Gene Editing in the Chagas Disease Vector Rhodnius prolixus by Cas9-Mediated ReMOT Control.

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing protocols to advance the field are still lacking. Here, we tested protocols for the maternal delivery of CRISPR-Cas9 (clustered regularly spaced palindromic repeats/Cas-9 associated) elements to developing R. prolixus oocytes and strategies for the identification of insertions and deletions (indels) in target loci of resulting gene-edited generation zero (G0) nymphs. We demonstrate successful gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) with the ovary-targeting BtKV ligand. These results provide proof of concepts for generating somatic mutations in R. prolixus and potentially for generating germ line-edited lines in triatomines, laying the foundation for gene editing protocols that could lead to the development of novel control strategies for vectors of Chagas disease.

Boosting life sciences research in Brazil: building a case for a local Drosophila stock center.

Drosophila melanogaster is undoubtedly one of the most useful model organisms in biology. Initially used in solidifying the principles of heredity, and establishing the basic concepts of population genetics and of the synthetic theory of evolution, it can currently offer scientists much more: the possibility of investigating a plethora of cellular and biological mechanisms, from development and function of the immune system to animal neurogenesis, tumorigenesis and beyond. Extensive resources are available for the community of Drosophila researchers worldwide, including an ever-growing number of mutant, transgenic and genomically-edited lines currently carried by stock centers in North America, Europe and Asia. Here, we provide evidence for the importance of stock centers in sustaining the substantial increase in the output of Drosophila research worldwide in recent decades. We also discuss the challenges that Brazilian Drosophila scientists face to keep their research projects internationally competitive, and argue that difficulties in importing fly lines from international stock centers have significantly stalled the progression of all Drosophila research areas in the country. Establishing a local stock center might be the first step towards building a strong local Drosophila community that will likely contribute to all areas of life sciences research.

Sulphur Amino Acids: How Important is it to Set Reference Ranges for Each Population?

INTRODUCTION: Metabolism of sulfur amino acids requires an optimal interplay between nutritional demand, enzymes, transporters, and adequate dietary intake of B vitamins. Insufficient intake and excess are detrimental, and concentrations depend on health status. However, plasma aminothiol concentrations, previously reported in healthy subjects using highly sensitive methods, vary considerably, and age- and gender differences were observed. Therefore, defining age- and gender-specific ranges for each population is crucial to evaluate the meaning of plasma thiol redox state in health and disease. METHODS: A healthy Portuguese pediatric population (n=90), aged 9- (n=38) and 17-year-old (n=52), was evaluated. Plasma aminothiols, total homocysteine (tHcy), cysteine (tCys), glutathione (tGSH) and γ-glutamylcysteine (tγ-Glu-Cys), were analysed as SBD-F derivatives by HPLC with fluorescence detection. RESULTS/CASE REPORT: Mean plasma concentrations (SD) for the 9- and the 17-year-old groups, were as following: tHcy = 4.58 (0.98); 8.13 (3.27) µM, p <0.001; tCys = 207.34 (32.07); 198.59 (21.24) µM, p = 0.274; tGSH = 4.54 (1.08); 5.20 (1.84) µM, p = 0.123 and tγ-Glu-Cys = 1.47 (0.30); 1.06 (0.28) µM, p < 0.001, respectively. No statistically significant differences were found between males and females in the 9-year-old group. However, in the 17-year-old group, significant differences between genders were observed for tHcys (p < 0.001) and tγ-Glu-Cys (p = 0.039), with males presenting the highest concentrations. When correlating the four thiols' plasma concentrations, only the precursors of glutathione, tγ-Glu-Cys and tCys, were positively correlated (r = 0.450, p < 0.001). CONCLUSION: Our results showed significant differences in tHcy and tγ-Glu-Cys levels across both age groups, which increased and decreased with age, respectively. It is interesting to highlight that in the 17-year-old group, tHcy and tγ-Glu-Cys levels were higher in males than in females. These observations showed that age and gender influence plasma levels of thiols, which may impact cellular oxidative status. In conclusion, setting age and gender distinct ranges for each specific population is of utmost importance for understanding disease mechanisms and the effectiveness of therapeutic interventions.

