GABAergic system and chloride cotransporters as potential therapeutic targets to mitigate cell death in ischemia.

Gamma aminobutyric acid (GABA) is a critical inhibitory neurotransmitter in the central nervous system that plays a vital role in modulating neuronal excitability. Dysregulation of GABAergic signaling, particularly involving the cotransporters NKCC1 and KCC2, has been implicated in various pathologies, including epilepsy, schizophrenia, autism spectrum disorder, Down syndrome, and ischemia. NKCC1 facilitates chloride influx, whereas KCC2 mediates chloride efflux via potassium gradient. Altered expression and function of these cotransporters have been associated with excitotoxicity, inflammation, and cellular death in ischemic events characterized by reduced cerebral blood flow, leading to compromised tissue metabolism and subsequent cell death. NKCC1 inhibition has emerged as a potential therapeutic approach to attenuate intracellular chloride accumulation and mitigate neuronal damage during ischemic events. Similarly, targeting KCC2, which regulates chloride efflux, holds promise for improving outcomes and reducing neuronal damage under ischemic conditions. This review emphasizes the critical roles of GABA, NKCC1, and KCC2 in ischemic pathologies and their potential as therapeutic targets. Inhibiting or modulating the activity of these cotransporters represents a promising strategy for reducing neuronal damage, preventing excitotoxicity, and improving neurological outcomes following ischemic events. Furthermore, exploring the interactions between natural compounds and NKCC1/KCC2 provides additional avenues for potential therapeutic interventions for ischemic injury.

Sex on steroids: how brassinosteroids shape reproductive development in plants.

Since the discovery of brassinolide in the pollen of rapeseed, brassinosteroids (BRs) have consistently been associated with reproductive traits. However, compared to what is known for how BRs shape vegetative development, the understanding of how these hormones regulate reproductive traits is comparatively still lacking. Nevertheless, there is now considerable evidence that BRs regulate almost all aspects of reproduction, from ovule and pollen formation to seed and fruit development. Here, we review the current body of knowledge on how BRs regulate reproductive processes in plants, and what is known about how these pathways are transduced at the molecular level. We then discuss how the manipulation of BR biosynthesis and signaling can be a promising avenue for improving crop traits which rely on efficient reproduction. We thus propose that BR hold an untapped potential for plant breeding, which could contribute to attain food security in the coming years.

Toxicity of particulate emissions from residential biomass combustion: An overview of in vitro studies using cell models.

This article aims to critically review the current state of knowledge on in vitro toxicological assessments of particulate emissions from residential biomass heating systems. The review covers various aspects of particulate matter (PM) toxicity, including oxidative stress, inflammation, genotoxicity, and cytotoxicity, all of which have important implications for understanding the development of diseases. Studies in this field have highlighted the different mechanisms that biomass combustion particles activate, which vary depending on the combustion appliances and fuels. In general, particles from conventional combustion appliances are more potent in inducing cytotoxicity, DNA damage, inflammatory responses, and oxidative stress than those from modern appliances. The sensitivity of different cell lines to the toxic effects of biomass combustion particles is also influenced by cell type and culture conditions. One of the main challenges in this field is the considerable variation in sampling strategies, sample processing, experimental conditions, assays, and extraction techniques used in biomass burning PM studies. Advanced culture systems, such as co-cultures and air-liquid interface exposures, can provide more accurate insights into the effects of biomass combustion particles compared to simpler submerged monocultures. This review provides critical insights into the complex field of toxicity from residential biomass combustion emissions, underscoring the importance of continued research and standardisation of methodologies to better understand the associated health hazards and to inform targeted interventions.

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus.

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

ConectAR: Collaborative network of patients with asthma and carers actively involved in health research: a protocol for patient and public involvement.

Summary: Background. Patients and Public Involvement in every stage of the patient-centered health research cycle is the key to the development of innovative solutions with an impact on patients' care. Methods. This protocol describes the development of ConectAR, a network to promote the involvement of patients with asthma and their carers in the health research cycle. Results. This protocol comprehends 4 tasks: 1) define the mission, vision, governance and activities of the network through focus groups; 2) establish the communication strategy and tools; 3) test the feasibility of the network in a Delphi study on the research priorities for asthma in Portugal; 4) coordination and dissemination activities. Conclusions. This network will improve research by ensuring that patients and carers have an active role in the co-creation of impactful solutions for asthma.

Caffeine and Its Neuroprotective Role in Ischemic Events: A Mechanism Dependent on Adenosine Receptors.

