Extracellular vesicles released by Trypanosoma evansi: induction analysis and proteomics.

Trypanosoma evansi is a unicellular protozoan responsible for causing a disease known as "surra," which is found in different regions of the world and primarily affects horses and camels. Few information is known about virulence factors released from the parasite within the animals. The organism can secrete extracellular vesicles (EVs), which transport a variety of molecules, including proteins. Before being considered exclusively as a means for eliminating unwanted substances, extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating interactions between cells, host cells, and parasites, and even between parasites themselves. Thus, they may be used as potential biomarkers. This study aimed to assess the induction of EVs production by Ca+2, conduct a proteomic analysis of the EVs released by T. evansi, and identify epitopes that could serve as biomarkers. The findings indicated that Ca+2 is not an effective promoter of vesiculation in T. evansi. Furthermore, the proteomic analysis has identified multiple proteins that have been investigated as biomarkers or vaccine antigens, previously. A total of 442 proteins were identified, with 7 of them specifically recognizing 9 epitopes that are unique to T. evansi. At least one of these epitopes of TevSTIB805.9.11580 have been previously identified, which increases the possibility of further investigating its potential as a biomarker.

Comparative study of three novel ion exchange resins with DEAE-cellulose for the purification of Trypanosoma evansi.

Ion exchange chromatography is a method that uses the different surface charges of trypanosomes and blood cells to separate them. This makes it possible to use molecular and immunological methods to diagnose or study these protozoans. DEAE-cellulose resin is commonly used to perform this method. The goal of this study was to compare three novel chromatographic resins designated as PURIFICA™ (Y-C2N®, Y-HONOH®, and Y-CNC3®). The resins were evaluated based on their ability to isolate the parasite, purification time, examination of parasite viability and morphology, and trypanosome recovery potential after passing through the columns. In terms of the evaluated parameters, there was no significant difference between DEAE-cellulose and the three tested resins in most experiments. However, PURIFICA™ (Y-C2N®, Y-HONOH®, and Y-CNC3®) resins are less expensive and easier to prepare than DEAE-Cellulose, making them an alternative for the purification of Trypanosoma evansi.

Temporal spread and evolution of SARS-CoV-2 in the second pandemic wave in Brazil.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic spread rapidly and this scenario is concerning in South America, mainly in Brazil that presented more than 21 million coronavirus disease 2019 cases and 590 000 deaths. The recent emergence of novel lineages carrying several mutations in the spike protein has raised additional public health concerns worldwide. The present study describes the temporal spreading and evolution of SARS-CoV2 in the beginning of the second pandemic wave in Brazil, highlighting the fast dissemination of the two major concerning variants (P.1 and P.2). A total of 2507 SARS-CoV-2 whole-genome sequences (WGSs) with available information from the country (Brazil) and sampling date (July 2020-February 2021), were obtained and the frequencies of the lineages were evaluated in the period of the growing second pandemic wave. The results demonstrated the increasing prevalence of P.1 and P.2 lineages in the period evaluated. P.2 lineage was first detected in the middle of 2020, but a high increase occurred only in the last trimester of this same year and the spreading to all Brazilian regions. P.1 lineage emerged even later, first in the North region in December 2020 and really fast dissemination to all other Brazilian regions in January and February 2021. All SARS-CoV-2 WGSs of P.1 and P.2 were further separately evaluated with a Bayesian approach. The rates of nucleotide and amino acid substitutions were statistically higher in P.1 than P.2 (p < 0.01). The phylodynamic analysis demonstrated that P.2 gradually spread in all the country from September 2020 to January 2021, while P.1 disseminated even faster from December 2020 to February 2021. Skyline plots of both lineages demonstrated a slight rise in the spreading for P.2 and exponential growth for P.1. In conclusion, these data demonstrated that the P.1 (recently renamed as Gamma) and P.2 lineages have predominated in the second pandemic wave due to the very high spreading across all geographic regions in Brazil at the end of 2020 and beginning of 2021.

