Critical Factors in Sample Collection and Preparation for Clinical Metabolomics of Underexplored Biological Specimens.

This review article compiles critical pre-analytical factors for sample collection and extraction of eight uncommon or underexplored biological specimens (human breast milk, ocular fluids, sebum, seminal plasma, sweat, hair, saliva, and cerebrospinal fluid) under the perspective of clinical metabolomics. These samples are interesting for metabolomics studies as they reflect the status of living organisms and can be applied for diagnostic purposes and biomarker discovery. Pre-collection and collection procedures are critical, requiring protocols to be standardized to avoid contamination and bias. Such procedures must consider cleaning the collection area, sample stimulation, diet, and food and drug intake, among other factors that impact the lack of homogeneity of the sample group. Precipitation of proteins and removal of salts and cell debris are the most used sample preparation procedures. This review intends to provide a global view of the practical aspects that most impact results, serving as a starting point for the designing of metabolomic experiments.

Sample Preparation in Microbial Metabolomics: Advances and Challenges.

Microbial metabolomics has gained significant interest as it reflects the physiological state of microorganisms. Due to the great variability of biological organisms, in terms of physicochemical characteristics and variable range of concentration of metabolites, the choice of sample preparation methods is a crucial step in the metabolomics workflow and will reflect on the quality and reliability of the results generated. The procedures applied to the preparation of microbial samples will vary according to the type of microorganism studied, the metabolomics approach (untargeted or targeted), and the analytical platform of choice. This chapter aims to provide an overview of the sample preparation workflow for microbial metabolomics, highlighting the pre-analytical factors associated with cultivation, harvesting, metabolic quenching, and extraction. Discussions focus on obtaining intracellular and extracellular metabolites. Finally, we introduced advanced sample preparation methods based on automated systems.

Physical Training Protects Against Brain Toxicity in Mice Exposed to an Experimental Model of Glioblastoma.

Glioma 261 (Gl261) cell-mediated neurotoxicity has been reported in previous studies examining glioblastoma (GBM), and the effects of physical exercise (PE) on this neurotoxicity have been poorly investigated. This study aimed to evaluate the effects of a PE program in animals with experimental GBM. Male C57BL/6J mice were randomized into sham or GBM groups and subjected to a PE program for four weeks. Gl261 cells were administered into the intraventricular region at 48 h after the last exercise session. Body weight, water and feed consumption, and behavior were all evaluated for 21 days followed by euthanasia. The right parietal lobe was removed for the analysis of glial fibrillary acidic protein (GFAP), epidermal growth factor receptor (EGFR), vimentin, C-myc, nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), hydrogen peroxide, the glutathione system, and oxidative damage to proteins. The results revealed changes in the behavioral patterns of the trained animals, and no anatomopathological changes were observed in response to PE training. In contrast, animals with GBM subjected to PE exhibited lower immunoexpression of c-MYC, vimentin, and GFAP. Although experimental GBM altered the redox profile and inflammatory mediators, no significant alterations were observed after PE. In conclusion, our data provide consistent evidence of the relationship between PE and the improvement of tumorigenic parameters against the neurotoxicity of GL261 cells.

Fatality due to COVID-19 associated with risk factors in the province of Buenos Aires, Argentina, 2020 / Letalidad por covid-19 asociada a factores de riesgo en la provincia de buenos aires, argentina, 2020

RESUMEN INTRODUCCIÓN : El objetivo principal fue evaluar el riesgo de defunción en pacientes con diagnóstico confirmado de COVID-19 según la presencia de uno o más factores de riesgo. MÉTODOS : Estudio transversal, descriptivo y correlacional de personas mayores de 18 años, residentes de la provincia de Buenos Aires, con diagnóstico de COVID-19 confirmado por laboratorio o criterio clínico-epidemiológico que hayan iniciado síntomas entre marzo y diciembre de 2020 (n = 622 084). Se utilizó información de fuente secundaria. Se consideraron cuatro escenarios de análisis con base en la calidad del dato y la condición del paciente. RESULTADOS : Se notificaron 21 706 casos fallecidos (letalidad 3,5%). La mayoría de los fallecidos tenía edad avanzada (72,8 ± 13,8 años en los fallecidos versus 41,2 ± 15,2 años en los no fallecidos) y eran de sexo masculino (56% versus 44%, p<0,05). Según el modelo multivariado, la edad de 60 años o más resultó ser el factor de mayor riesgo (razón de momios OR, por su sigla en inglés: 8,1), seguida por la hepatopatía crónica (OR: 2,3). DISCUSIÓN : Los resultados muestran que la mayoría de los casos sintomáticos tuvieron una evolución favorable. La tasa de letalidad provincial es similar a la tasa nacional. Los factores de riesgo que resultaron relevantes se condicen con estudios realizados tanto en Argentina como en otros países. Si bien tener 60 años o más fue el mayor factor de riesgo, la presencia concomitante de enfermedades crónicas no transmisibles también contribuyó al agravamiento de la infección por COVID-19. Esta información es relevante para pensar políticas de salud en dicha población.