Flying under the radar - impact and factors influencing asymptomatic DENV infections.

The clinical outcome of DENV and other Flaviviruses infections represents a spectrum of severity that ranges from mild manifestations to severe disease, which can ultimately lead to death. Nonetheless, most of these infections result in an asymptomatic outcome that may play an important role in the persistent circulation of these viruses. Also, although little is known about the mechanisms that lead to these asymptomatic infections, they are likely the result of a complex interplay between viral and host factors. Specific characteristics of the infecting viral strain, such as its replicating efficiency, coupled with host factors, like gene expression of key molecules involved in the immune response or in the protection against disease, are among crucial factors to study. This review revisits recent data on factors that may contribute to the asymptomatic outcome of the world's widespread DENV, highlighting the importance of silent infections in the transmission of this pathogen and the immune status of the host.

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.

MicroRNAs and exosomes: promising new biomarkers in acute myeloid leukemias?

Despite advances in understanding of carcinogenesis and of treatment of acute myeloid leukemia, this neoplasm still has a lethality of at least 30%. The search for biomarkers that can predict the response to treatment in the early stages of the disease is still necessary. In recent years, a new form of cellular communication between tumor and non-neoplastic cells has been discovered: the exchange of information through extracellular vesicles. These are small vesicles released by membrane-coated cells that carry proteins, lipids, messenger RNAs, microRNA and DNA, which can be internalized and promote biological changes in target cells. Exosomes are qualified as a type of extracellular vesicle and, in tumors, carry immunoinhibitory signals that promote the escape of immune control. Recent studies have showed their involvement in communication with the cells of the tumor microenvironment and with chemoresistance in several tumors. To date, there is no information about immunoregulatory microRNAs transported by exosomes and their correlation with clinical evolution during chemotherapy for acute myeloid leukemia. Knowledge about immunomodulatory microRNAs obtained by leukemic cells and transported by exosomes can direct us towards the design of new diagnostic and treatment tools in this type of leukemia.

Patterns of energetic substrate modifications in response to feeding in boas, Boa constrictor (Serpentes, Boidae).

Ambush-foraging snakes that ingest large meals might undergo several months without eating when they use the internal reserves to support the energetic costs of living. Then, morphological and physiological processes might be orchestrated during the transition from fasting to the postprandial period to rapidly use the energetic stores while the metabolic rate is elevated in response to food intake. To understand the patterns of substrates deposition after feeding, we accessed the morphological and biochemical response in Boa constrictor snakes after two months of fasting and six days after feeding. We followed the plasma levels of glucose, total proteins, and total lipids, and we performed the stereological ultrastructural analysis of the liver and the proximal region of the intestine to quantify glycogen granules and lipid droplets. In the same tissues and stomach, we measured the activity of the enzyme fructose-1,6-biphosphatase (FBPase1) involved in the gluconeogenic pathway, and we measured pyruvate kinase (PK) and lactate dehydrogenase (LDH) enzymatic activities involved in the anaerobic pathway in the liver. Briefly, our results indicated an increase in boas' plasma glucose one day after meal intake compared to unfed snakes. The hepatic glycogen reserves were continuously restored within days after feeding. Also, the enzymes involved in the energetic pathways increased activity six days after feeding in the liver. These findings suggest a quick restoring pattern of energetic stores during the postprandial period.

Evolution of the dorsoventral axis in insects: the changing role of Bone Morphogenetic Proteins.

Embryonic dorsal-ventral (DV) patterning by Bone Morphogenetic Proteins (BMPs) is a conserved feature of Bilateria, based on graded BMP activity set up by diffusible BMP ligands and Chordin/Sog antagonists. In the fly Drosophila melanogaster BMP function is secondary to patterning by the Toll pathway, suggesting a more restricted role for BMPs in insects. With widespread genome sequencing technologies allied to functional analysis in a growing number of species, recent work has shown that BMP's role in DV patterning relative to Toll varies among insect orders. Further, the role of BMP antagonists to set up BMP gradients is also greatly diversified. Here we review the recent findings concerning the role of BMP in the DV patterning of insects and address the potential aspects that may have co-evolved with BMPs to attain this functional divergence.

