A versatile inhibitor of digestive enzymes in Aedes aegypti larvae selected from a pacifastin (TiPI) phage display library.

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 105 cfu with random mutations in the reactive site (P2-P2'). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies.

Rotenoids from Clitoria fairchildiana R. Howard (Fabaceae) seeds affect the cellular metabolism of larvae of Aedes aegypti L. (Culicidae).

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-ß-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves' ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain.

MAIN CONCLUSION: Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant's intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20-25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors' activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs' complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

Topical capsaicin 8% patch in peripheral neuropathic pain: Efficacy and quality of life.

Introduction: Peripheral neuropathic pain (PNP) is one of the most challenging diseases to treat with a significant negative impact on the patients' health-related quality of life (HRQoL). Capsaicin 8% patch has arisen in the last decades as an alternative to oral drugs in the treatment of PNP with fewer side effects and promising results in efficacy. Objectives: This work aims to evaluate the effectiveness of the topical application of capsaicin in PNP and its impact on patients' HRQoL based on the use of capsaicin in a tertiary hospital of Oporto. Methods: This study included 100 patients with localized PNP with poor pain control and without improvement with previous treatments that were treated at least once with an 8% capsaicin patch. Effectiveness on pain relief, number of treatments needed, safety and impact on HRQoL were assessed through a set of questionnaires. Results: Regarding the aetiology of PNP, 67.6% (N = 46) have post-surgery or trauma induced PNP with 64.7% (N = 44) of patients reporting pain in the lower limb. After the treatment, 30.9% (N = 21) felt minimally improved, 22.1 (N = 15) felt much improved and 13.2 (N = 9) felt very much improved. On a scale from 1 to 10, in the week prior to the survey, the median intensity of pain was 6 and the median interference in quality of life was 7. The majority of patients still report limitations in mobility and daily activities and moderate pain. Conclusion: Capsaicin 8% patch is effective in PNP treatment at least in the short term. Repeated applications may be important for long-term analgesia. The low systemic dose and few side effects mean that the treatment is generally well tolerated by patients. Due to the analgesic effect, capsaicin can improve the HRQoL of patients with PNP.

Peripheral muscle oxygenation, pain, and disability indices in individuals with and without nonspecific neck pain, before and after myofascial reorganization®: A double-blind randomized controlled trial.

To investigate whether myofascial reorganization® in the trapezius muscle (MRT) improves peripheral muscle oxygenation and pain tolerance and decreases neck disability index (NDI) scores in individuals with and without nonspecific neck pain (NP) using a double-blind randomized controlled trial. Seventy-five subjects were equally and randomly assigned to three groups: the intervention groups (experimental [EG] and sham sSG]) and the control group (CG). Several inclusion criteria were applied to the intervention groups: male or female, aged 18-32 years, self-reported NP in the last 3 months without a defined cause; at least "soft" pain in session 1 of the NDI, and at least a score of 1 on the Visual Analogue Scale (VAS). The CG was required to have NDI and VAS scores of 0 at recruitment. Intervention: The EG underwent MRT for 10 min, once a week for 6 weeks. Patients with NP in the SG underwent classical massage for the same duration and frequency. Patients in the CG had no pain and underwent no intervention. Data collection was performed using the NDI Questionnaire, a pressure algometer for pain evaluation, and near-infrared spectroscopy for muscle oxygenation measurements. It was registered as NCT03882515 at ClinicalTrials.gov. The NDI score in both the EG (p<0.001) and SG (p<0.001) decreased after 6 weeks of intervention compared to the CG. The CG demonstrated a lower basal tissue saturation (TSI) index than the EG (p<0.001) and SG (p = 0.02). The EG demonstrated higher oxyhemoglobin values than the SG (p<0.001) and CG (p = 0.03). The CG had higher pain tolerance than the EG (p = 0.01) and SG (p<0.001) post-intervention. MRT increased trapezius muscle oxygenation after 6 weeks of intervention.

Acute effects of myofascial reorganization on trapezius muscle oxygenation in individuals with nonspecific neck pain.