ABSTRACT INTRODUCTION . The main objective was to evalúate the risk of death in patients according to the presence of one or more risk factors in confirmed cases of COVID-19. METHODS : Cross-sectional, descriptive, correlational study of COVID-19 cases in people over 18 years of age, residents of the Province of Buenos Aires, confirmed by laboratory or clinical-epidemiological criteria that have started symptoms from March to December 2020 (n = 622,084). Secondary source information. Four analysis scenarios were considered taking into account the quality of the data and the patient's condition. RESULTS : 21,706 deceased cases were reported (3.5% fatality rate). Age (72.8 ± 13.8 vs 41.2 ± 15.2) and male patients (56% vs 44%; p <0.05) were higher in the deceased. According to the multivariate model, being 60 years or older was the highest risk factor (OR: 8.1) followed by chronic liver disease (OR: 2.3). DISCUSSION : The results show that most of the symptomatic cases had a favorable evolution. The provincial fatality rate is similar to the national rate. The risk factors that were relevant are consistent with studies carried out both in our country and in other countries. Although being 60 years or older was the greatest risk factor, most of the CNCDs analyzed also contributed to the COVID-19 infection becoming worse. This information is relevant to think about health policies for this population.

Letalidad por COVID-19 asociada a factores de riesgo en la provincia de Buenos Aires, Argentina, 2020 / Fatality due to COVID-19 associated with risk factors in the province of Buenos Aires, Argentina, 2020

INTRODUCCIÓN: El objetivo principal fue evaluar el riesgo de defunción en pacientes con diagnóstico confirmado de COVID-19 según la presencia de uno o más factores de riesgo. MÉTODOS: Estudio transversal, descriptivo y correlacional de personas mayores de 18 años, residentes de la provincia de Buenos Aires, con diagnóstico de COVID-19 confirmado por laboratorio o criterio clínico-epidemiológico que hayan iniciado síntomas entre marzo y diciembre de 2020 (n = 622 084). Se utilizó información de fuente secundaria. Se consideraron cuatro escenarios de análisis con base en la calidad del dato y la condición del paciente. RESULTADOS: Se notificaron 21 706 casos fallecidos (letalidad 3,5%). La mayoría de los fallecidos tenía edad avanzada (72,8 ± 13,8 años en los fallecidos versus 41,2 ± 15,2 años en los no fallecidos) y eran de sexo masculino (56% versus 44%, p<0,05). Según el modelo multivariado, la edad de 60 años o más resultó ser el factor de mayor riesgo (razón de momios [OR, por su sigla en inglés]: 8,1), seguida por la hepatopatía crónica (OR: 2,3). DISCUSIÓN: Los resultados muestran que la mayoría de los casos sintomáticos tuvieron una evolución favorable. La tasa de letalidad provincial es similar a la tasa nacional. Los factores de riesgo que resultaron relevantes se condicen con estudios realizados tanto en Argentina como en otros países. Si bien tener 60 años o más fue el mayor factor de riesgo, la presencia concomitante de enfermedades crónicas no transmisibles también contribuyó al agravamiento de la infección por COVID-19. Esta información es relevante para pensar políticas de salud en dicha población.

Infrared Thermography Applied to Monitoring Musculoskeletal Adaptation to Training in Thoroughbred Race Horses.