MicroRNAs and exosomes: promising new biomarkers in acute myeloid leukemias?

ABSTRACT Despite advances in understanding of carcinogenesis and of treatment of acute myeloid leukemia, this neoplasm still has a lethality of at least 30%. The search for biomarkers that can predict the response to treatment in the early stages of the disease is still necessary. In recent years, a new form of cellular communication between tumor and non-neoplastic cells has been discovered: the exchange of information through extracellular vesicles. These are small vesicles released by membrane-coated cells that carry proteins, lipids, messenger RNAs, microRNA and DNA, which can be internalized and promote biological changes in target cells. Exosomes are qualified as a type of extracellular vesicle and, in tumors, carry immunoinhibitory signals that promote the escape of immune control. Recent studies have showed their involvement in communication with the cells of the tumor microenvironment and with chemoresistance in several tumors. To date, there is no information about immunoregulatory microRNAs transported by exosomes and their correlation with clinical evolution during chemotherapy for acute myeloid leukemia. Knowledge about immunomodulatory microRNAs obtained by leukemic cells and transported by exosomes can direct us towards the design of new diagnostic and treatment tools in this type of leukemia.

Feeding effects on liver mitochondrial bioenergetics of Boa constrictor (Serpentes: Boidae).

Snakes are interesting examples of taxa that can overcome energy metabolism challenges, as many species can endure long periods without feeding, and their eventual meals are of reasonably large sizes, thus exhibiting dual extreme adaptations. Consequently, metabolic rate increases considerably to attend to the energetic demand of digestion, absorption and protein synthesis. These animals should be adapted to transition from these two opposite states of energy fairly quickly, and therefore we investigated mitochondrial function plasticity in these states. Herein, we compared liver mitochondrial bioenergetics of the boid snake Boa constrictor during fasting and after meal intake. We fasted the snakes for 60 days, and then we fed a subgroup with 30% of their body size and evaluated their maximum postprandial response. We measured liver respiration rates from permeabilized tissue and isolated mitochondria. From isolated mitochondria, we also measured Ca2+ retention capacity and redox status. Mitochondrial respiration rates were maximized after feeding, reaching an approximately 60% increase from fasting levels when energized with complex I-linked substrates. Interestingly, fasting and fed snakes exhibited similar respiratory control ratios and citrate synthase activity. Furthermore, we found no differences in Ca2+ retention capacity, indicating no increase in susceptibility to mitochondrial permeability transition, and no changes in mitochondrial redox state, although fed animals exhibited increases in the release of H2O2. Thus, we conclude that liver mitochondria from B. constrictor snakes increase respiration rates during the postprandial period and quickly improve the bioenergetic capacity without compromising redox balance.

A reaction-diffusion network model predicts a dual role of Cactus/IκB to regulate Dorsal/NFκB nuclear translocation in Drosophila.

Dorsal-ventral patterning of the Drosophila embryo depends on the NFκB superfamily transcription factor Dorsal (Dl). Toll receptor activation signals for degradation of the IκB inhibitor Cactus (Cact), leading to a ventral-to-dorsal nuclear Dl gradient. Cact is critical for Dl nuclear import, as it binds to and prevents Dl from entering the nuclei. Quantitative analysis of cact mutants revealed an additional Cact function to promote Dl nuclear translocation in ventral regions of the embryo. To investigate this dual Cact role, we developed a predictive model based on a reaction-diffusion regulatory network. This network distinguishes non-uniform Toll-dependent Dl nuclear import and Cact degradation, from the Toll-independent processes of Cact degradation and reversible nuclear-cytoplasmic Dl flow. In addition, it incorporates translational control of Cact levels by Dl. Our model successfully reproduces wild-type data and emulates the Dl nuclear gradient in mutant dl and cact allelic combinations. Our results indicate that the dual role of Cact depends on the dynamics of Dl-Cact trimers along the dorsal-ventral axis: In the absence of Toll activation, free Dl-Cact trimers retain Dl in the cytoplasm, limiting the flow of Dl into the nucleus; in ventral-lateral regions, Dl-Cact trimers are recruited by Toll activation into predominant signaling complexes and promote Dl nuclear translocation. Simulations suggest that the balance between Toll-dependent and Toll-independent processes are key to this dynamics and reproduce the full assortment of Cact effects. Considering the high evolutionary conservation of these pathways, our analysis should contribute to understanding NFκB/c-Rel activation in other contexts such as in the vertebrate immune system and disease.