BACKGROUND AND PURPOSE: Nonspecific neck pain (NNP) is a common idiopathic disorder in the general population that affects the trapezius muscle (TM) and blood supply, thereby compromising the fascial system. Myofascial reorganization (MR) is a physical therapy technique that can influence the dynamics of local fluids reducing excessive muscle tension and capillary constriction, and increasing local blood flow. This study aimed to investigate whether MR improves peripheral muscle oxygenation. METHODS: This was a quasi-experimental study with an intentional non-probability sample. Fifty participants (women: 36, men: 14) with and without NNP were assigned to either the experimental group (EG: n = 25, with NNP, subjected to MR) or the control group (CG: without NNP, no MR intervention). TM oxygenation was measured using near-infrared spectroscopy (NIRS) before and after a single intervention. All participants were evaluated and reassessed after 10 min. RESULTS: The results revealed that immediately after 10 min of MR, the EG exhibited an increase in the oxyhemoglobin level of the medium fibers of the TM (0.72 ± 1.47 vs. -0.14 ± 1.33 mmol/dL, p = 0.01). In addition, functional disability (CG: 5.48 ± 5.58%/EG: 21.12 ± 7.73%) and neck pain were measured using the neck disability index. The pain pressure threshold (CG: 70.49 ± 32.29 kgf/EG: 51.08 ± 27.65 kgf) and pain intensity (CG: 0.76 ± 1.56/EG: 3.28 ± 2.35) were also measured. CONCLUSION: The findings indicate that application of MR for 10 min increases the tissue oxyhemoglobin level in the TM of the group with NNP compared to the CG without NNP.

Performance of a cutinase membrane reactor for the production of biodiesel in organic media.

The enzymatic transesterification of oils with an alcohol, using recombinant cutinase of Fusarium solani pisi microencapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane reversed micelles, was performed in a membrane bioreactor (MBR). A tubular ceramic membrane with a nominal molecular weight cut off of 15,000 Da was used to retain the enzyme, and characterized in terms of rejection coefficients of the reaction components by transmission experiments. The performance of the MBR in a total recirculation-batch mode was compared with results obtained in a stirred batch tank reactor. The continuous operation of the MBR was also evaluated and the influence of the alcohol type and permeate flow rate on conversion degree and productivity (up to 500 g(product) /day/g(enzyme) was attained) were analyzed. Cutinase wild type and mutant T179C were tested for this process and the high long-term operational stability of the cutinase mutant demonstrated its potential as biocatalyst for the enzymatic continuous production of biodiesel.

Kinetics and mechanism of the cutinase-catalyzed transesterification of oils in AOT reversed micellar system.

The kinetics of the enzymatic transesterification between a mixture of triglycerides (oils) and methanol for biodiesel production in a bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane reversed micellar system, using recombinant cutinase from Fusarium solani pisi as a catalyst, was investigated. In order to describe the results that were obtained, a mechanistic scheme was proposed, based on the literature and on the experimental data. This scheme includes the following reaction steps: the formation of the active enzyme-substrate complex, the addition of an alcohol molecule to the complex followed by the separation of a molecule of the fatty acid alkyl ester and a glycerol moiety, and release of the active enzyme. Enzyme inhibition and deactivation effects due to methanol and glycerol were incorporated in the model. This kinetic model was fitted to the concentration profiles of the fatty acid methyl esters (the components of biodiesel), tri-, di- and monoglycerides, obtained for a 24 h transesterification reaction performed in a stirred batch reactor under different reaction conditions of enzyme and initial substrates concentration.

Germ band retraction as a landmark in glucose metabolism during Aedes aegypti embryogenesis.