Infrared thermography has been used to help in diagnosing lameness. It is hypothesized that, if used in a routine basis, it could help in understanding musculoskeletal modifications during race training. This study aimed to evaluate thermal variation in the musculoskeletal regions of young Thoroughbred (TB) horses during their initial months of race training. Thermographic examinations were performed once every 2 weeks on 16 (10 male, 6 female) two-year-old TB racehorses, from arrival to the racetrack in June 2016, until January 2017, for a total of 16 evaluations. Thermographic imaging was performed using the appropriate protocol. Temperature (°C) was measured at the dorsal and palmar/plantar aspects of specific regions of interest (fetlock, metacarpal, metatarsal, carpal, tarsal, thoracolumbar, sacroiliac spine, and both hips). Initially, we found a thermal balance and all regions demonstrated a positive correlation with one another. However, a significant difference was noted between the left and right sides as training progressed. Four horses were withdrawn from the study after 50% of evaluations because of metacarpal conditions associated with training. Thermographic examination revealed changes before the clinical manifestation of these conditions. In conclusion, this study demonstrated that infrared thermography is an image technique that can facilitate understanding of musculoskeletal system modifications to race training and should be further investigated as a predictive tool to anticipate the occurrence of lesions.

Angiostrongylus cantonensis and the giant African snail ­ threat or not?

Objective: Angiostrongylus cantonensis or the rat lungworm can cause eosinophilic meningitis in humans. The Giant African snail has been reported to be a suitable intermediate host for this parasite. As the population of Giant African snails has recently exploded, there is an increased risk of transmission of this helminths to humans residing in this country. Therefore the objective of this study is to detect the presence of the rat lungworm in the Giant African Snails in Trinidad by conventional polymerase chain reaction (PCR). Design and Methodology: A total of 178 Giant African snails were collected from ten different locations throughout Trinidad. DNA was extracted from 25 mg of the mantle of each Giant African snail using the DNeasy® PoweSoil® Kit. Conventional PCR was performed using the primers AngioF1 and AngioR1 to amplify a 1,134bp fragment of the 18S rRNA gene of Angiostrongylus spp. The PCR reactions and conditions were are adapted from Qvarnstrom et al in 2007(1). Results: Six of the DNA extracted samples were positive for Angiostrongylus spp. by conventional PCR. Conclusion: Giant African snails in Trinidad are a suitable intermediate host for the rat lungworm and can increase transmission of these helminths to humans. Therefore Angiostrongylus cantonensis infection should be considered to be a differential for eosinophilic meningitis in humans.

Hand Function in Muscular Dystrophies.

The aim of this study was to investigate the relationship between Performance of Upper Limb (PUL) and Jebsen-Taylor Test (JTT) to assess and monitor upper limb function progression in patients with muscular dystrophy. Thirty patients diagnosed with Duchenne muscular dystrophy, limb-girdle muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy Type 1, and fascioscapulohumeral dystrophy were submitted to the shoulder, elbow, and wrist domains of PUL, and to JTT subtests. Spearman tests investigated the relationships between PUL and JTT total scores and domains. Correlations were classified as strong ( r ≥ 0.70), moderate (0.40 ≤ r < 0.70), or weak ( r ≤ 0.40). There were strong correlations between the PUL and JTT total scores ( r = -0.706). Although JTT measures time and PUL provides kinesiologic scores, these measures were related. Therefore, muscle synergies, which control the compensatory movements and motor functions involving mainly shoulder, elbow, wrist, and finger movements, are related to timed performance in patients with muscular dystrophies.

Human herpesvirus 8 can be transmitted through blood in drug addicts.

Human Herpes virus type-8 (HHV-8) seroprevalence was studied in a population of HIV positive intravenous drug users (IVDUs) from Argentina. Analysis of this population also indirectly made it possible to study HHV-8 blood transmission, because these individuals frequently engage in needle sharing behavior and are capable of acquiring a broad array of blood borne pathogens, including Hepatitis B/C virus. The seroprevalence of HHV-8 in IVDUs was compared to a group of non-IVDUs and HIV negative individuals. Of the 223 individuals tested, 13.45% were HHV-8 positive, 16.99% in the IVDUs group, and 5.71% in the non-IVDUs. Among HIV positive IVDUs, 25/144 (17.36%) were also HHV-8 seropositive. The seropositivity rate of HHV-8 in HIV negative IVDUs was 11.1%. In contrast, HHV-8 seroprevalence in HIV negative heterosexual individuals without drug usage behavior was even lower (5.71%). The rate of HHV-8 infection in HIV positive IVDUs was three times as high compared to the non IVDU HIV negative individuals, suggesting that IVDU is a risk for HHV-8 infection. Furthermore, it was found that IVDUs showed a very high rate of Hepatitis B/C (52.77%), which also correlate with HHV-8 infection in this population (23.68%). All Hepatitis B/C positive individuals were also HIV positive. Our data confirm other studies showing that individuals who share needles are at risk for acquiring Hepatitis B/C and HIV infections. In addition, our results suggest that they are also at risk to acquiring HHV-8 infection by the same route.