Teledermatology in Times of COVID-19 Confinement: Comparing Patients' and Physicians' Satisfaction by the Standardized Brest Teledermatology Questionnaire.

The French government imposed the first COVID-19 pandemic lockdown from March 17 until May 11, 2020. Only emergency cases and teledermatology (TD) were allowed in outpatient settings. A standardized questionnaire was developed to compare the satisfaction level of patients and their treating physicians. Our main question was whether the patients would perceive TD as a valid alternative for direct physical face-to-face consultation. Eighty-two patients and their 4 treating dermatologists from one dermatology department participated in the study (43 females, 39 males) with a mean age of 46.6 years (SD ±23.9). The reason for TD was a chronic disease in the majority (87.8%), and mainly as a follow-up (96.3%). Regarding satisfaction, almost all categories rated around 9 on a 0-10 verbal analogue scale. The same level of global satisfaction could be seen between the patients and the physicians as well as for the quality of the patient-physician relation and whether all questions could be addressed during the TC. Physicians showed significantly higher scores than patients only for the category of "length" of the consultation. Gender, age, as well as distance between the clinic and home of the patient were not influencing factors for satisfaction. Regarding the technical parameters, the evaluation was mostly comparable for patients and physicians, but overall lower than the relational satisfaction parameters, especially for image quality. Patients were significantly more motivated to continue the TD after the lockdown than their treating dermatologists. We see an interest for implementing TD in specialized centers with chronic patients coming from remote places for regular follow-ups. TD cannot replace in-person patient-physician interaction, but was helpful during the lockdown. As a result, TD might become part of dermatology training to prepare for future lockdown situations.

Analysis of ovarian transcriptomes reveals thousands of novel genes in the insect vector Rhodnius prolixus.

Rhodnius prolixus is a Triatominae insect species and a primary vector of Chagas disease. The genome of R. prolixus has been recently sequenced and partially assembled, but few transcriptome analyses have been performed to date. In this study, we describe the stage-specific transcriptomes obtained from previtellogenic stages of oogenesis and from mature eggs. By analyzing ~ 228 million paired-end RNA-Seq reads, we significantly improved the current genome annotations for 9206 genes. We provide extended 5' and 3' UTRs, complete Open Reading Frames, and alternative transcript variants. Strikingly, using a combination of genome-guided and de novo transcriptome assembly we found more than two thousand novel genes, thus increasing the number of genes in R. prolixus from 15,738 to 17,864. We used the improved transcriptome to investigate stage-specific gene expression profiles during R. prolixus oogenesis. Our data reveal that 11,127 genes are expressed in the early previtellogenic stage of oogenesis and their transcripts are deposited in the developing egg including key factors regulating germline development, genome integrity, and the maternal-zygotic transition. In addition, GO term analyses show that transcripts encoding components of the steroid hormone receptor pathway, cytoskeleton, and intracellular signaling are abundant in the mature eggs, where they likely control early embryonic development upon fertilization. Our results significantly improve the R. prolixus genome and transcriptome and provide novel insight into oogenesis and early embryogenesis in this medically relevant insect.

Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands.