BACKGROUND: The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown. RESULTS: Glucose metabolism was investigated throughout Aedes aegypti (Diptera) embryonic development. Both cellular blastoderm formation (CBf, 5 h after egg laying - HAE) and germ band retraction (GBr, 24 HAE) may be considered landmarks regarding glucose 6-phosphate (G6P) destination. We observed high levels of glucose 6-phosphate dehydrogenase (G6PDH) activity at the very beginning of embryogenesis, which nevertheless decreased up to 5 HAE. This activity is correlated with the need for nucleotide precursors generated by the pentose phosphate pathway (PPP), of which G6PDH is the key enzyme. We suggest the synchronism of egg metabolism with carbohydrate distribution based on the decreasing levels of phosphoenolpyruvate carboxykinase (PEPCK) activity and on the elevation observed in protein content up to 24 HAE. Concomitantly, increasing levels of hexokinase (HK) and pyruvate kinase (PK) activity were observed, and PEPCK reached a peak around 48 HAE. Glycogen synthase kinase (GSK3) activity was also monitored and shown to be inversely correlated with glycogen distribution during embryogenesis. CONCLUSIONS: The results herein support the hypothesis that glucose metabolic fate changes according to developmental embryonic stages. Germ band retraction is a moment that was characterized as a landmark in glucose metabolism during Aedes aegypti embryogenesis. Furthermore, the results also suggest a role for GSK3 in glycogen balance/distribution during morphological modifications.

Transesterification of oil mixtures catalyzed by microencapsulated cutinase in reversed micelles.

Recombinant cutinase from Fusarium solani pisi was used to catalyze the transesterification reaction between a mixture of triglycerides (oils) and methanol in reversed micelles of bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane for the purposes of producing biodiesel. The use of a bi-phase lipase-catalyzed system brings advantages in terms of catalyst re-use and the control of water activity in the medium and around the enzyme micro-environment. Small-scale batch studies were performed to study the influence of the initial enzyme and alcohol concentrations, and the substrates molar ratio. Conversions in excess of 75 were obtained with reaction times under 24 h, which makes this enzymatic process highly competitive when compared to similar lipase catalyzed reactions for biodiesel production using methanol.

Comparative larvicidal activity of essential oils from three medicinal plants against Aedes aegypti L.

This work aimed at comparing larvicidal activity of essential oils extracted from the dried leaves of Alpinia speciosa, Cymbopogon citratus, and Rosmarinus officinalis against Ae. aegypti larvae. The larvae were observed for 4 h and at 24 h according to a completely randomized design with three replications and the following concentrations [µl/ml]: 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, and controls were distilled water, and commercial standard citral, camphor, eucalyptol, α-pinene, and ß-myrcene. The essential oil of C. citratus had the lowest LC50 (0.28) and LC90 (0.56) values, followed by that of A. speciosa (0.94 and 1.2, resp.) and of R. officinalis (1.18 and 1.67, resp.), and only the commercial standards citral and α-pinene were larvicidal.

Neem oil increases the persistence of the entomopathogenic fungus Metarhizium anisopliae for the control of Aedes aegypti (Diptera: Culicidae) larvae.

BACKGROUND: The entomopathogenic fungus Metarhizium anisopliae is a candidate for the integrated management of the disease vector mosquito Aedes aegypti. Metarhizium anisopliae is pathogenic and virulent against Ae. aegypti larvae; however, its half-life is short without employing adjuvants. Here, we investigated the use of neem oil to increase virulence and persistence of the fungus under laboratory and simulated field conditions. METHODS: Neem was mixed with M. anisopliae and added to recipients. Larvae were then placed in recipients at 5-day intervals for up to 50 days. Survival rates were evaluated 7 days after exposing larvae to each treatment. The effect of neem on conidial germination following exposure to ultraviolet radiation was evaluated under laboratory conditions. Statistical tests were carried out using ANOVA and regression analysis. RESULTS: Laboratory bioassays showed that the fungus alone reduced survival to 30% when larvae were exposed to the treatment as soon as the suspension had been prepared (time zero). A mixture of fungus + neem resulted in 11% survival at time zero. The combination of fungus + neem significantly reduced larval survival rates even when suspensions had been maintained for up to 45 days before adding larvae. For simulated-field experiments 1% neem was used, even though this concentration is insecticidal, resulting in 20% survival at time zero. However, this toxic effect was reduced over time. When used alone under simulated-field conditions the fungus rapidly lost virulence. The formulation fungus + neem effectively maintained fungal virulence, with larval survival rates significantly reduced for up to 45 days after preparation of the suspensions. The effective half-life of the fungus or neem when used separately was 6 and 13 days, respectively. The half-life of fungus formulated in 1% neem was 34 days. Conidia suspended in neem maintained high levels of germination even following a 2-h exposure to ultraviolet radiation. CONCLUSIONS: A combination of the entomopathogenic fungus M. anisopliae with neem oil effectively increases the half-life and virulence of the fungus when tested against Ae. aegypti larvae, even under simulated field conditions. Neem oil also protected the fungus from the damaging effects of ultraviolet radiation.