Neuroinflammation in the NTS is associated with changes in cardiovascular reflexes during systemic inflammation.

BACKGROUND: Lipopolysaccharide (LPS)-induced systemic inflammation (SI) is associated with neuroinflammation in the brain, hypotension, tachycardia, and multiple organs dysfunctions. Considering that during SI these important cardiovascular and inflammatory changes take place, we measured the sensitivity of the cardiovascular reflexes baroreflex, chemoreflex, and Bezold-Jarisch that are key regulators of hemodynamic function. We also evaluated neuroinflammation in the nucleus tractus solitarius (NTS), the first synaptic station that integrates peripheral signals arising from the cardiovascular and inflammatory status. METHODS: We combined cardiovascular recordings, immunofluorescence, and assays of inflammatory markers in male Wistar rats that receive iv administration of LPS (1.5 or 2.5 mg kg-1) to investigate putative interactions of the neuroinflammation in the NTS and in the anteroventral preoptic region of the hypothalamus (AVPO) with the short-term regulation of blood pressure and heart rate. RESULTS: LPS induced hypotension, tachycardia, autonomic disbalance, hypothermia followed by fever, and reduction in spontaneous baroreflex gain. On the other hand, during SI, the bradycardic component of Bezold-Jarisch and chemoreflex activation was increased. These changes were associated with a higher number of activated microglia and interleukin (IL)-1ß levels in the NTS. CONCLUSIONS: The present data are consistent with the notion that during SI and neuroinflammation in the NTS, rats have a reduced baroreflex gain, combined with an enhancement of the bradycardic component of Bezold-Jarisch and chemoreflex despite the important cardiovascular impairments (hypotension and tachycardia). These changes in the cardiac component of Bezold-Jarisch and chemoreflex may be beneficial during SI and indicate that the improvement of theses reflexes responsiveness though specific nerve stimulations may be useful in the management of sepsis.

Sex differences and the role of ovarian hormones in site-specific nociception of SHR.

Accurate diagnosis and treatment of pain is dependent on knowledge of the variables that might alter this response. Some of these variables are the locality of the noxious stimulus, the sex of the individual, and the presence of chronic diseases. Among these chronic diseases, hypertension is considered a serious and silent disease that has been associated with hypoalgesia. The main goal of this study was to evaluate the potential nociceptive differences in spontaneously hypertensive rats (SHR) regarding the locality of the stimulus, i.e., the temporomandibular joint or paw, the sex, and the role of ovarian hormones in a model of mechanical nociception (Von Frey test) or formalin-induced inflammatory nociception. Our results indicate that SHR had lower orofacial mechanical nociception beyond the lower mechanical nociception in the paw compared with WKY rats. In a model of formalin-induced inflammatory nociception, SHR also had decreased nociception compared with normotensive rats. We also sought to evaluate the influence of sex and ovarian hormones on orofacial mechanical nociception in SHR. We observed that female SHR had higher mechanical nociception than male SHR only in the paw, but it had higher formalin-induced orofacial nociception than male SHR. Moreover, the absence of ovarian hormones caused an increase in mean arterial pressure and a decrease in paw nociception in female SHR.

Central serotonin prevents hypotension and hypothermia and reduces plasma and spleen cytokine levels during systemic inflammation.

