Comparison between Measured and Predicted Resting Metabolic Rate Equations in Cross-Training Practitioners.

This study aimed to investigate the resting metabolic rate (RMR) in cross-training practitioners (advanced and novice) using indirect calorimetry (IC) and compare it with predictive equations proposed in the scientific literature. METHODS: A cross-sectional and comparative study analyzed 65 volunteers, both sexes, practicing cross-training (CT). Anthropometry and body composition were assessed, and RMR was measured by IC (FitMate PRO®), bioimpedance (BIA-InBody 570®), and six predictive equations. Data normality was tested by the Kolgomorov-Smirnov test and expressed as mean ± standard deviation with 95% confidence intervals (CI), chi-square test was performed to verify ergogenic resources, and a Bland-Altman plot (B&A) was made to quantify the agreement between two quantitative measurements. One-way ANOVA was applied to body composition parameters, two-way ANOVA with Bonferroni post hoc was used to compare the RMR between groups, and two-way ANCOVA was used to analyze the adjusted RMR for body and skeletal muscle mass. The effect size was determined using Cohen's d considering the values adjusted by ANCOVA. If a statistical difference was found, post hoc Bonferroni was applied. The significance level was p < 0.05 for all tests. RESULTS: The main results indicated that men showed a higher RMR than women, and the most discrepant equations were Cunningham, Tinsley (b), and Johnstone compared to IC. Tinsley's (a) equation indicated greater precision in measuring the RMR in CM overestimated it by only 1.9%, and BIA and the Harris-Benedict in CW overestimated RMR by only 0.1% and 3.4%, respectively. CONCLUSIONS: The BIA and Harris-Benedict equation could be used reliably to measure the RMR of females, while Tinsley (a) is the most reliable method to measure the RMR of males when measuring with IC is unavailable. By knowing which RMR equations are closest to the gold standard, these professionals can prescribe a more assertive diet, training, or ergogenic resources. An assertive prescription increases performance and can reduce possible deleterious effects, maximizing physical sports performance.

In the fed state, autophagy plays a crucial role in assisting the insect vector Rhodnius prolixus mobilize TAG reserves under forced flight activity.

Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%-50% higher autophagic activation in the first 2 weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.

Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus.

In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

Evolution of innate immunity: lessons from mammalian models shaping our current view of insect immunity.

The innate immune system, a cornerstone for organismal resilience against environmental and microbial insults, is highly conserved across the evolutionary spectrum, underpinning its pivotal role in maintaining homeostasis and ensuring survival. This review explores the evolutionary parallels between mammalian and insect innate immune systems, illuminating how investigations into these disparate immune landscapes have been reciprocally enlightening. We further delve into how advancements in mammalian immunology have enriched our understanding of insect immune responses, highlighting the intertwined evolutionary narratives and the shared molecular lexicon of immunity across these organisms. Therefore, this review posits a holistic understanding of innate immune mechanisms, including immunometabolism, autophagy and cell death. The examination of how emerging insights into mammalian and vertebrate immunity inform our understanding of insect immune responses and their implications for vector-borne disease transmission showcases the imperative for a nuanced comprehension of innate immunity's evolutionary tale. This understanding is quintessential for harnessing innate immune mechanisms' potential in devising innovative disease mitigation strategies and promoting organismal health across the animal kingdom.

Western diet consumption by host vertebrate promotes altered gene expression on Aedes aegypti reducing its lifespan and increasing fertility following blood feeding.

BACKGROUND: The high prevalence of metabolic syndrome in low- and middle-income countries is linked to an increase in Western diet consumption, characterized by a high intake of processed foods, which impacts the levels of blood sugar and lipids, hormones, and cytokines. Hematophagous insect vectors, such as the yellow fever mosquito Aedes aegypti, rely on blood meals for reproduction and development and are therefore exposed to the components of blood plasma. However, the impact of the alteration of blood composition due to malnutrition and metabolic conditions on mosquito biology remains understudied. METHODS: In this study, we investigated the impact of whole-blood alterations resulting from a Western-type diet on the biology of Ae. aegypti. We kept C57Bl6/J mice on a high-fat, high-sucrose (HFHS) diet for 20 weeks and followed biological parameters, including plasma insulin and lipid levels, insulin tolerance, and weight gain, to validate the development of metabolic syndrome. We further allowed Ae. aegypti mosquitoes to feed on mice and tracked how altered host blood composition modulated parameters of vector capacity. RESULTS: Our findings identified that HFHS-fed mice resulted in reduced mosquito longevity and increased fecundity upon mosquito feeding, which correlated with alteration in the gene expression profile of nutrient sensing and physiological and metabolic markers as studied up to several days after blood ingestion. CONCLUSIONS: Our study provides new insights into the overall effect of alterations of blood components on mosquito biology and its implications for the transmission of infectious diseases in conditions where the frequency of Western diet-induced metabolic syndromes is becoming more frequent. These findings highlight the importance of addressing metabolic health to further understand the spread of mosquito-borne illnesses in endemic areas.

