, the acute exanthematous illness outbreak could be associated with the congenital microcephaly outbreak. We could not distinguish which virus caused the acute exanthematous illness in the study subjects (Zika, dengue or chikungunya), but these results could help to reduce the misquided speculation in regards to the cause of the microcephaly and could have expedited public health policies intended for controlling the mosquito vector. In addition to the lower head circumference, microcephalic neonates also had lower thoracic circumference, lower height and lower weight compared to non-microcephalic babies suggesting intrauterine growth restriction. Additionally, we found borderline association between mothers classified as homemakers and, who had past dengue infections with microcephaly. Prior contraction of dengue virus seems to play a role in the risk for the condition reflecting the domestication of the Aedes Aegypti and the enhancement of the Zika virus infection by dengue antibodies, respectively. The limitations of this study are: (a) participants recall bias, (b) absence of laboratory test results for Zika virus and other arboviruses and (c) incomplete test results for other pathogens that could lead to microcephaly. The study protocol was registered at ClinicalTrial.gov under the identifier NCT02741882. Registered on April 13th, 2016.