Ischemia is characterized by a transient, insufficient, or permanent interruption of blood flow to a tissue, which leads to an inadequate glucose and oxygen supply. The nervous tissue is highly active, and it closely depends on glucose and oxygen to satisfy its metabolic demand. Therefore, ischemic conditions promote cell death and lead to a secondary wave of cell damage that progressively spreads to the neighborhood areas, called penumbra. Brain ischemia is one of the main causes of deaths and summed with retinal ischemia comprises one of the principal reasons of disability. Although several studies have been performed to investigate the mechanisms of damage to find protective/preventive interventions, an effective treatment does not exist yet. Adenosine is a well-described neuromodulator in the central nervous system (CNS), and acts through four subtypes of G-protein-coupled receptors. Adenosine receptors, especially A1 and A2A receptors, are the main targets of caffeine in daily consumption doses. Accordingly, caffeine has been greatly studied in the context of CNS pathologies. In fact, adenosine system, as well as caffeine, is involved in neuroprotection effects in different pathological situations. Therefore, the present review focuses on the role of adenosine/caffeine in CNS, brain and retina, ischemic events.

Personal exposure assessment of pesticides in residents: The association between hand wipes and urinary biomarkers.

BACKGROUND: Residential exposure to pesticides may occur via inhalation of airborne pesticides, direct skin contacts with pesticide-contaminated surfaces, and consumption of food containing pesticide residues. The aim was to study the association of dermal exposure to pesticides between the use and non-use periods, between farmer and non-farmer families and between dermal exposure and the excretion of metabolites from urine in residents living close to treated agricultural fields. METHODS: In total, 112 hand wipes and 206 spot urine samples were collected from 16 farmer and 38 non-farmer participants living within 50 m from an agricultural field in the Netherlands. The study took place from May 2016 to December 2017 during the use as well as the non-use periods of pesticides. Hand wipes were analysed for the parent compound and urines samples for the corresponding urinary metabolite of five applied pesticides: asulam, carbendazim (applied as thiophanate-methyl), chlorpropham, prochloraz and tebuconazole. Questionnaire data was used to study potential determinants of occurrence and levels of pesticides in hand wipes according to univariate and multivariate analysis. RESULTS: Carbendazim and tebuconazole concentrations in hand wipes were statistically significantly higher in the pesticide-use period compared to the non-use period. In addition, especially during the use periods, concentrations were statistically significantly higher in farmer families compared to non-farmer families. For asulam, chlorpropham and prochloraz, the frequency of non-detects was too high (57-85%) to be included in this analysis. The carbendazim contents in urine samples and hand wipes were correlated on the first and second day after taking the hand wipe, whereas chlorpropham was only observed to be related on the second day following the spray event. CONCLUSIONS: Concentrations in hand wipes were overall higher in pesticide use periods compared to non-use periods and higher in farmer families compared to non-farmer families. Only for carbendazim a strong correlation between concentrations in hand wipes and its main metabolite in urine was observed, indicating dermal exposure via contaminated indoor surfaces. We expect this to be related to the lower vapour pressure and longer environmental lifetime of carbendazim compared to the other pesticides studies.

Microcephaly epidemic in Brazil: An earlier chapter.

OBJECTIVE: Identify risk factors for microcephaly and evaluate historical trends of microcephaly and arboviruses to recognize patterns and anomalies that indicate the beginning of the microcephaly epidemic associated with Zika infection. METHODS: The head circumferences of 62,298 newborns was analyzed to identify cases of microcephaly between 2014 and 2017. We compared the groups of newborns with normal head circumferences and those with microcephaly to identify risk factors. A time series with the incidences of microcephaly was analyzed to assess the appearance of anomalous values in order to identify the beginning of the microcephaly epidemic. Data on the incidence of dengue fever was used to develop a control chart, aiming to identify changes in incidence and seasonality that could suggest the circulation of a new arbovirus. FINDINGS: Premature newborns, children of mothers under 20 years of age and those born in 2014 and 2015 had a higher risk of microcephaly. Three quarters with anomalous incidences of microcephaly were identified, the first in 2014 and the others in 2015. The dengue fever epidemic curve in 2013 shows persistence of high incidences in atypical periods, suggesting the entry of a new virus in the 3rd and 4th quarters. CONCLUSIONS: These findings represent epidemiological evidence of the existence of cases of Zika virus between the 2nd quarter of 2013 and the beginning of 2014. The results add new elements to understanding the Zika virus epidemic in the Americas.