Deficiency of Brummer lipase disturbs lipid mobilization and locomotion, and impairs reproduction due to defects in the eggshell ultrastructure in the insect vector Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.

Lipid metabolism dynamic in Triatomine Rhodnius prolixus during acute Trypanosoma rangeli infection.

During its life cycle, Trypanosoma rangeli invades the hemolymph of its invertebrate host and colonizes hemocytes and salivary glands. The parasite cannot synthesize some lipid classes, and during its cycle, it depends on the uptake of these molecules from its vertebrate and invertebrate hosts to meet growth and differentiation requirements. However, until now, knowledge on how the parasite affects the lipid physiology of individual insect organs has been largely unknown. Herein, the biochemical and molecular dynamics of triatomine R. prolixus lipid metabolism in response to acute T. rangeli infection were investigated. Biochemical and microscopic assays revealed the lipid droplet profile and the levels of the different identified lipid classes. In addition, a qRT‒PCR approach was used to determine the expression profile of 6 protein-coding genes involved in the R. prolixus lipid physiology. We observed that triacylglycerol (TAG), monoacylglycerol (MAG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) levels in the fat body decreased in infected insects. On the other hand, high levels of free fatty acids were observed in the hemolymph during infection. Analysis by confocal microscopy revealed a decrease in lipid droplets size from infected fat bodies, and investigations by scanning electron microscopy revealed a significant number of parasites adhered to the surface of the organ. T. rangeli infection upregulated the transcript levels of the protein-coding gene for the acetyl-CoA carboxylase, the first enzyme in the de novo fatty acid synthesis pathway, responsible for the production of malonyl-CoA. On the other hand, downregulation of lipophorin receptor was observed. In conclusion, this study reveals a new set of molecular events that occur within the vector in response to the challenge imposed by the parasite.

Knockdown of carnitine palmitoyltransferase I (CPT1) reduces fat body lipid mobilization and resistance to starvation in the insect vector Rhodnius prolixus.

The energy stored in fatty acids is essential for several critical activities of insects, such as embryogenesis, oviposition, and flight. Rhodnius prolixus is an obligatory hematophagous hemipteran and vector of Chagas disease, and it feeds infrequently on very large blood meals. As digestion slowly occurs, lipids are synthesized and accumulate in the fat body, mainly as triacylglycerol, in lipid droplets. Between feeding bouts, proper mobilization and oxidation of stored lipids are crucial for survival, and released fatty acids are oxidized by mitochondrial ß-oxidation. Carnitine palmitoyl transferase I (CPT1) is the enzyme that catalyzes the first reaction of the carnitine shuttle, where the activated fatty acid, acyl-CoA, is converted to acyl-carnitine to be transported into the mitochondria. Here, we investigated the role of CPT1 in lipid metabolism and in resistance to starvation in Rhodnius prolixus. The expression of the CPT1 gene (RhoprCpt1) was determined in the organs of adult females on the fourth day after a blood meal, and the flight muscle showed higher expression levels than the ovary, fat body, and anterior and posterior midgut. RhoprCpt1 expression in the fat body dramatically decreased after feeding, and started to increase again 10 days later, but no changes were observed in the flight muscle. ß-oxidation rates were determined in flight muscle and fat body homogenates with the use of 3H-palmitate, and in unfed females, they were higher in the flight muscle. In the fat body, lipid oxidation activity did not show any variation before or at different days after feeding, and was not affected by the presence of etomoxir or malonyl-CoA. We used RNAi and generated RhoprCPT1-deficient insects, which surprisingly did not show a decrease in measured 3H-palmitate oxidation rates. However, the RNAi-knockdown females presented increased amounts of triacylglycerol and larger lipid droplets in the fat body, but not in the flight muscle. When subjected to starvation, these insects had a shorter lifespan. These results indicated that the inhibition of RhoprCpt1 expression compromised lipid mobilization and affected resistance to starvation.

