Lifestyles during the First Wave of COVID-19: A Cross-Sectional Study of 16,811 Adults from Spanish-Speaking Countries in South America.

The aim of this research was to analyze the lifestyles of adults from Spanish-speaking countries in South America during the COVID-19 pandemic using a cross-sectional, analytical, and multicenter study. The target population was composed of people aged 18 and older who resided in South America during the pandemic; the final sample comprised 16,811 participants who were predominantly female, with ages ranging from 18 to 79 years. The results showed that approximately six out of ten respondents did not engage in any physical activity; only one in four respondents indicated that their diet was sufficient and balanced; and most washed their hands frequently and showered every day. Regarding the type of isolation, half reported that it was voluntary and the rest mandatory or restricted. Regarding mobility, six out of ten surveyed leave their residence on a weekly basis. Regarding the use of personal protective equipment, the majority used masks and a smaller proportion used gloves. In conclusion, the majority of respondents did not perform any physical activity; moreover, one in four reported having a sufficient balanced diet. We therefore recommend an improvement of public policies to promote better lifestyles in South America, in particular the reorientation of the health system to prevent similar situations.

Perception of memory in university students of Venezuela during COVID-19 / [Percepción de la memoria en estudiantes universitarios de Venezuela durante la COVID-19]

Introduction: University students need memory to manage the learning processes based on metacognition and in this way they can respond to future demands as professionals. Methods: was structured with a quantitative approach, comparative type, non-experimental cross-sectional design, the sample consisted of 237 responses from students. Results: the age was 26.45 ± 8.96 years, 66% female and 34% male, students older than 27 years had a higher average in the use of memory strategies (p = 0.003); a longer semester was obtained, the average perception of the strategy increased (p = 0.027). When using ICT, the perception of satisfaction (p = 0.014), competence (p <0.001), strategy (p = 0.004) and general memory (p = 0.001) decrease. Conclusions: the subjects showed a perception of memory approximately the same according to sex, it was found that, at an older age, there was a better perception of the strategy and in terms of regions of origin, the students of the University of Los Andes reported an average of satisfaction lower than that of students from other study houses, and a higher average in relation to the perception of competence. Regarding the use of ICT and the perception of memory, it shows that students who do not use mobile devices before going to sleep have a more satisfactory perception of their general memory, as well as their perception of competence and strategy.

Introducción: Los estudiantes universitarios necesita de la memoria para el manejo de los procesos de aprendizaje basados en la metacognición y de esta manera puedan responder a las demandas en el futuro como profesionales. Métodos: se estructuró con enfoque cuantitativo, tipo comparativo, diseño no experimental transversal, la muestra estuvo conformada por 237 respuestas de estudiantes. Resultados: la edad fue de 26,45 ± 8,96 años, 66% del sexo femenino y 34% masculino, los estudiantes mayores de 27 años poseen un mayor promedio en el uso de estrategias de memoria (p=0,003); se obtuvo que a mayor semestre aumenta el promedio de la percepción de la estrategia (p=0,027). Cuando se utilizan las TIC disminuye en promedio la percepción de satisfacción (p=0,014), competencia (p<0,001), estrategia (p=0,004) y memoria general (p=0,001). Conclusiones: los sujetos mostraron una percepción de la memoria aproximadamente igual según sexo, se encontró que, a mayor edad, hubo una mejor percepción de la estrategia y en cuanto a regiones de procedencia, los estudiantes de la Universidad de Los Andes reportaron un promedio de satisfacción menor al de los estudiantes provenientes de otras casas de estudio, y un promedio mayor en relación a la percepción de la competencia. En cuanto al uso de las TIC y la percepción de la memoria muestra que los estudiantes que no utilizan dispositivos móviles antes de dormir tienen una percepción más satisfactoria de su memoria general, al igual que de su percepción de competencia y estrategia.

Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from the blood of Lyme disease patients by digital PCR.

Lyme disease patients would greatly benefit from a timely, sensitive, and specific molecular diagnostic test that can detect the causal agent Borrelia burgdorferi at the onset of symptoms. Currently available diagnostic methods recommended by the Centers for Disease Control and Prevention for Lyme disease involve indirect serological tests that rely on the detection of a host-antibody response, which often takes more than three weeks to develop. With this process, many positive cases are not detected within a timely manner, preventing a complete cure. In this study, we have developed a digital polymerase chain reaction (PCR) assay that detects Lyme disease on clinical presentation with a sensitivity two-fold higher than that of the currently available diagnostic methods, using a cohort of patient samples collected from the Lyme disease endemic state of Connecticut, USA, in 2016-2018. Digital PCR technology was chosen as it is more advanced and sensitive than other PCR techniques in detecting rare targets. The analytical detection sensitivity of this diagnostic assay is approximately three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients has hindered the clinical adoption of PCR-based diagnostic tests. However, this drawback was overcome by using a comparatively larger sample volume, applying pre-analytical processing to the blood samples, and implementing a pre-amplification step to enrich for B. burgdorferi-specific gene targets before the patient samples are analyzed via digital PCR technology. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma rather than whole blood. If detected in a timely manner, Lyme disease can be completely cured, thus limiting antibiotic overuse and associated morbidities.