Synthesis and bactericide activity of nanofiltration composite membranes - Cellulose acetate/silver nanoparticles and cellulose acetate/silver ion exchanged zeolites.

The present work addresses the synthesis of nanofiltration composite membranes with bactericide properties. The cellulose acetate based membranes with polyvinylpyrrolidone coated silver nanoparticles, silver ion-exchanged ß-zeolite and ß-zeolite are casted by the phase inversion technique and subjected to an annealing post-treatment. They are characterized in terms of the nanofiltration permeation performance and antibacterial properties. The incorporation of silver nanoparticles produces a threefold increase in the membrane hydraulic permeability when compared to the silver-free membranes and the incorporation of silver ion loaded zeolite resulted in a 56.3% increase in hydraulic permeability. In contrast to the influence of silver presence, either in nanometric or in the ionic form, the presence of zeolite does not significantly influence the hydraulic permeability. The rejection coefficients to salts range from 83% to 93% for the silver ion-exchanged zeolite membrane and from 84% to 97% for the polyvinylpyrrolidone coated silver nanoparticles membrane. They are higher for sulfate salts than for chloride salts. The antibacterial properties of the membranes were evaluated against Escherichia coli. The results have shown that the silver ion-exchanged ß-zeolite membrane was effective in inactivating Escherichia coli after just 210 min of contact time. No bacterial activity was detected following 24 h of contact time with the membrane containing polyvinylpyrrolidone coated silver nanoparticles. A reduction of more than 6-log, in the number of Escherichia coli, was achieved for both membranes. The different patterns of bactericide activity are associated to the silver speciation in metallic or ionic form. The high flux nanofiltration composite membranes with bactericidal properties represent a strong asset in water treatment biofouling control.

Hypometabolic strategy and glucose metabolism maintenance of Aedes aegypti egg desiccation.

The mosquito Aedes aegypti is vector of several viruses including yellow fever virus, dengue virus chikungunya virus and Zika virus. One of the major problems involving these diseases transmission is that A. aegypti embryos are resistant to desiccation at the end of embryogenesis, surviving and remaining viable for several months inside the egg. Therefore, a fine metabolism control is essential to support these organisms throughout this period of resistance. The carbohydrate metabolism has been shown to be of great importance during arthropod embryogenesis, changing dramatically in order to promote growth and differentiation and in periods of resistance. This study investigated fundamental aspects of glucose metabolism in three stages of A. aegypti egg development: pre-desiccated, desiccated, and rehydrated. The activities of regulatory enzymes in carbohydrate metabolism such as pyruvate kinase, hexokinase and glucose 6-phosphate dehydrogenase were evaluated. We show that these activities were reduced in A. aegypti desiccated eggs, suggesting a decreased activity of glycolytic and pentose phosphate pathway. In contrast, gluconeogenesis increased in desiccated eggs, which uses protein as substrate to synthesize glucose. Accordingly, protein amount decreased during this stage, while glucose levels increased. Glycogen content, a major carbohydrate reserve in mosquitoes, was evaluated and shown to be lower in desiccated and rehydrated eggs, indicating it was used to supply energy metabolism. We observed a reactivation of carbohydrate catabolism and an increased gluconeogenesis after rehydration, suggesting that controlling glucose metabolism was essential not only to survive the period of desiccation, but also for subsequent larvae hatch. Taken together, these results contribute to a better understanding of metabolism regulation in A. aegypti eggs during desiccation periods. Such regulatory mechanisms enable higher survival rate and consequently promote virus transmission by these important disease vectors, making them interesting subjects in the search for novel control methods.