Human herpesvirus 8 can be transmitted through blood in drug addicts.

Human Herpes virus type-8 (HHV-8) seroprevalence was studied in a population of HIV positive intravenous drug users (IVDUs) from Argentina. Analysis of this population also indirectly made it possible to study HHV-8 blood transmission, because these individuals frequently engage in needle sharing behavior and are capable of acquiring a broad array of blood borne pathogens, including Hepatitis B/C virus. The seroprevalence of HHV-8 in IVDUs was compared to a group of non-IVDUs and HIV negative individuals. Of the 223 individuals tested, 13.45

were HHV-8 positive, 16.99

in the IVDUs group, and 5.71

in the non-IVDUs. Among HIV positive IVDUs, 25/144 (17.36

) were also HHV-8 seropositive. The seropositivity rate of HHV-8 in HIV negative IVDUs was 11.1

. In contrast, HHV-8 seroprevalence in HIV negative heterosexual individuals without drug usage behavior was even lower (5.71

). The rate of HHV-8 infection in HIV positive IVDUs was three times as high compared to the non IVDU HIV negative individuals, suggesting that IVDU is a risk for HHV-8 infection. Furthermore, it was found that IVDUs showed a very high rate of Hepatitis B/C (52.77

), which also correlate with HHV-8 infection in this population (23.68

). All Hepatitis B/C positive individuals were also HIV positive. Our data confirm other studies showing that individuals who share needles are at risk for acquiring Hepatitis B/C and HIV infections. In addition, our results suggest that they are also at risk to acquiring HHV-8 infection by the same route.

Reversible cardiomyopathy in an infant with unrecognized congenital adrenal hyperplasia.

We describe an infant who had a dilated cardiomyopathy and who was later found to have congenital adrenal hyperplasia. The cardiomyopathy resolved after replacement of glucocorticoid and mineralocorticoid. We believe that glucocorticoid deficiency may have played a direct role in the evolution of this cardiomyopathy.

Thyroxine-binding globulin deficiency detected by newborn screening.

We examined the results of the Northwest Regional Screening Program from May 1975 to June 1991 to determine the prevalence of inherited thyroxine-binding globulin (TBG) deficiency and its effect on thyroid hormone concentrations in infants. Serum thyroxine (T4), triiodothyronine resin uptake (T3RU), and thyrotropin values were requested of physicians caring for all infants with a single filter paper T4 level < 38.6 nmol/L (3 micrograms/dl) or a T4 level < 3rd percentile on two filter paper tests (at birth and 2 to 6 weeks of age). From 1,367,724 infants screened in five states, TBG deficiency, an X-linked disorder, was identified in 317 infants (285 boys). For the entire screening program the calculated frequency of TBG deficiency was 1:4315 infants (1:2400 for boys). In Oregon, where 95% of infants have two screening tests performed, the calculated frequency was somewhat higher (1:3080 infants; 1712 boys) and is probably more accurate. The mean serum T4 concentration for TBG-deficient boys was 41.9 nmol/L (3.26 micrograms/dl); 31% had values < 25.7 nmol/L (2.0 micrograms/dl). The mean serum T4 concentration for TBG-deficient girls was 60.2 nmol/L (4.68 micrograms/dl), with none < 2.0 micrograms/dl. The mean T3RU value was 0.472 in TBG-deficient boys, and 0.412 in TBG-deficient girls; the T3RU value was > 0.55 in 24% of TBG-deficient boys but was > 0.55 in only one girl. Free serum T4 levels were normal in all 56 TBG-deficient infants studied, and TBG levels were low in all 20 infants studied. Inherited TBG deficiency is common in boys in the Northwest, with a frequency of 1:1700 and a male/female ratio of 8.9:1. Boys with TBG deficiency have mild, moderate, or severe alterations in total T4 and T3RU values, but severe deficiency is rare in girls.