Gene identification and RNAi-silencing of p62/SQSTM1 in the vector Rhodnius prolixus reveals a high degree of sequence conservation but no apparent deficiency-related phenotypes in vitellogenic females.

Autophagy and the ubiquitin-proteasome system (UPS) are important cellular mechanisms that coordinate protein degradation essential for proteostasis. P62/SQSTM1 is a receptor cargo protein able to deliver ubiquitinated targets to the proteasome proteolytic complex and/or to the autophagosome. In the insect vector of Chagas disease, Rhodnius prolixus, previous works have shown that the knockdown of different autophagy-related genes (ATGs) and ubiquitin-conjugating enzymes resulted in abnormal oogenesis phenotypes and embryo lethality. Here, we investigate the role of the autophagy/UPS adaptor protein p62 during the oogenesis and reproduction of this vector. We found that R. prolixus presents one isoform of p62 encoded by a non-annotated gene. The predicted protein presents the domain architecture anticipated for p62: PB1 (N-term), ZZ-finger, and UBA (C-term) domains, and phylogenetic analysis showed that this pattern is highly conserved within insects. Using parental RNAi, we found that although p62 is expressed in the ovary, midgut, and fat body of adult females, systemic silencing of this gene did not result in any apparent phenotypes under in-house conditions. The insects' overall levels of blood meal digestion, lifespan, yolk protein production, oviposition, and embryo viability were not altered when compared to controls. Because it is known that autophagy and UPS can undergo compensatory mechanisms, we asked whether the silencing of p62 was triggering adaptative changes in the expression of genes of the autophagy, UPS, and the unfolded protein response (UPR) and found that only ATG1 was slightly up regulated in the ovaries of silenced females. In addition, experiments to further investigate the role of p62 in insects previously silenced for the E1-conjugating enzyme (a condition known to trigger the upregulation of p62), also did not result in any apparent phenotypes in vitellogenic females.

Silencing of the 20S proteasomal subunit-α6 triggers full oogenesis arrest and increased mRNA levels of the selective autophagy adaptor protein p62/SQSTM1 in the ovary of the vector Rhodnius prolixus.

The high reproductive rates of insects contribute significantly to their ability to act as vectors of a variety of vector-borne diseases. Therefore, it is strategically critical to find molecular targets with biotechnological potential through the functional study of genes essential for insect reproduction. The ubiquitin-proteasome system is a vital degradative pathway that contributes to the maintenance of regular eukaryotic cell proteostasis. This mechanism involves the action of enzymes to covalently link ubiquitin to proteins that are meant to be delivered to the 26S proteasome and broken down. The 26S proteasome is a large protease complex (including the 20S and 19S subcomplexes) that binds, deubiquitylates, unfolds, and degrades its substrates. Here, we used bioinformatics to identify the genes that encode the seven α and ß subunits of the 20S proteasome in the genome of R. prolixus and learned that those transcripts are accumulated into mature oocytes. To access proteasome function during oogenesis, we conducted RNAi functional tests employing one of the 20S proteasome subunits (Prosα6) as a tool to suppress 20S proteasomal activity. We found that Prosα6 silencing resulted in no changes in TAG buildup in the fat body and unaffected availability of yolk proteins in the hemolymph of vitellogenic females. Despite this, the silencing of Prosα6 culminated in the impairment of oocyte maturation at the early stages of oogenesis. Overall, we discovered that proteasome activity is especially important for the signals that initiate oogenesis in R. prolixus and discuss in what manner further investigations on the regulation of proteasome assembly and activity might contribute to the unraveling of oogenesis molecular mechanisms and oocyte maturation in this vector.

Functional characterization of maternally accumulated hydrolases in the mature oocytes of the vector Rhodnius prolixus reveals a new protein phosphatase essential for the activation of the yolk mobilization and embryo development.