BACKGROUND: The invasion of the mosquito salivary glands by Plasmodium sporozoites is a critical step that defines the success of malaria transmission and a detailed understanding of the molecules responsible for salivary gland invasion could be leveraged towards control of vector-borne pathogens. Antibodies directed against the mosquito salivary gland protein SGS1 have been shown to reduce Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands, but the specific role of this protein in sporozoite invasion and in other stages of the Plasmodium life cycle remains unknown. METHODS: RNA interference and CRISPR/Cas9 were used to evaluate the role of A. aegypti SGS1 in the P. gallinaceum life cycle. RESULTS: Knockdown and knockout of SGS1 disrupted sporozoite invasion of the salivary gland. Interestingly, mosquitoes lacking SGS1 also displayed fewer oocysts. Proteomic analyses confirmed the abolishment of SGS1 in the salivary gland of SGS1 knockout mosquitoes and revealed that the C-terminus of the protein is absent in the salivary gland of control mosquitoes. In silico analyses indicated that SGS1 contains two potential internal cleavage sites and thus might generate three proteins. CONCLUSION: SGS1 facilitates, but is not essential for, invasion of A. aegypti salivary glands by P. gallinaceum and has a dual role as a facilitator of parasite development in the mosquito midgut. SGS1 could, therefore, be part of a strategy to decrease malaria transmission by the mosquito vector, for example in a transgenic mosquito that blocks its interaction with the parasite.

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae), an important vector of Leishmaniosis in South America, described by scanning electron microscopy.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes-trichoidea, basiconica, coelonica and campanoformia-were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species.

Myelodysplastic syndrome: validation of flow cytometry multilineage score system.

OBJECTIVE: To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. METHODS: A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. RESULTS: Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. CONCLUSION: The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.

The first characterization of a cystatin and a cathepsin L-like peptidase from Aedes aegypti and their possible role in DENV infection by the modulation of apoptosis.

Recently, a salivary gland transcriptome study demonstrated that the transcripts of a putative cystatin gene (SeqID AAEL013287; Aacystatins) from Aedes aegypti were increased in DENV2-infected mosquitoes and that silencing of the Aacystatin gene resulted in an increase in DENV titres. In this work, Aacystatin was biochemically characterized; the purified recombinant inhibitor was able to inhibit typical cysteine proteases with a Ki in the nM range. Pulldown assays using Aag2 cell extracts identified a cathepsin L-like peptidase (AaCatL) as a possible target of Aacystatin. Purified recombinant AaCatL had an optimal pH of 5.0 and displayed a preference for Leu, Val and Phe residues at P2, which is common for other cathepsin L-like peptidases. Transcription analysis of Aacystatin and AaCatL in the salivary glands and midgut of DENV2-infected mosquitoes revealed a negative correlation between DENV2 titres and levels of the inhibitor and peptidase, suggesting their involvement in DENV2-mosquito interactions. Considering that apoptosis may play an important role during viral infections, the possible involvement of Aacystatin in staurosporine-induced apoptosis in Aag2 cells was investigated; the results showed higher expression of the inhibitor in treated cells; moreover, pre incubation with rAacystatin was able to increase Aag2 cell viability.

Myelodysplastic syndrome: validation of flow cytometry multilineage score system / Síndrome mielodisplásica: validação de ficha de escore multilinhagem por citometria de fluxo

ABSTRACT Objective To validate multilineage score system correlating results of flow cytometry, cytogenetics, cytomorphology and histology from samples of patients with suspected myelodysplastic syndrome or cytopenia of unknown origin. Methods A retrospective study analyzing laboratory data of 49 patients with suspected myelodysplastic syndrome or cytopenia of unknown origin, carried out between May and September 2017. The inclusion criteria were availability of flow cytometry results, and at least one more method, such as morphology, histology or cytogenetics. Thirty-eight patients were classified as diagnosis of myelodysplastic syndromes, whereas 11 were classified as normal. Patients were evaluated based on score systems, Ogata score and flow cytometry multilineage score. Results Comparing the scores obtained in the Ogata score and the multilineage score, it was observed that in four cases the Ogata score was zero or 1 point, while the multilineage score was higher than 3 points. In addition, in 12 cases with Ogata score of 2, the multilineage score was greater than 3. Conclusion The flow cytometry multilineage score system demonstrated to be more effective in dysplasia analysis, by assessing the erythroid, monocytic, granulocytic and precursor cell lineages, apart from the parameters evaluated by the Ogata score.