An exceptionally high mortality rate is observed in sepsis and septic shock. Systemic administration of lipopolysaccharide (LPS) has been used as an experimental model for sepsis resulting in an exacerbated immune response, brain neurochemistry adjustments, hypotension, and hypothermia followed by fever. Central serotonergic pathways not only modulate systemic inflammation (SI) but also are affected by SI, including in the anteroventral region of the hypothalamus (AVPO), which is the hierarchically most important region for body temperature (Tb) control. In this study, we sought to determine if central serotonin (5-HT) plays a role in SI induced by intravenous administration of LPS (1.5 mg/kg) in male Wistar rats (280-350 g) by assessing 5-HT levels in the AVPO, mean arterial pressure, heart rate, and Tb up to 300 min after LPS administration, as well as assessing plasma and spleen cytokine levels, nitric oxide (NO) plasma levels, and prostaglandin (PG) E2 levels in the AVPO at 75 min and 300 min after LPS administration. We observed reduced AVPO 5-HT levels, hypotension, tachycardia, hypothermia followed by fever, as well as observing increased plasma NO, plasma and spleen cytokines and AVPO PGE2 levels in SI. Intracerebroventricular (icv) administration of 5-HT 30 min before LPS administration prevented hypotension and hypothermia, which were accompanied by reduced plasma NO, as well as plasma TNF-α, IL-1ß, IL-6, and IL-10 and spleen TNF-α and IL-10 levels. We suggest that SI reduced 5-HT levels in the AVPO favor an increased pro-inflammatory status both centrally and peripherally that converge to hypotension and hypothermia. Moreover, our results are consistent with the notion that exogenous 5-HT given icv prevents hypotension and hypothermia probably activating the splenic anti-inflammatory pathway.

Molecular hydrogen potentiates hypothermia and prevents hypotension and fever in LPS-induced systemic inflammation.

Molecular hydrogen (H2) exerts anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Here we tested the hypothesis that H2 modulates cardiovascular, inflammatory, and thermoregulatory changes in systemic inflammation (SI) induced by lipopolysaccharide (LPS) at different doses (0.1 or 1.5 mg/kg, intravenously, to induce mild or severe SI) in male Wistar rats (250-300 g). LPS or saline was injected immediately before the beginning of 360-minute inhalation of H2 (2% H2, 21% O2, balanced with nitrogen) or room air (21% O2, balanced with nitrogen). Deep body temperature (Tb) was measured by dataloggers pre-implanted in the peritoneal cavity. H2 caused no change in cardiovascular, inflammatory parameters, and Tb of control rats (treated with saline). During mild SI, H2 reduced plasma surges of proinflammatory cytokines (TNF-α and IL-6) while caused an increase in plasma IL-10 (anti-inflammatory cytokine) and prevented fever. During severe SI, H2 potentiated hypothermia, and prevented fever and hypotension, which coincided with reduced plasma nitric oxide (NO) production. Moreover, H2 caused a reduction in surges of proinflammatory cytokines (plasma TNF-α and IL-1ß) and prostaglandin E2 [(PGE2), in plasma and hypothalamus], and an increase in plasma IL-10. These data are consistent with the notion that H2 blunts fever in mild SI, and during severe SI potentiates hypothermia, prevents hypotension reducing plasma NO production, and exerts anti-inflammatory effects strong enough to prevent fever by altering febrigenic signaling and ultimately down-modulating hypothalamic PGE2 production.

Effect of green juice and their bioactive compounds on genotoxicity induced by alkylating agents in mice.

Kale juice (Brassica oleracea L. var. acephala D.C.) is a reliable source of dietary carotenoids and typically contains the highest concentrations of lutein (LT) and beta-carotene (BC) among green leafy vegetables. As a result of their antioxidant properties, dietary carotenoids are postulated to decrease the risk of disease occurrence, particularly certain cancers. The present study aimed to (1) examine the genotoxic and antigenotoxic activity of natural and commercially available juices derived from Brassica oleracea and (2) assess influence of LT or BC against DNA damage induced by alkylating agents such as methyl methanesulfonate (MS) or cyclophosphamide (CP) in vivo in mice. Male Swiss mice were divided into groups of 6 animals, which were treated with water, natural, or commercial Brassica oleraceae juices (kale), LT, BC, MMS, or CP. After treatment, DNA damage was determined in peripheral blood lymphocytes using the comet assay. Results demonstrated that none of the Brassica oleraceae juices or carotenoids produced genotoxic effects. In all examined cell types, kale juices or carotenoids inhibited DNA damage induced by MMS or CP administered either pre- or posttreatment by 50 and 20%, respectively. Under our experimental conditions, kale leaf juices alone exerted no marked genotoxic or clastogenic effects. However, a significant decrease in DNA damage induced by MMS or CP was noted. This effect was most pronounced in groups that received juices, rather than carotenoids, suggesting that the synergy among constituents present in the food matrix may be more beneficial than the action of single compounds. Data suggest that the antigenotoxic properties of kale juices may be of therapeutic importance.