A prospective cohort study on the effects of RME in the mandibular dentition of cleft subjects.

PURPOSE: Rapid maxillary expansion (RME) is the most frequent orthopedic procedure in cleft subjects. However, little is known about its effects on the mandible. The aim of this study was to investigate the spontaneous response of the mandibular teeth following RME. METHODS: This prospective cohort study was carried out with a sample of thirty participants with unilateral cleft lip and palate (UCLP), 8-15 years old, who had transverse maxillary deficiency. Two participants were excluded. They were allocated into three groups: G1 (n = 10), G2 (n = 10), and G3 (n = 8). G1 was treated with a Fan-type expander; G2 with an iMini expander; and G3 with a Hyrax expander. Measurements were performed in Cone Beam CT scans obtained before treatment (T1) and 3 months post-expansion (T2). The primary outcomes were buccolingual inclination of mandibular first molars and canines, and intercanine and intermolar width at different levels. RESULTS: Dental changes were significant (P < 0.05) for intercanine width, increasing in G1 and G2, and for intermolar width, increasing in G2 and G3. There were no significant differences among groups (P > 0.05). CONCLUSION: RME in UCLP subjects performed with these expanders may lead to significant spontaneous changes in both anterior and posterior region of the mandible.

Caffeine exposure ameliorates acute ischemic cell death in avian developing retina.

In infants, the main cause of blindness is retinopathy of prematurity that stems in a hypoxic-ischemic condition. Caffeine is a psychoactive compound that at low to moderate concentrations, selectively inhibits adenosine A1 and A2A receptors. Caffeine exerts beneficial effects in central nervous system of adult animal models and humans, whereas it seems to have malefic effect on the developing tissue. We observed that 48-h exposure (during synaptogenesis) to a moderate dose of caffeine (30 mg/kg of egg) activated pro-survival signaling pathways, including ERK, CREB, and Akt phosphorylation, alongside BDNF production, and reduced retinal cell death promoted by oxygen glucose deprivation in the chick retina. Blockade of TrkB receptors and inhibition of CREB prevented caffeine protection effect. Similar signaling pathways were described in previously reported data concerning chemical preconditioning mechanism triggered by NMDA receptors activation, with low concentrations of agonist. In agreement to these data, caffeine increased NMDA receptor activity. Caffeine decreased the levels of the chloride co-transporter KCC2 and delayed the developmental shift on GABAA receptor response from depolarizing to hyperpolarizing. These results suggest that the caffeine-induced delaying in depolarizing effect of GABA could be facilitating NMDA receptor activity. DPCPX, an A1 adenosine receptor antagonist, but not A2A receptor inhibitor, mimicked the effect of caffeine, suggesting that the effect of caffeine occurs through A1 receptor blockade. In summary, an in vivo caffeine exposure could increase the resistance of the retina to ischemia-induced cell death, by triggering survival pathways involving CREB phosphorylation and BDNF production/TrkB activation.

Aqueous leaf extract of Passiflora alata Curtis promotes antioxidant and anti-inflammatory effects and consequently preservation of NOD mice beta cells (non-obese diabetic).

Passiflora alata Curtis (P. alata) leaves have anti-inflammatory properties; the present study aimed to investigate the anti-diabetogenic properties of P. alata aqueous leaf extract. HPLC analysis identified the phenolic compounds catechin, epicatechin and rutin. The aqueous extract was administered for 30weeks to non-obese diabetic (NOD) mice presenting a decrease of 28.6% in diabetes incidence and the number of inflammatory cells in pancreatic islets, when compared with the control group (water). The P. alata group presented an antioxidant effect and decreased lipid peroxidation in the serum of NOD mice. Increased numbers of insulin-positive cells were also observed in the pancreatic islets of the treated group. The diabetic group exhibited higher levels in the glucose tolerance test and glycemic index, in comparison to the P. alata-treated group and non-diabetic control BALB/c mice. In addition, the P. alata extract reduced the percentage and the proliferation index of NOD mice lymphocytes submitted to in vitro dose/response mitogenic stimulation assays. These results suggest that the aqueous extract of P. alata has anti-inflammatory properties, contributing to the protection of beta cells in pancreatic islets in NOD mice, and presents potential for use a supporting approach to treat type 1 diabetes.

Invasive Trichosporon infection in solid organ transplant patients: a report of two cases identified using IGS1 ribosomal DNA sequencing and a review of the literature.