Yolk biogenesis and consumption have been well conserved in oviparous animals throughout evolution. Most egg-laying animals store yolk proteins within the oocytes' yolk granules (Ygs). Following fertilization, the Ygs participate in controlled pathways of yolk breakdown to support the developing embryo's anabolic metabolism. While the unfolding of the yolk degradation program is a crucial process for successful development in many species, the molecular mechanisms responsible for yolk mobilization are still mysterious and have mostly not been explored. Here, we investigate the functional role of the oocyte maternally accumulated mRNAs of a protein phosphatase (PP501) and two aspartic proteases (cathepsin-D 405, CD405 and cathepsin-D 352, CD352) in the yolk degradation and reproduction of the insect vector of Chagas disease Rhodnius prolixus. We found that PP501 and CD352 are highly expressed in the vitellogenic ovary when compared to the other organs of the adult insect. Parental RNAi silencing of PP501 resulted in a drastic reduction in oviposition and increased embryo lethality whereas the silencing of CD352 resulted only in a slight decrease in oviposition and embryo viability. To further investigate the PP501-caused high reproduction impairment, we investigated the Ygs biogenesis during oocyte maturation and the activation of the yolk degradation program at early development. We found that the Ygs biogenesis was deficient during oogenesis, as seen by flow cytometry, and that, although the PP501-silenced unviable eggs were fertilized, the Ygs acidification and acid phosphatase activity were affected, culminating in a full impairment of the yolk proteins degradation at early embryogenesis. Altogether we found that PP501 is required for the oocyte maturation and the activation of the yolk degradation, being, therefore, essential for this vector reproduction.

Influence of active games and play on children's cognition: a systematic review / Influência dos jogos e brincadeiras ativos na cognição de crianças: uma revisão sistemática

ABSTRACT Games and play are strategies to assist in the cognitive development of children. However, there are still gaps about their effects and associations when it comes to children's cognition. Thus, the objective is to synthesize the influence of active games and play on the cognition of children. The databases searched were: Lilacs, PsycINFO, Pubmed, Scielo and Scopus. Articles with observational or experimental design were included. Their quality was evaluated by means of RoB 2. Five studies were included, in accordance with the eligibility criteria. All studies had an experimental design, with two being conducted chronically, and three, acutely. Three studies showed positive effects of games and play on attention, reaction time, and executive functions: inhibitory control, logical reasoning, verbal factor, numerical factor, spatial factor, and general intelligence. Regarding the methodological quality of the studies, it is necessary to better describe and use the randomization allocation process, be more careful with data analysis, and avoid multiple assessments for a single variable. It is concluded that active games and play can generate a positive effect on the attention and executive functions of children. Further research is needed for an understanding of the relationship that parameters such as intensity, length and types of activities have with cognition.

RESUMO Jogos e brincadeiras são estratégias para auxiliar no desenvolvimento cognitivo do público infantil. Contudo ainda existem lacunas sobre os efeitos e associações dos jogos e brincadeiras ativos na cognição de crianças. Assim, o objetivo é sintetizar a influência dos jogos e brincadeiras ativos na cognição de crianças. As bases de dados foram: Lilacs, PsycINFO, Pubmed, Scielo e Scopus. Foram incluídos artigos com delineamento observacional ou experimental. A qualidade dos artigos foi avaliada com a RoB 2. Cinco estudos foram incluídos seguindo os critérios de elegibilidade. Todos os estudos apresentaram delineamento experimental, dois realizados de forma crônica e três de forma aguda. Três estudos apresentaram efeitos positivos dos jogos e brincadeiras na atenção, tempo de reação e funções executivas: controle inibitório, raciocínio lógico, fator verbal, fator numérico, fator espacial e inteligência geral. Quanto à qualidade metodológica dos estudos é necessário melhor descrição e utilização do processo de alocação da randomização, maior cuidado na análise de dados e evitar múltiplas avaliações para uma variável. Conclui-se que jogos e brincadeiras ativos podem gerar efeito positivo para a atenção e funções executivas de crianças. Mais estudos são necessários para compreensão da relação de parâmetros como intensidade, duração e tipos de atividades com a cognição.