ATM-Mutated Pancreatic Cancer: Clinical and Molecular Response to Gemcitabine/Nab-Paclitaxel After Genome-Based Therapy Resistance.

Metastatic pancreatic cancer (PC) is an aggressive malignancy, with most patients deriving benefit only from first-line chemotherapy. Increasingly, the recommended treatment for those with a germline mutation in a gene involved in homologous recombination repair is with a platinum drug followed by a poly (ADP-ribose) polymerase (poly adenosine phosphate-ribose polymerase [PARP]) inhibitor. Yet, this is based largely on studies of BRCA1/2 or PALB2 mutated PC. We present the case of a 44-year-old woman with ATM-mutated PC who achieved stable disease as the best response to first-line fluorouracil, leucovorin, irinotecan, and oxaliplatin, followed by progression on a PARP inhibitor. In the setting of jaundice, painful hepatomegaly, and a declining performance status, she experienced rapid disease regression with the nonplatinum regimen, gemcitabine plus nab-paclitaxel. Both physical stigmata and abnormal laboratory values resolved, imaging studies showed a reduction in metastases and her performance status returned to normal. Measurement of circulating tumor DNA for KRAS G12R by digital droplet polymerase chain reaction confirmed a deep molecular response. This case highlights that first-line treatment with a platinum-containing regimen followed by PARP inhibition may not be the best choice for individuals with ATM-mutated pancreatic cancer. Additional predictors of treatment response are needed in this setting.

Vitamin D-dependent rickets: a resurgence of the rachitic lung in the 21st century.

Respiratory complications of rickets may be life-threatening particularly in developing countries. A 7-month-old boy presented with recurrent infections, seizures, failure to thrive, wheezing and respiratory distress progressing to global respiratory failure. Several antimicrobial regimens, bronchodilators and corticosteroids resulted in only short-term improvement. He was transferred from Cape Verde to a third-care hospital in Portugal. He was hypotonic and undernourished, with respiratory anguish and classical skeletal signs of rickets, despite vitamin D supplementation. Hypocalcaemia, normal phosphate levels and normal vitamin D status 25(OH)D3 and 1.25(OH)2D3) pointed to vitamin D-dependent rickets type II. Treatment with high doses of calcium and calcitriol allowed progressive respiratory, musculoskeletal and neurological recovery. Although respiratory manifestations of rickets were described many years ago, the present case raises relevant issues about the level of diagnostic support, the risk of complications and how they should be assessed and monitored.

Fatty acid flux in adipocytes: the in's and out's of fat cell lipid trafficking.

The trafficking of fatty acids into and out of adipocytes is regulated by a complex series of proteins and enzymes and is under control by a variety of hormonal and metabolic factors. The biochemical basis of fatty acid influx, despite its widespread appreciation, remains enigmatic with regard to the biophysical and biochemical properties that facilitate long-chain fatty acid uptake. Fatty acid efflux is initiated by hormonally controlled lipolysis of the droplet stores and produces fatty acids that must transit from their site of production to the plasma membrane and subsequently out of the cells. This review will focus on the "in's and out's" of fatty acid trafficking and summarize the current concepts in the field.

FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes.

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.

Functional analysis of long-chain acyl-CoA synthetase 1 in 3T3-L1 adipocytes.

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a approximately 15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase C and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4.

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.

Fatty acid transport in adipocytes and the development of insulin resistance.

Fatty acid influx into adipocytes is a complex multifactoral process driven by biochemical and biophysical processes linking transmembrane flux to the ATP-dependent esterification of fatty acids. Adipocyte proteins implicated in free fatty acid (FFA) influx include CD36 functioning as a general lipid receptor, caveolin 1 functioning as a component of an endocytotic/exocytotic vesicular cycle and the acyl CoA synthetases (FATP1, ACSL1) catalysing esterification of lipids producing acyl CoAs. In adipocytes, CD36, ACSL1 and FATP1 translocate from intracellular sites to the plasma membrane in response to insulin thereby positioning these key proteins to facilitate FFA esterification. Lentiviral delivery of shRNA targeting FATP1 in 3T3-L1 adipocytes results in a complete loss of insulin-stimulated FFA uptake, decreased accumulation of TAG/DAG/MAG and potentiated insulin-stimulated 2-deoxyglucose uptake. Increased insulin-stimulated hexose uptake in FATP1 knockdown adipocytes is correlated with increased tyrosine phosphorylation and abundance of IRS1 protein. Evaluation of the lipid activated serine kinases implicated in insulin signalling reveals that S6K and JNK1 were not altered in abundance or phosphorylation in FATP1 knockdown adipocytes but that the phosphorylation of PKCtheta and abundance of IKKalpha/beta were significantly reduced. These results suggest lipid droplet pools in the adipocyte play a major role in regulating kinase cascades controlling insulin action.

DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras.

Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H- and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 co-distribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2.Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9.GCP16 exhibits substrate specificity, palmitoylating H- and N-Ras but not myristoylated G (alphai1) or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9.GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

Palmitoylation and plasma membrane localization of Ras2p by a nonclassical trafficking pathway in Saccharomyces cerevisiae.

Subcellular localization of Ras proteins to the plasma membrane is accomplished in part by covalent attachment of a farnesyl moiety to the conserved CaaX box cysteine. Farnesylation targets Ras to the endoplasmic reticulum (ER), where additional processing steps occur, resulting in translocation of Ras to the plasma membrane. The mechanism(s) by which this occurs is not well understood. In this report, we show that plasma membrane localization of Ras2p in Saccharomyces cerevisiae does not require the classical secretory pathway or a functional Golgi apparatus. However, when the classical secretory pathway is disrupted, plasma membrane localization requires Erf2p, a protein that resides in the ER membrane and is required for efficient palmitoylation of Ras2p. Deletion of ERF2 results in a Ras2p steady-state localization defect that is more severe when combined with sec-ts mutants or brefeldin A treatment. The Erf2p-dependent localization of Ras2p correlates with the palmitoylation of Cys-318. An Erf2p-Erf4p complex has recently been shown to be an ER-associated palmitoyltransferase that can palmitoylate Cys-318 of Ras2p (S. Lobo, W. K. Greentree, M. E. Linder, and R. J. Deschenes, J. Biol. Chem. 277:41268-41273, 2002). Erf2-dependent palmitoylation as well as localization of Ras2p requires a region of the hypervariable domain adjacent to the CaaX box. These results provide evidence for the existence of a palmitoylation-dependent, nonclassical endomembrane trafficking system for the plasma membrane localization of Ras proteins.

Erf4p and Erf2p form an endoplasmic reticulum-associated complex involved in the plasma membrane localization of yeast Ras proteins.

Ras oncogene proteins are plasma membrane-associated signal transducers that are found in all eukaryotes. Posttranslational addition of lipid to a carboxyl-terminal CaaX box (where "C" represents a cysteine, "a" is generally an aliphatic residue, and X can be any amino acid) is required to target Ras proteins to the cytosolic surface of the plasma membrane. The pathway by which Ras translocates from the endoplasmic reticulum to the plasma membrane is currently not clear. We have performed a genetic screen to identify components of the Ras plasma membrane localization pathway. Mutations in two genes, ERF2 and ERF4/SHR5, have been shown to affect the palmitoylation and subcellular localization of Ras proteins. In this report, we show that Erf4p is localized on the endoplasmic reticulum as a peripheral membrane protein in a complex with Erf2p, an integral membrane protein that was identified from the same genetic screen. Erf2p has been shown to be required for the plasma membrane localization of GFP-Ras2p via a pathway distinct from the classical secretory pathway (X. Dong and R. J. Deschenes, manuscript in preparation). We show here that Erf4p, like Erf2p, is involved in the plasma membrane localization of Ras2p. Erf2p and Erf4p represent components of a previously uncharacterized subcellular transport pathway involved in the plasma membrane targeting of Ras proteins.

Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae.

Most Ras proteins are posttranslationally modified by a palmitoyl lipid moiety through a thioester linkage. However, the mechanism by which this occurs is not known. Here, evidence is presented that the Ras2 protein of Saccharomyces cerevisiae is palmitoylated by a Ras protein acyltransferase (Ras PAT) encoded by the ERF2 and ERF4 genes. Erf2p is a 41-kDa protein localized to the membrane of the endoplasmic reticulum and contains a conserved DHHC cysteine-rich domain (DHHC-CRD). Erf2p co-purifies with Erf4p (26 kDa) when it is expressed in yeast or in Escherichia coli. The Erf2p/Erf4p complex is required for Ras PAT activity, and mutations within conserved residues (Cys(189), His(201), and Cys(203)) of the Erf2p DHHC-CRD domain abolish Ras PAT activity. Furthermore, a palmitoyl-Erf2p intermediate is detected suggesting that Erf2p is directly involved in palmitate transfer. ERF2 and ERF4 are the first genes identified that encode a palmitoyltransferase for a Ras GTPase.