Trichosporon species are rare etiologic agents of invasive fungal infection in solid organ transplant (SOT) recipients. We report 2 well-documented cases of Trichosporon inkin invasive infection in SOT patients. We also conducted a detailed literature review of Trichosporon species infections in this susceptible population. We gathered a total of 13 cases of Trichosporon species infections. Any type of organ transplantation can be complicated by Trichosporon infection. Bloodstream infections and disseminated infections were the most common clinical presentations. Liver recipients with bloodstream or disseminated infections had poor prognoses. Although the most common species was formerly called Trichosporon beigelii, this species name should no longer be used because of the changes in the taxonomy of this genus resulting from the advent of molecular approaches, which were also used to identify the strains isolated from our patients. Antifungal susceptibility testing highlights the possibility of multidrug resistance. Indeed, Trichosporon has to be considered in cases of breakthrough infection or treatment failure under echinocandins or amphotericin therapy. Voriconazole seems to be the best treatment option.

Usefulness of matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry for identifying clinical Trichosporon isolates.

Trichosporon spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for therapeutic management, but conventional identification based on biochemical traits is hindered by the lack of updates to the species databases provided by the different commercial systems. In this study, 93 strains, or isolates, belonging to 16 Trichosporon species were subjected to both molecular identification using IGS1 gene sequencing and matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) analysis. Our results confirmed the limits of biochemical systems for identifying Trichosporon species, because only 27 (36%) of the isolates were correctly identified using them. Different protein extraction procedures were evaluated, revealing that incubation for 30 min with 70% formic acid yields the spectra with the highest scores. Among the six different reference spectra databases that were tested, a specific one composed of 18 reference strains plus seven clinical isolates allowed the correct identification of 67 of the 68 clinical isolates (98.5%). Although until recently it has been less widely applied to the basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level.

Antioxidant and anti-diabetic potential of Passiflora alata Curtis aqueous leaves extract in type 1 diabetes mellitus (NOD-mice).

Leaves of Passiflora alata Curtis were characterized for their antioxidant capacity. Antioxidant analyses of DPPH, FRAP, ABTS, ORAC and phenolic compounds were made in three different extracts: aqueous, methanol/acetone and ethanol. Aqueous extract was found to be the best solvent for recovery of phenolic compounds and antioxidant activity, when compared with methanol/acetone and ethanol. To study the anti-inflammatory properties of this extract in experimental type 1 diabetes, NOD mice were divided into two groups: the P. alata group, treated with aqueous extract of P. alata Curtis, and a non-treated control group, followed by diabetes expression analysis. The consumption of aqueous extract and water ad libitum lasted 28 weeks. The treated-group presented a decrease in diabetes incidence, a low quantity of infiltrative cells in pancreatic islets and increased glutathione in the kidney and liver (p<0.05), when compared with the diabetic and non-diabetic control-groups. In conclusion, our results suggest that the consumption of aqueous extract of P. alata may be considered a good source of natural antioxidants and compounds found in its composition can act as anti-inflammatory agents, helping in the control of diabetes.

Utilization of wasted sardine oil as co-substrate with pig slurry for biogas production--a pilot experience of decentralized industrial organic waste management in a Portuguese pig farm.

This work aimed to demonstrate in a pig farm and in real conditions, the possibilities to co-digest wasted sardine oil (WSO) and pig slurry (PS) at farm scale. A biogas mobile pilot plant, was set up in the farm and operated in real conditions during 4 months. Dynamic mesophilic (35-37 °C) continuous pilot trials were performed during four different periods of time. In each period a different organic loading rate (OLR) based on the chemical oxygen demand (COD) was operated sequentially, with pig slurry (PS) (OLR = 1.6 kg COD/m(3) d(-1)) and with mixtures of WSO:PS with a volumetric composition (% v/v) of 2:98 (OLR = 3.0 kg COD/m(3) d(-1)), 3:97 (OLR = 3.7 kg COD/m(3) d(-1)) and 5:95 (OLR = 5.2 kg COD/m(3) d(-1)). Biomass adapted very fast in metabolise the WSO and biogas productivity was raised substantially for different compositions of WSO:PS. Process stability indicators pH and Total volatile fatty acids/bicarbonate alkalinity (T-VFA/BA) ratio, suggests that the co-digestion process was robust. It was concluded that WSO could be easily co-digested in farm scale biogas plants.

Genomics and proteomics approaches to the study of cancer-stroma interactions.

BACKGROUND: The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. METHODS: The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells. RESULTS: We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR. CONCLUSIONS: A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.