Upper extremities functional disability and pain intensity in female volleyball athletes: cross-sectional study / Incapacidade funcional de membro superior e intensidade de dor em atletas de voleibol do sexo feminino: estudo transversal

ABSTRACT BACKGROUND AND OBJECTIVES: In volleyball, the high demand for shoulder sports makes it susceptible to injuries due to the repetition of sporting gestures. The presence of pain in the shoulder can lead to the disability to perform movements correctly, resulting in reduced sports performance. The study aimed to evaluate and correlate the presence of pain and functional disability of the upper extremity in volleyball athletes. METHODS: Cross-sectional descriptive study, developed with 30 female volleyball athletes. Functional disability was assessed using the Disabilities of the Arm, Shoulder, and Hand Questionnaire (DASH) and pain intensity using the Visual Numeric Scale (VNS). The study was conducted in an online format. The IBM SPSS 21.0 statistical package for Windows was used, where descriptive measures, Wilcoxon, and Spearman correlation tests were performed, adopting p≤0.05. RESULTS: Nineteen athletes reported mild to moderate pain to perform daily activities, and 21 athletes reported pain to perform sporting gestures, with pain being significantly more intense in the dominant shoulder in the spike movement (p<0.001), serve (p<0.001), and blocking (p=0.03). Athletes did not present functional limitations in the DASH, but the opposite, outside hitter, and middle blocker, presented mild limitations in the DASH sport. There was a strong correlation between DASH and day-to-day pain (rs=0.79; p<0.001) and a moderate correlation between sports DASH and pain in the dominant shoulder in the spike (rs=0.67; p<0.001) and in the serve (rs=0.60; p<0.001) movements. CONCLUSION: Evaluated athletes presented shoulder pain, higher functional disability to performer the sports activity, and pain and functional disability were positively correlated HIGHLIGHTS Overhead movements such as serve and spike contribute to shoulder pain in volleyball players; The functional disability of the upper limbs is positively correlated with the intensity of pain to perform daily activities and sports; The kinetic-functional diagnosis is important to minimize the risk of injury, rehabilitate injured athletes and provide an adequate return of the athlete to sports practice.

RESUMO JUSTIFICATIVA E OBJETIVOS: No voleibol, a alta exigência da articulação do ombro torna-o suscetível a lesões devido à repetição dos gestos esportivos. A presença de dor no ombro pode gerar incapacidade para executar os movimentos corretamente, ocasionando redução do desempenho esportivo. O estudo teve como objetivo avaliar e correlacionar a presença de dor e incapacidade funcional de membro superior em atletas de voleibol. METODOS: Estudo transversal, descritivo, desenvolvido com 30 mulheres atletas de voleibol de quadra. A incapacidade funcional foi avaliada por meio do Disabilities of the Arm, Shoulder and Hand Questionnaire (DASH) e a intensidade de dor pela Escala Visual Numérica (EVN) de 10 pontos. A pesquisa foi conduzida no formato on-line. Utilizou-se o programa estatístico IBM SPSS 21.0 para Windows, pelo qual foram realizadas as medidas descritivas, o teste de Wilcoxon e a correlação de Spearman adotando p≤0,05. RESULTADOS: Dezenove atletas relataram dor leve a moderada para realizar as atividades diárias, e 21 atletas para realizar os gestos esportivos, sendo a dor significativamente mais intensa no ombro dominante em movimentos para ataque (p<0,001), saque (p<0,001) e bloqueio (p=0,03). As atletas não apresentaram limitação funcional no DASH geral, mas no DASH esporte as opostas, ponteiras e centrais foram classificadas com limitação leve. Houve correlação forte entre DASH e dor no dia a dia (rs=0,79; p<0,001) e correlação moderada entre DASH esporte e dor no ombro dominante no ataque (rs=0,67; p<0,001) e no saque (rs=0,60; p<0,001). CONCLUSAO: Atletas avaliadas apresentaram dor no ombro, maior incapacidade funcional na atividade esportiva, e dor e incapacidade funcional estavam positivamente correlacionadas. DESTAQUES Movimentos aéreos como saque e ataque contribuem para a dor no ombro em jogadores de voleibol; A incapacidade funcional dos membros superiores correlaciona-se positivamente com a intensidade da dor para a realização de atividades cotidianas e esportivas; O diagnóstico cinético-funcional é importante para minimizar o risco de lesão, reabilitar atletas lesionados e proporcionar um retorno adequado do atleta à prática esportiva.