RESUMO Objetivo Validar ficha de escore multilinhagem correlacionando resultados obtidos de citometria de fluxo, citogenética, citomorfologia e histologia de amostras de pacientes com suspeita de síndrome mielodisplásica ou citopenias a esclarecer. Métodos Estudo retrospectivo de análise de dados laboratoriais de 49 pacientes com suspeita clínica de síndrome mielodisplásica ou citopenias a esclarecer realizado entre maio e setembro de 2017. Os critérios de inclusão foram a disponibilidade de resultados de citometria de fluxo e de, pelo menos, outra metodologia, entre morfologia, histologia, ou citogenética. Trinta e oito pacientes foram classificados como diagnosticados com síndromes mielodisplásicas enquanto 11 foram classificados como normais. Os pacientes foram avaliados utilizando sistemas de escore, escore de Ogata e ficha multilinhagem. Resultados Comparando as pontuações obtidas no escore de Ogata e na ficha multilinhagem, observou-se que, em quatro casos, o score de Ogata foi zero ou 1 ponto, enquanto, pela ficha multilinhagem, a pontuação foi superior a 3 pontos. Além disso, em 12 casos com escore de Ogata 2, a pontuação pela ficha multilinhagem foi superior a 3. Conclusão A ficha multilinhagem demonstrou ser mais eficaz na análise de displasia por avaliar as linhagens eritroide, monocítica, granulocítica e células precursoras, além dos parâmetros avaliados no escore de Ogata.

Homocysteine Metabolism in Children and Adolescents: Influence of Age on Plasma Biomarkers and Correspondent Genotype Interactions.

BACKGROUND: Imbalance of homocysteine (Hcy) metabolism links with several pathologies; nevertheless, it is poorly characterized in pediatric populations. This study investigated the impact of age on plasma concentrations of Hcy and relevant biomarkers along with correspondent genotype interactions. METHODS: A healthy pediatric cohort aged 9 (n = 195) and 17 (n = 128) years old (yo) was studied. Immunoassays and GC-MS-SIM-mode quantified plasma levels of Hcy and biomarkers. PCR-RFLP or quantitative-PCR assays assessed common variations in related genes. RESULTS: Age impacted on levels of Hcy and metabolic markers: older children presented with the lowest folates and total-cobalamin (tCbl), while with the highest Hcy concentrations, whereas methylmalonic acid (MMA) and holotranscobalamin (Holo-TC) levels remained similar in 9-yo and 17-yo children. The relationships between B-vitamins and metabolic markers were also dependent on age. Only in the older children, MMA correlated with tCbl and Holo-TC, and MMA levels were markedly higher in the 17-yo subjects presenting with the lowest quartiles of Holo-TC concentrations. Lastly, age also impacted on the correlations between genotype and biomarkers. In the 17-yo group, however not in the 9-yo children, tHcy differed between MTHFR 677 genotypes, with subjects who had the MTHFR 677TT genotype displaying the highest tHcy concentrations. CONCLUSIONS: Age impacts on the Hcy metabolism dynamics in a pediatric population.

Expression and Activity of Calpain A in Drosophila melanogaster.

Detecting calpain activity in Drosophila tissues is a fundamental tool to study calpain function. We use differential centrifugation to prepare membrane- versus cytosol-enriched fractions for measuring calpain activity with the fluorogenic substrate N-LY-AMC. With this method one can measure calpain A activity in wild-type flies and in several mutant fly backgrounds, revealing a strong correlation between in situ membrane distribution and in vitro determined activity measurements. Here we describe the steps for tissue preparation and calpain activity measurement in the Drosophila embryo.