Impact of Bariatric Surgery on the Pharmacokinetics Parameters of Amoxicillin.

BACKGROUND: Bariatric surgery leads to several anatomo-physiological modifications that may affect pharmacokinetic parameters and consequently alter the therapeutic effect of drugs, such as antibiotics. The pharmacokinetics of oral amoxicillin after Roux-en-Y gastric bypass (RYGB) surgery is unknown. OBJECTIVES: The objective of this study was to evaluate the impact of bariatric surgery on the pharmacokinetics of amoxicillin. METHODS: This study was performed as a randomized, open-label, single-dose clinical trial, with two periods of treatment, in which obese subjects (n = 8) received an amoxicillin 500 mg capsule orally before and 2 months after the RYGB surgery. The amoxicillin plasma concentration was determined by liquid chromatography coupled to mass spectrometry (LC-MS/MS). RESULTS: After the surgery, the mean weight loss was 17.03 ± 5.51 kg, and mean body mass index (BMI) decreased from 46.21 ± 2.82 to 38.82 ± 3.32 kg/m2. The mean amoxicillin area under the plasma concentration versus time curve from time zero to the time of the last quantifiable concentration (AUC0-tlast) increased significantly (3.5-fold); the maximum plasma concentration (Cmax) increased 2.8-fold after the bariatric surgery. No correlation was found between amoxicillin absorption, BMI, and weight loss percentage. CONCLUSION: The alterations observed in the amoxicillin pharmacokinetics suggest that obese subjects included in this trial had a substantially increase in amoxicillin systemic exposure after RYGB surgery. However, despite this increase, its exposure was lower than the values reported for non-obese volunteers. TRIAL REGISTRATION: Identifiers: NCT03588273.

Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length.

In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.

New phenyl-nickel complexes of bulky 2-iminopyrrolyl chelates: synthesis, characterisation and application as aluminium-free catalysts for the production of hyperbranched polyethylene.

A family of mono(2-iminopyrrolyl) complexes with the general formula [Ni{κ2N,N'-5-(aryl)-NC4H2-2-C(H)[double bond, length as m-dash]N-2,6-(aryl)}(C6H5)(PPh3)] were obtained from the reaction of the sodium salts of the newly synthesised 5-aryl-2-(N-arylformimino)pyrroles with the square planar complex trans-[Ni(C6H5)(PPh3)2Cl]. These new iminopyrrole ligand precursors, designed with increasing bulkiness and different electronic properties, and their corresponding nickel(ii) complexes were characterised by NMR spectroscopy and elemental analysis, and their structural features were analysed by single crystal X-ray diffraction. The nickel complexes were tested as aluminium-free catalysts for the polymerisation of ethylene, at low to moderate pressures and different temperatures and in the absence or presence of the phosphine scavenger [Ni(COD)2], giving rise to catalytic activities in the range of 3.61-73.12 kgPE molNi-1 h-1 bar-1. The polyethylene products formed in these catalytic reactions were characterised by GPC/SEC and NMR spectroscopy. Generally, low molecular weight (Mn 510-1300 g mol-1) low viscosity oils were obtained, presenting high branching degrees (80-125 branches per 1000 C atoms), which are characteristic of hyperbranched polyethylene products. In particular, polymerisation reactions using catalyst 7 led to higher viscosity oils with molecular weights between 11 000 and 20 000 g mol-1, and branching degrees of 100-120 branches per 1000 C atoms.

Functional characterization of a serine protease inhibitor modulated in the infection of the Aedes aegypti with dengue virus.