De Novo Nonepileptic Seizures after Cranial Surgery for Epilepsy: Incidence and Risk Factors.

We evaluated the incidence of de novo nonepileptic seizures (NES), confirmed by EEG monitoring, after cranial surgery for intractable epilepsy in 228 surgery patients. Eight patients (3.5%) developed de novo NES at 6 weeks to 6 years (mean, 23 months) after surgery. Six had undergone a resection and two complete callosotomy. They did not differ from a larger surgical group with respect to sex, side of surgery, age at onset, or duration of epilepsy, Full Scale Intelligence Quotient, seizure outcome, or preoperative interictal dysphoric disorder (IDD), but there was a significant excess of postoperative IDD and operative complications (bone flap infections); the callosotomy patients had marked hemisphere disconnection syndromes. Repeat EEG videotelemetry monitoring is important to detect postoperative NES so that inappropriate therapeutic measures may be avoided. Risk factors may be exacerbation or persistence of IDD and surgical complications. The etiology of NES is discussed.

Genes encoding acyl-CoA dehydrogenase (AcdH) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched-chain fatty acids and macrolide antibiotic production.

The cloning, using a PCR approach, of genes from both Streptomyces coelicolor and Streptomyces avermitilis encoding an acyl-CoA dehydrogenase (AcdH), putatively involved in the catabolism of branched-chain amino acids, is reported. The deduced amino acid sequences of both genes have a high similarity to prokaryotic and eukaryotic short-chain acyl-CoA dehydrogenases. When the S. coelicolor and S. avermitilis acyl-CoA dehydrogenase genes (acdH) were expressed in Escherichia coli, each of the AcdH flavoproteins was able to oxidize the branched-chain acyl-CoA derivatives isobutyryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA, as well as the short straight-chain acyl-CoAs n-butyryl-CoA and n-valeryl-CoA in vitro. NMR spectral data confirmed that the oxidized product of isobutyryl-CoA is methacrylyl-CoA, which is the expected product at the acyl-CoA dehydrogenase step in the catabolism of valine in streptomycetes. Disruption of the S. avermitilis acdH produced a mutant unable to grow on solid minimal medium containing valine, isoleucine or leucine as sole carbon sources. Feeding studies with 13C triple-labelled isobutyrate revealed a significant decrease in the incorporation of label into the methylmalonyl-CoA-derived positions of avermectin in the acdH mutant. In contrast the mutation did not affect incorporation into the malonyl-CoA-derived positions of avermectin. These results are consistent with the acdH gene encoding an acyl-CoA dehydrogenase with a broad substrate specificity that has a role in the catabolism of branched-chain amino acids in S. coelicolor and S. avermitilis.

Adequacy of different respiratory specimens and culture methods for bacteriological diagnosis of cystic fibrosis

Para avaliar a sensibilidade de diferentes espécimes respiratórios e métodos de cultivo para o monitoramento bacterilógico das infecçöes pulmonares em pacientes com fibrose cística (FC), comparamos 41 espécimes de orofaringe (OF), nasofaringe (NF) e escarro, coletados simultaneamente. Analisamos também 144 culturas de escarro pelos métodos convencional e quantitativo. A P. aeruginosa mucóide (M) foi isolada em 70 por cento das amostras de escarro, 46 por cento de OF e 4,9 por cento de NF (p < 0,05). Para P. aeruginosa näo mucóide (NM) os índices foram de 32 por cento no escarro, 12 por cento no OF e 7 por cento no NF (p < 0,05). Para S. aureus e H. influenzae os índices foram semelhantes nos 3 espécimes (p < 0,05). O percentual de resultados falso-negativos em OF e NF foi alto para todos oa patógenos respiratórios. P. aeruginosa NM, M e H. influenzae näo foram detectados em algumas culturas de NF (77 por cento, 93 por cento e 78 por cento, respectivamente), näo tendo sido encontrados em cerca de 60 por cento das culturas de OF. As culturas de escarro convencional e quantitativa tiveram resultados similares, em mais de 70 por cento dos casos, porém ambos mostraram resultados falso-negativos. A técnica convencional foi menos sensível para H. influenzae (16,6 por cento) e P. aeruginosa NM (15,9 por cento, enquanto que o método quantitativo foi pior para S. aureus (13,1 por cento de falsos-negativos). Ambas as culturas deixaram de identificar 6 por cento dos casos positivos de P. aeruginosa M. Nossos resultados sugerem que a análise microbiológica em pacientes com FC deve ser criteriosa, uma vez que a escolha do espécime clínico e a metodologia de cultivo empregada säo determinantes na frequência de resultados falso-negativos