Deficiency of Acetyl-CoA Carboxylase Impairs Digestion, Lipid Synthesis, and Reproduction in the Kissing Bug Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, vector of Chagas disease. After feeding, as blood is slowly digested, amino acids are used as substrates to fuel lipid synthesis, and adult females accumulate lipids in the fat body and produce eggs. In order to evaluate the importance of de novo fatty acid synthesis for this insect metabolism, we generated acetyl-CoA carboxylase (ACC) deficient insects. The knockdown (AccKD) females had delayed blood digestion and a shorter lifespan. Their fat bodies showed reduced de novo lipogenesis activity, did not accumulate triacylglycerol during the days after blood meal, and had smaller lipid droplets. At 10 days after feeding, there was a general decrease in the amounts of neutral lipids and phospholipids in the fat body. In the hemolymph, no difference was observed in lipid composition at 5 days after blood meal, but at day ten, there was an increase in hydrocarbon content and a decrease in phospholipids. Total protein concentration and amino acid composition were not affected. The AccKD females laid 60% fewer eggs than the control ones, and only 7% hatched (89% for control), although their total protein and triacylglycerol contents were not different. Scanning electron microscopy of the egg surface showed that chorion (eggshell) from the eggs laid by the AccKD insects had an altered ultrastructural pattern when compared to control ones. These results show that ACC has a central role in R. prolixus nutrient homeostasis, and its appropriate activity is important to digestion, lipid synthesis and storage, and reproductive success.

Knockdown of E1- and E2-ubiquitin enzymes triggers defective chorion biogenesis and modulation of autophagy-related genes in the follicle cells of the vector Rhodnius prolixus.

In insects, the last stage of oogenesis is the process where the chorion layers (eggshell) are synthesized and deposited on the surface of the oocytes by the follicle cells. Protein homeostasis is determined by the fine-tuning of translation and degradation pathways, and the ubiquitin-proteasome system is one of the major degradative routes in eukaryotic cells. The conjugation of ubiquitin to targeted substrates is mediated by the ordered action of E1-activating, E2-conjugating, and E3-ligase enzymes, which covalently link ubiquitin to degradation-targeted proteins delivering them to the proteolytic complex proteasome. Here, we found that the mRNAs encoding polyubiquitin (pUbq), E1, and E2 enzymes are highly expressed in the ovaries of the insect vector of Chagas Disease Rhodnius prolixus. RNAi silencing of pUbq was lethal whereas the silencing of E1 and E2 enzymes resulted in drastic decreases in oviposition and embryo viability. Eggs produced by the E1- and E2-silenced insects presented particular phenotypes of altered chorion ultrastructure observed by high-resolution scanning electron microscopy as well as readings for dityrosine cross-linking and X-ray elemental microanalysis, suggesting a disruption in the secretory routes responsible for the chorion biogenesis. In addition, the ovaries from silenced insects presented altered levels of autophagy-related genes as well as a tendency of upregulation in ER chaperones, indicating a disturbance in the general biosynthetic-secretory pathway. Altogether, we found that E1 and E2 enzymes are essential for chorion biogenesis and that their silencing triggers the modulation of autophagy genes suggesting a coordinated function of both pathways for the progression of choriogenesis.

Atypical strategies for cuticle pigmentation in the blood-feeding hemipteran Rhodnius prolixus.

Pigmentation in insects has been linked to mate selection and predator evasion, thus representing an important aspect for natural selection. Insect body color is classically associated to the activity of tyrosine pathway enzymes, and eye color to pigment synthesis through the tryptophan and guanine pathways, and their transport by ATP-binding cassette proteins. Among the hemiptera, the genetic basis for pigmentation in kissing bugs such as Rhodnius prolixus, that transmit Chagas disease to humans, has not been addressed. Here, we report the functional analysis of R. prolixus eye and cuticle pigmentation genes. Consistent with data for most insect clades, we show that knockdown for yellow results in a yellow cuticle, while scarlet and cinnabar knockdowns display red eyes as well as cuticle phenotypes. In addition, tyrosine pathway aaNATpreto knockdown resulted in a striking dark cuticle that displays no color pattern or UV reflectance. In contrast, knockdown of ebony and tan, that encode N-beta-alanyl dopamine hydroxylase branch tyrosine pathway enzymes, did not generate the expected dark and light brown phenotypes, respectively, as reported for other insects. We hypothesize that R. prolixus, which requires tyrosine pathway enzymes for detoxification from the blood diet, evolved an unusual strategy for cuticle pigmentation based on the preferential use of a color erasing function of the aaNATpreto tyrosine pathway branch. We also show that genes classically involved in the generation and transport of eye pigments regulate red body color in R. prolixus. This is the first systematic approach to identify the genes responsible for the generation of color in a blood-feeding hemiptera, providing potential visible markers for future transgenesis.

Open questions on the functional biology of the yolk granules during embryo development.