Elemental composition of vegetables cultivated over coal-mining waste.

We assessed elemental composition of the liver in mice subjected to one-time or chronic consumption of the juice of vegetables cultivated in a vegetable garden built over deposits of coal waste. Lactuca sativa L. (lettuce), Beta vulgaris L. (beet), Brassica oleracea L. var. italica (broccoli) and Brassica oleracea L. var. acephala (kale) were collected from the coal-mining area and from a certified organic farm (control). Elemental composition was analyzed by particle-induced X-ray emission (PIXE) method. Concentrations of Mg, S, and Ca of mice subjected to one-time consumption of broccoli and concentrations of these same elements plus Si of mice receiving kale were higher in the coal-mining area. Concentrations of P, K, and Cu were increase after chronic consumption of lettuce from the coal-mining area, whereas the levels of Si, P, K, Fe, and Zn were higher in the group consuming kale from the coal-mining area. Our data suggests that people consuming vegetables grown over coal wastes may ingest significant amounts of chemical elements that pose a risk to health, since these plants contain both essential and toxic metals in a wide range of concentrations, which can do more harm than good.

L-tyrosine induces DNA damage in brain and blood of rats.

Mutations in the tyrosine aminotransferase gene have been identified to cause tyrosinemia type II which is inherited in an autosomal recessive manner. Studies have demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. Considering that the L-tyrosine may promote oxidative stress, the main objective of this study was to investigate the in vivo effects of L-tyrosine on DNA damage determined by the alkaline comet assay, in brain and blood of rats. In our acute protocol, Wistar rats (30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. For chronic administration, the animals received two subcutaneous injections of L-tyrosine (500 mg/kg, 12-h intervals) or saline administered for 24 days starting at postnatal day (PD) 7 (last injection at PD 31), 12 h after the last injection, the animals were killed by decapitation. We observed that acute administration of L-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood when compared to control group. Moreover, we observed that chronic administration of L-tyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood when compared to control group. In conclusion, the present work demonstrated that DNA damage can be encountered in brain from animal models of hypertyrosinemia, DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder and to reinforce the role of oxidative stress in the pathophysiology of tyrosinemia type II.

Electronic von Frey associated with the Mouse Grimace Scale to Assess Allodynia and Pain in Trypanosoma evansi-Infected Mice.

Infectious disease pathogenesis is still a complex field to study. The course of several clinical signs, such as allodynia and pain, may be observed in domestic animals. However, the knowledge of their pathways and correct treatment need controlled experiments, many of them using laboratory animals. Measuring changes in mechanical thresholds of the hind paw and viscera is a useful technique to observe changes in pain perception in rodents. Withdrawal response can be measured first in baseline tests, which creates better control of experimental groups. Subsequent tests can be performed after inducing infection and adding drugs to the protocol. The use of an electronic von Frey apparatus associated with the use of a facial scale to observe pain-like changes allows a simple, precise, and consistent assessment to evaluate allodynia and pain in mice. Thus, experiments using the present methodology for Trypanosoma evansi infection represent a useful method to evaluate allodynia and pain in laboratory-infected animals, which can be applied to the conventional treatment for livestock animals.

Reliability of roadside oral fluid testing devices for ∆9 -tetrahydrocannabinol (∆9 -THC) detection.