Metabolic evaluation of obese and nonobese siblings.

OBJECTIVE: To test the hypotheses that obese adolescents have a lower resting metabolic rate and less aerobic endurance than their nonobese siblings. DESIGN: Case-referent study of obese and nonobese siblings from the same kindred. SETTING: Tertiary referral center. PARTICIPANTS: Telephone screening of community volunteers resulted in a consecutive sample of 16 kindreds. Obese and nonobese siblings were similar in age, height, and pubertal status. Significantly more female subjects were in the obese group (p less than 0.01). MEASUREMENTS AND MAIN RESULTS: Body composition studies revealed that the obese siblings had higher body fat (p less than 0.001) but that fat-free mass was similar to that of the lean siblings. Resting metabolic rates determined by indirect calorimetry for the obese and nonobese pairs did not differ. Although the obese siblings appeared less fit when maximal oxygen consumption was measured in relation to total weight, maximal oxygen consumption did not differ when values were standardized for fat-free mass. CONCLUSIONS: The obese adolescents did not have a reduced resting metabolic rate. As in adults, the relationship between resting metabolic rate and fat-free mass was similar for obese and nonobese children and adolescents. Any decreased sport participation by the obese siblings was not due to inherent reductions in aerobic capacity.

Detection of congenital hypopituitary hypothyroidism: ten-year experience in the Northwest Regional Screening Program.

We examined the results of the Northwest Regional Screening Program (NWRSP) over its first 10 years to determine whether the detection of hypopituitary hypothyroidism is a justified advantage of the primary thyroxine (T4)-supplemental thyroid-stimulating hormone (TSH) screening strategy, and to determine whether all such infants will be detected by this screening approach. Between May 1975 and May 1985, the NWRSP screened 850,431 infants, detecting 192 infants with primary hypothyroidism (1:4429) and eight with hypopituitary hypothyroidism (1:106,304). In 11 additional infants, TSH deficiency, not detected by the screening program, was diagnosed on recognition of clinical features over the same period. Thyroid hormone treatment was begun in seven of the 11 infants prior to obtaining the screening sample results because of clinical symptoms of hypopituitarism, including hypoglycemia, persistent jaundice, microgenitalia, diabetes insipidus, midface hypoplasia, cleft lip or palate, or abnormalities of vision. The other four infants were not detected despite clinical features of hypopituitarism (in retrospect) and low serum T4 with TSH concentration below assay sensitivity on at least one screening sample. The most accurate assessment of total cases comes from Oregon, where all cases of congenital hypopituitarism are referred to our center; we estimate a frequency of 1:29,000. In our experience, a combination of newborn T4-supplemental TSH screening measurements and recognition of clinical features of hypopituitarism is the optimal strategy for detecting infants with congenital hypopituitary hypothyroidism.

Effect of growth hormone replacement on development of hypothyroidism and hyperlipidemia.

To determine the frequency with which hypothyroidism develops during human growth hormone therapy and to corroborate its onset with blood lipid changes, we measured growth rate, serum T4 and T3, and plasma cholesterol, triglyceride, and lipoprotein concentrations at 4-month intervals for a year in two subgroups of hGH-deficient children. The first group was initially euthyroxinemic (n = 16), and the second was TSH deficient and therefore already receiving thyroxine (n = 15). Basal plasma concentrations of total and low-density lipoprotein cholesterol and, to a lesser extent, plasma triglycerides were increased in both groups compared with an age-matched reference group. Basal plasma cholesterol levels were not statistically different in the euthyroxinemic and thyroxine-treated subgroups, and hGH treatment for a year did not lower lipid values in either subgroup. With hGH replacement, 25% of the euthyroxinemic patients experienced a slowdown in growth rate (3.2 +/- 0.7 cm/yr) associated with decreasing T4 (4.8 +/- 1.1 micrograms/dl) and increasing cholesterol concentrations (218 +/- 23 mg/dl); with thyroxine treatment, the growth rate improved (6.9 +/- 2.2 cm/yr), T4 increased (10.0 +/- 4.0 micrograms/dl), and cholesterol decreased (173 +/- 44 mg/dl, P less than 0.05). Although our results do not justify routine thyroid replacement, they do indicate that hypothyroxinemia and hypercholesterolemia may precede the growth slowdown during hGH treatment, and the need to monitor thyroid function at this time.