Biogenesis and consumption of the yolk are well-conserved aspects of the reproductive biology in oviparous species. Most egg-laying animals accumulate yolk proteins within the oocytes thus creating the source of nutrients and energy that will feed embryo development. Yolk accumulation drives the generation of a highly specialized oocyte cytoplasm with maternal mRNAs, ribosomes, mitochondria, and, mainly, a set of organelles collectively referred to as yolk granules (Ygs). Following fertilization, the Ygs are involved in regulated mechanisms of yolk degradation to fuel the anabolic metabolism of the growing embryo. Thus, yolk accumulation and degradation are essential processes that allow successful development in many species. Nevertheless, the molecular machinery and mechanisms dedicated to the programmed yolk mobilization throughout development are still enigmatic and remain mostly unexplored. Moreover, while the Ygs functional biology as a nutritional source for the embryo has been acknowledged, several reports have suggested that Ygs cargoes and functions go far beyond yolk storage. Evidence of the role of Ygs in gene expression, microbiota harboring, and paracrine signaling has been proposed. In this study, we summarize the current knowledge of the Ygs functional biology pointing to open questions and where further investigation is needed.

The transition from vitellogenesis to choriogenesis triggers the downregulation of the UPR sensors IRE1 and PERK and alterations in the ER architecture in the follicle cells of the vector Rhodnius prolixus.

In insects, the follicle cells (FCs) give rise to a single-layered tissue of binucleated professional secretory cells that surround the oocytes during oogenesis. In the latest stage of oocyte development, the FCs rapidly synthesize and secrete the chorion (eggshell) immediately before degenerating through apoptosis. Here, we used RT-qPCR, electron microscopy, and RNAi silencing to explore the role of the main unfolded protein response (UPR) receptors IRE1 and PERK, as well as the ultrastructure dynamics of the FCs during oogenesis of the insect vector of Chagas disease Rhodnius prolixus. We found that IRE1 and PERK mRNAs are highly expressed in the ovaries of vitellogenic females. Interestingly, we observed that IRE1 and PERK, as well as different isoforms of the chaperones Bip and PDI, have their FCs gene expression levels decreased during the vitellogenesis to choriogenesis transition. Using transmission electron microscopy, we observed that the downregulation of the UPR gene expression is accompanied by dramatic changes in the FCs ultrastructure, with an 80% reduction in the mean area of the ER tubules, and circularization and enlargement of the mitochondria. Additionally, we found that parental RNAi silencing of both IRE1 and PERK resulted in minor changes in the chorion protein composition and ultrastructure, accessed by urea extraction of the chorion proteins and scanning electron microscopy, respectively, but did not impact the overall levels of oviposition and F1 embryo development.

Retenção das Habilidades de Ressuscitação Cardiopulmonar nos Estudantes de Medicina / Retention of Cardiopulmonary Resuscitation Skills in Medical Students

Resumo Fundamento A redução da mortalidade e das sequelas de uma vítima de parada cardíaca depende de um atendimento eficaz, rápido e iniciado o mais precocemente possível. O suporte básico de vida (SBV) compreende uma série de etapas que podem ser iniciadas fora do ambiente hospitalar, e ensinadas para qualquer pessoa em cursos específicos. Porém, é importante que o socorrista retenha o conhecimento e as habilidades, pois nunca se sabe quando será necessário realizar uma ressuscitação cardiopulmonar (RCP). Entretanto, estudos mostram que existe uma perda das habilidades em executar uma RCP já com 30 dias após o treinamento, com variações segundo algumas características das pessoas e da atividade profissional. Objetivo Avaliar se os estudantes de medicina são capazes de reter as habilidades por mais de seis meses. Métodos Estudo prospectivo, caso controle, observacional. Estudantes de medicina realizaram um curso sobre morte súbita e parada cardíaca de 40 horas. A avaliação das habilidades foi realizada imediatamente após o curso e seis meses depois. Foram comparadas as notas individuais entre dois momentos, foi avaliada a porcentagem de acerto em cada etapa e uma análise global do atendimento foi classificado como ótimo, bom e ruim. Os avaliadores e critérios foram os mesmos nos dois momentos. Os dados foram analisados pelos teste-t pareado e teste de McNemar, onde para um nível de confiança de 95% o critério para significância foi p < 0,05. Resultados Cinquenta estudantes (27 do sexo feminino) do primeiro ano, com idade entre 18 e 24 anos (média 21), realizaram o curso. O número de etapas cumpridas de forma correta após seis meses foi significativamente menor que logo após o curso (10,8 vs 12,5 p < 0,001). O sexo e idade não interferiram nos resultados. A qualidade global foi considerada ótima em 78% dos atendimentos realizados logo após o curso, significativamente, maior que os 40% após seis meses (p < 0,01). Após seis meses, maior número de erros foi observado nas etapas relacionadas às habilidades mais práticas (como posicionamento das mãos). Conclusão Seis meses após o curso observamos uma perda significativa das habilidades, entre estudantes de medicina, prejudicando a eficácia global do atendimento.