Driving under the influence of cannabis (DUIC) is increasing worldwide, and cannabis is the most prevalent drug after alcohol in impaired driving cases, emphasizing the need for a reliable traffic enforcement strategy. ∆9 -tetrahydrocannabinol (THC) detection in oral fluid has great potential for identifying recent cannabis use; however, additional data are needed on the sensitivities, specificities, and efficiencies of different oral fluid devices for detecting cannabinoids at the roadside by police during routine traffic safety enforcement efforts. At the roadside, 8945 oral fluid THC screening tests were performed with four devices: AquilaScan®, Dräger DrugTest®, WipeAlyser Reader®, and Druglizer®. A total of 530 samples screened positive for THC (5.9%) and were analyzed by liquid chromatography-tandem mass spectrometry at multiple cutoff concentrations (2 ng/mL, 10 ng/mL, and manufacturers' recommended device cutoffs) to investigate device performance. Results varied substantially, with sensitivities of 0%-96.8%, specificities of 89.8%-98.5%, and efficiencies of 84.3%-97.8%. The Dräger DrugTest® outperformed the other devices with a 96.8% sensitivity, 97.1% specificity, and 97.0% efficiency at a 5-ng/mL LC-MS/MS confirmation cutoff. The WipeAlyser Reader® had good performance with a 91.4% sensitivity, 97.2% specificity, and 96.4% efficiency. AquilaScan® and Druglizer® had unacceptable performance for cannabinoid detection, highlighted by sensitivity <13%. The choice of roadside oral fluid testing device must offer good analytical performance for cannabinoids because of its high prevalence of use and impact on road safety.

Moderate-intensity aerobic exercise training improves CD8+ tumor-infiltrating lymphocytes effector function by reducing mitochondrial loss.

Aerobic exercise training (AET) has emerged as a strategy to reduce cancer mortality, however, the mechanisms explaining AET on tumor development remain unclear. Tumors escape immune detection by generating immunosuppressive microenvironments and impaired T cell function, which is associated with T cell mitochondrial loss. AET improves mitochondrial content and function, thus we tested whether AET would modulate mitochondrial metabolism in tumor-infiltrating lymphocytes (TIL). Balb/c mice were subjected to a treadmill AET protocol prior to CT26 colon carcinoma cells injection and until tumor harvest. Tissue hypoxia, TIL infiltration and effector function, and mitochondrial content, morphology and function were evaluated. AET reduced tumor growth, improved survival, and decreased tumor hypoxia. An increased CD8+ TIL infiltration, IFN-γ and ATP production promoted by AET was correlated with reduced mitochondrial loss in these cells. Collectively, AET decreases tumor growth partially by increasing CD8+ TIL effector function through an improvement in their mitochondrial content and function.

Effects of acute and chronic administration of fenproporex on DNA damage parameters in young and adult rats.

Obesity is a chronic and multifactorial disease, whose prevalence is increasing in many countries. Pharmaceutical strategies for the treatment of obesity include drugs that regulate food intake, thermogenesis, fat absorption, and fat metabolism. Fenproporex is the second most commonly consumed amphetamine-based anorectic worldwide; this drug is rapidly converted in vivo into amphetamine, which is associated with neurotoxicity. In this context, the present study evaluated DNA damage parameters in the peripheral blood of young and adult rats submitted to an acute administration and chronic administration of fenproporex. In the acute administration, both young and adult rats received a single injection of fenproporex (6.25, 12.5 or 25 mg/kg i.p.) or vehicle. In the chronic administration, both young and adult rats received one daily injection of fenproporex (6.25, 12.5, or 25 mg/kg i.p.) or Tween for 14 days. 2 h after the last injection, the rats were killed by decapitation and their peripheral blood removed for evaluation of DNA damage parameters by alkaline comet assay. Our study showed that acute administration of fenproporex in young and adult rats presented higher levels of damage index and frequency in the DNA. However, chronic administration of fenproporex in young and adult rats did not alter the levels of DNA damage in both parameters of comet assay. The present findings showed that acute administration of fenproporex promoted damage in DNA, in both young and adult rats. Our results are consistent with other reports which showed that other amphetamine-derived drugs also caused DNA damage. We suggest that the activation of an efficient DNA repair mechanism may occur after chronic exposition to fenproporex. Our results are consistent with other reports that showed some amphetamine-derived drugs also caused DNA damage.