During feeding with blood meal, female Aedes aegypti can transmit infectious agents, such as dengue, yellow fever, chikungunya and Zika viruses. Dengue virus causes human mortality in tropical regions of the world, and there is no specific treatment or vaccine with maximum efficiency being used for these infections. In the vector-virus interaction, the production of several molecules is modulated by both mosquitoes and invading agents. However, little information is available about these molecules in the Ae. aegypti mosquito during dengue infection. Inhibitors of the pacifastin family have been described to participate in the immune response of insects and Pac2 is the only gene of this family present in Ae. aegypti being then chosen for investigation. Pac2 was expressed in E. coli, purified and analyzed by mass spectrometry and SDS-PAGE. The Pac2 transcript was detected by qPCR, and its protein levels were assessed by Western blotting. The inhibitory activity of Pac2 was measured using its Ki, IC50 and zymography. Mosquito infections with DENV were introduced with the Brazilian ACS-46 DENV-2 strain propagated in C6/36 cells. In the present work, we showed that it is possibly involved in the interaction of the mosquitoes with the dengue virus. The Pac2 transcript was detected in larvae and in both the salivary gland and midgut of Ae. aegypti females, while the native protein was identified in females 3 h post-blood meal. Pac2 is a strong inhibitor of trypsin-like and thrombin-like proteases, which are present in 4th instar larvae midgut and females 24 h after blood meal. During DENV infection, up regulation of Pac2 expression occurs in the salivary gland and midgut. Pac2 is the first Pacifastin inhibitor member described in mosquitoes. Our results suggest that Pac2 acts on mosquito serine proteases, mainly the trypsin-like type, and is under transcriptional control by virus infection signals to allow its survival in the vector or by the mosquito as a defense mechanism against virus infection.

Induction of actin gene expression in the mosquito midgut by blood ingestion correlates with striking changes of cell shape.

Ingestion of a blood meal by the female mosquito Anopheles gambiae (L., Diptera: Culicidae), results in a dramatic distention of the midgut epithelium. Here, we report that these events correlate with a transient increase of actin mRNA and protein abundance. The newly synthesized actin may provide a pool of actin protein needed to remodel epithelial cell cytoarchitecture. We also document changes in midgut epithelial cell morphology. Upon blood ingestion, the columnar cells flatten accompanied by the loss of microvilli on the lumenal side and the unfolding of the labyrinth on the basal side. These changes correlate with the large increase of epithelial surface area needed to accommodate the blood meal. Actin gene expression, actin synthesis and cell morphology all return to the pre-feeding state by 24 h after blood intake.

Effects of bariatric surgery on night eating and depressive symptoms: a prospective study.

BACKGROUND: Night eating syndrome (NES) is characterized by a delayed pattern of food intake and is clinically defined by the presence of evening hyperphagia, nocturnal ingestion, morning anorexia, and sleep problems. Although most studies report an association of obesity, depressive mood, and NES, very little is known about the impact of bariatric surgery in the course of this syndrome, particularly in patients with depressive symptoms. OBJECTIVES: To assess the effects of bariatric surgery on night eating and depressive symptoms. SETTING: Tertiary hospital, Brazil. METHODS: Sixty patients consecutively submitted to bariatric surgery were included in the study. Baseline depressive symptoms were assessed by the Beck Depression Inventory-Short Form (BDI-SF) and night eating by the Night Eating Questionnaire. Measurements were repeated postoperatively for comparison. RESULTS: Participants were predominantly female (M/F = 9/51). Mean (±SD) age was 34.7±9.2 years and body mass index was 46.04±7.52 kg/m². On average, BDI-SF scores improved after bariatric surgery (9.77±7.01 versus 4.70±4.60; P = .001). Patients with baseline depressive symptoms (BDI-SF>4), in contrast to those without, showed a significant improvement in Night Eating Questionnaire scores (16.03±7.73 versus 12.80±7.76; P = .01), despite a similar postoperative reduction in weight. CONCLUSION: Bariatric surgery is associated with a reduction in depressive symptoms. After the surgical intervention, improvement of night eating can be observed predominantly in patients with preoperative depressive symptoms. These findings are consistent with the hypothesis of an important role of mood problems in NES.