Abstract Background Reduction of mortality and sequelae of cardiac arrest depends on an effective and fast intervention, started as soon as possible. Basic life support involves a series of steps that may be initiated out of the hospital setting and taught to any person in specific courses. However, it is important that the rescuers retain the knowledge and skills to perform cardiopulmonary resuscitation (CPR), as one never knows when they will be required. Studies have shown that a loss of skills occurs as early as 30 days after the training course, with variations according to personal and professional characteristics. Objectives to assess whether medical students are able to retain skills acquired in a BLS course for more than six months. Methods Prospective, case-control, observational study. Medical students attended a 40-hour course on sudden death and cardiac arrest. Skills acquired during the course were evaluated immediately after and six months after the course. Students' individual scores were compared between these time points, the percentage of correct answers was evaluated, and overall performance was rated as excellent, good, and poor. Observers and evaluation criteria were the same immediately after the course and six months later. Data were analyzed using the paired t-test and the McNemar test. The 95% confidence interval was established, and a p < 0.05 was set as statistically significant. Results Fifty students (27 female) in the first year of medical school aged from 18 to 24 years (mean of 21 years) attended the course. The number of steps successfully completed by the students at six months was significantly lower than immediately after the course (10.8 vs 12.5 p < 0.001). Neither sex nor age affected the results. Overall performance of 78% of the students was considered excellent immediately after the course, and this percentage was significantly higher than six months later (p < 0.01). After six months, the steps that the students failed to complete at six months were those related to practical skills (such as a correct hand positioning). Conclusion A significant loss of skills was detected six months after the BLS course among medical students, compromising their overall performance.

VPS38/UVRAG and ATG14, the variant regulatory subunits of the ATG6/Beclin1-PI3K complexes, are crucial for the biogenesis of the yolk organelles and are transcriptionally regulated in the oocytes of the vector Rhodnius prolixus.

In insects the reserve proteins are stored in the oocytes into endocytic-originated vesicles named yolk organelles. VPS38/UVRAG and ATG14 are the variant regulatory subunits of two class-III ATG6/Beclin1 PI3K complexes that regulate the recruitment of the endocytic (complex II) and autophagic (complex I) machineries. In a previous work from our group, we found that the silencing of ATG6/Beclin1 resulted in the formation of yolk-deficient oocytes due to defects in the endocytosis of the yolk proteins. Because ATG6/Beclin1 is present in the two above-described PI3K complexes, we could not identify the contributions of each complex to the yolk defective phenotypes. To address this, here we investigated the role of the variant subunits VPS38/UVRAG (complex II, endocytosis) and ATG14 (complex I, autophagy) in the biogenesis of the yolk organelles in the insect vector of Chagas Disease Rhodnius prolixus. Interestingly, the silencing of both genes phenocopied the silencing of ATG6/Beclin1, generating 1) accumulation of yolk proteins in the hemolymph; 2) white, smaller, and yolk-deficient oocytes; 3) abnormal yolk organelles in the oocyte cortex; and 4) unviable F1 embryos. However, we found that the similar phenotypes were the result of a specific cross-silencing effect among the PI3K subunits where the silencing of VPS38/UVRAG and ATG6/Beclin1 resulted in the specific silencing of each other, whereas the silencing of ATG14 triggered the silencing of all three PI3K components. Because the silencing of VPS38/UVRAG and ATG6/Beclin1 reproduced the yolk-deficiency phenotypes without the cross silencing of ATG14, we concluded that the VPS38/UVRAG PI3K complex II was the major contributor to the previously observed phenotypes in silenced insects. Altogether, we found that class-III ATG6/Beclin1 PI3K complex II (VPS38/UVRAG) is essential for the yolk endocytosis and that the subunits of both complexes are under an unknown transcriptional regulatory system.