A multi-analyte LC-MS/MS method for the determination of 57 pharmaceuticals and illicit drugs in plasma, and its application to poisoning cases.

The diagnostic methods in an emergency scenario must be simple, fast, and efficient to provide an effectiveness and efficient treatment, thus reducing the consequences of exposure. Considering the sample analysis, the protein precipitation combined with LC-MS/MS has been shown to be a good strategy for the simultaneous determination of compounds of toxicological interest, such as medicines and drugs of abuse. In this study, a rapid and simple multi-analyte method was developed and validated for the quantification of 57 pharmaceuticals and illicit drugs in plasma samples. Sample pre-treatment consists of protein precipitation of 50 µL of the sample with 240 µL of organic solvent mixture (MeOH:ACN, 3:1, v/v), centrifugation, and injection into the LC-MS/MS, with a chromatographic run time of 7 min. The method was validated considering lower limit of quantification (LLOQ), interferences, linearity, precision, accuracy, dilution integrity, carryover, and matrix effect. The LLOQs ranged from 5 to 20 ng/mL and all analytes were linear (r2>0.99) in the tested concentration ranges. The method proved to be precise and accurate, presenting QC concentrations for all analytes within acceptable limits by the guideline used (CV % ≤20 % and bias ± 20 %). The developed method was successfully applied in 470 plasma samples of real cases of poisoning. A total of 80 % of the samples were positive for at least one substance, with acetaminophen (32.1 %), diazepam (25.1 %), and lidocaine (18.9 %) being the most detected. The most prevalent exposure circumstance among the cases was suicide attempt. The most frequent age groups were young adults between 20 and 29 years old and children under 5 years old. The methodology developed proved to be efficient in the simultaneous determination of 57 substances of toxicological interest, contributing to a correct diagnosis and, consequently, to the most appropriate management and treatment of the intoxicated patient. Furthermore, it is possible to observe the most commonly involved toxic agents in the Rio Grande do Sul, southern Brazil, helping to trace a profile of the poisoning patient, important in toxicovigilance actions.

A new approach with dried plasma spots: a rapid determination of antidepressants and antipsychotics by fast GC-MS.

Background: The increasing prevalence of poisoning cases related to antidepressants and antipsychotics has raised concerns. Methods: To address this issue, a new adaptation of the dried plasma spot technique was developed using a 24-well plate and fast gas chromatography-mass spectrometry. The method involves the optimization of extraction variables and sample preparation, and was successfully validated. Results: The limits of quantitation ranged from 20 to 60 ng/ml, and accuracy ranged from 87.8% to 112.2%. The technique was applied to 102 human plasma samples from suspected poisoning cases, with positivity of 90.2%. Conclusion: This method provides a cheap, easy to implement and fast approach, making it ideal for toxicological emergency laboratories and promoting valuable support for healthcare professionals managing poisoning cases involving antidepressants and antipsychotics.

Increased sympathetic nervous system impairs prognosis in lung cancer patients: a scoping review of clinical studies.

Aim: To summarize current knowledge, gaps, quality of the evidence and show main results related to the role of the autonomic nervous system in lung cancer. Methods: Studies were identified through electronic databases (PubMed, Scopus, Embase and Cochrane Library) in October 2023, and a descriptive analysis was performed. Twenty-four studies were included, and most were observational. Results: Our data indicated an increased expression of ß-2-adrenergic receptors in lung cancer, which was associated with poor prognosis. However, the use of ß-blockers as an add-on to standard treatment promoted enhanced overall survival, recurrence-free survival and reduced metastasis occurrence. Conclusion: Although the results herein seem promising, future research using high-quality prospective clinical trials is required to draw directions to guide clinical interventions.

Lung cancer is one of the most common causes of cancer-related deaths in the world, which often goes undiagnosed until it is in an advanced stage. Recently, the autonomic nervous system (sympathetic and parasympathetic nervous systems) has been identified as a regulator of cancer growth and spread, including lung cancer. In fact, preclinical studies have demonstrated that autonomic innervation in lung cancer can trigger tumor progression, metastasis, and resistance to treatment, worsening the prognosis. In this sense, add-on strategies to standard cancer treatments have been investigating and one of them has stood out: the incidental use of ß-blockers (patients who used ß-blockers for the treatment of hypertension and/or cardiovascular diseases or anxiety) before surgeries or during chemotherapy, which has been associated with improved clinical outcomes. Thus, a scoping review was conducted to summarizing the current knowledge about the quality of evidence, gaps and main results related to the role of the autonomic nervous system in human lung cancer. Data from this review indicated an increase in sympathetic nervous system receptors associated with a worse prognosis in patients with lung cancer. Indeed, those patients who took ß-blockers along with lung cancer treatment showed an increase in survival and a reduction in the occurrence of metastases. Although the results herein seem promising, further prospective clinical studies are needed to investigate the effect of the intentional and controlled use of ß-blockers as an add-on strategy on the treatment of different types and stages of lung cancer.

DNA damage in an animal model of maple syrup urine disease.

Maple syrup urine disease is an inborn error of metabolism caused by a severe deficiency of the branched chain alpha-ketoacid dehydrogenase complex. Neurological dysfunction is a common finding in patients with maple syrup urine disease. However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly understood. In this study, we investigated whether acute or chronic administration of a branched chain amino acid pool (leucine, isoleucine and valine) causes transient DNA damage, as determined by the alkaline comet assay, in the brain and blood of rats during development and whether antioxidant treatment prevented the alterations induced by branched chain amino acids. Our results showed that the acute administration of branched chain amino acids increased the DNA damage frequency and damage index in the hippocampus. However, the chronic administration of branched chain amino acids increased the DNA damage frequency and damage index in both the hippocampus and the striatum, and the antioxidant treatment was able to prevent DNA damage in the hippocampus and striatum. The present study demonstrated that metabolite accumulation in MSUD induces DNA damage in the hippocampus and striatum and that it may be implicated in the neuropathology observed in the affected patients. We demonstrated that the effect of antioxidant treatment (N-acetylcysteine plus deferoxamine) prevented DNA damage, suggesting the involvement of oxidative stress in DNA damage.

Nek1-inhibitor and temozolomide-loaded microfibers as a co-therapy strategy for glioblastoma treatment.

Malignant glioblastoma (GB) is the predominant primary brain tumour in adults, but despite the efforts towards novel therapies, the median survival of GB patients has not significantly improved in the last decades. Therefore, localised approaches that treat GB straight into the tumour site provide an alternative to enhance chemotherapy bioavailability and efficacy, reducing systemic toxicity. Likewise, the discovery of protein targets, such as the NIMA-related kinase 1 (Nek1), which was previously shown to be associated with temozolomide (TMZ) resistance in GB, has stimulated the clinical development of target therapy approaches to treat GB patients. In this study, we report an electrospun polyvinyl alcohol (PVA) microfiber (MF) brain-implant prepared for the controlled release of Nek1 protein inhibitor (iNek1) and TMZ or TMZ-loaded nanoparticles. The formulations revealed adequate stability and drug loading, which prolonged the drugs' release allowing a sustained exposure of the GB cells to the treatment and enhancing the drugs' therapeutic effects. TMZ-loaded MF provided the highest concentration of TMZ within the brain of tumour-bearing rats, and it was statistically significant when compared to TMZ via intraperitoneal (IP). All animals treated with either co-therapy formulation (TMZ + iNek1 MF or TMZ nanoparticles + iNek1 MF) survived until the endpoint (60 days), whereas the Blank MF (drug-unloaded), TMZ MF and TMZ IP-treated rats' median survival was found to be 16, 31 and 25 days, respectively. The tumour/brain area ratio of the rats implanted with either MF co-therapy was found to be reduced by 5-fold when compared to Blank MF-implanted rats. Taken together, our results strongly suggest that Nek1 is an important GB oncotarget and the inhibition of Nek1's activity significantly decreases GB cells' viability and tumour size when combined with TMZ treatment.