Post-weaning social isolation modifies neonatal anoxia-induced changes in energy metabolism and growth of rats.

Neonatal oxygen deficiency in rats may disturb growth and long-term metabolic homeostasis. In order to facilitate metabolic evaluation, the subjects are usually housed individually. However, social isolation associated with individually housed conditions alters animal behavior, which may influence the experimental results. This study investigated the effects of social isolation on neonatal anoxia-induced changes in growth and energy metabolism. Male and female Wistar rats were exposed, on postnatal day 2 (P2), to either 25-min of anoxia or control treatment. From P27 onward, part of the subjects of each group was isolated in standard cages, and the remaining subjects were housed in groups. At P34 or P95, the subjects were fasted for 18 h, refeed for 1 h, and then perfused 30 min later. Glycemia, leptin, insulin, and morphology of the pancreas were evaluated at both ages. For subjects perfused at P95, body weight and food intake were recorded up to P90, and the brain was collected for Fos and NeuN immunohistochemistry. Results showed that male rats exposed to neonatal anoxia and social isolation exhibited increased body weight gain despite the lack of changes in food intake. In addition, social isolation (1) decreased post-fasting weight loss and post-fasting food intake and (2) increased glycemia, insulin, and leptin levels of male and female rats exposed to anoxia and control treatments, both at P35 and P95. Furthermore, although at P35, anoxia increased insulin levels of males, it decreased the area of the ß-positive cells in the pancreas of females. At P95, anoxia increased post-prandial weight loss of males, post-fasting food intake, insulin, and leptin, and decreased Fos expression in the arcuate nucleus (ARC) of males and females. Hyperphagia was associated with possible resistance to leptin and insulin, suspected by the high circulating levels of these hormones and poor neuronal activation of ARC. This study demonstrated that continuous social isolation from weaning modifies, in a differentiated way, the long-term energy metabolism and growth of male and female Wistar rats exposed to neonatal anoxia or even control treatments. Therefore, social isolation should be considered as a factor that negatively influences experimental results and the outcomes of the neonatal injury. These results should also be taken into account in clinical procedures, since the used model simulates the preterm babies' conditions and some therapeutic approaches require isolation.

Gephyrin phosphorylation facilitates sexually dimorphic development and function of parvalbumin interneurons in the mouse hippocampus.

The precise function of specialized GABAergic interneuron subtypes is required to provide appropriate synaptic inhibition for regulating principal neuron excitability and synchronization within brain circuits. Of these, parvalbumin-type (PV neuron) dysfunction is a feature of several sex-biased psychiatric and brain disorders, although, the underlying developmental mechanisms are unclear. While the transcriptional action of sex hormones generates sexual dimorphism during brain development, whether kinase signaling contributes to sex differences in PV neuron function remains unexplored. In the hippocampus, we report that gephyrin, the main inhibitory post-synaptic scaffolding protein, is phosphorylated at serine S268 and S270 in a developmentally-dependent manner in both males and females. When examining GphnS268A/S270A mice in which site-specific phosphorylation is constitutively blocked, we found that sex differences in PV neuron density in the hippocampal CA1 present in WT mice were abolished, coincident with a female-specific increase in PV neuron-derived terminals and increased inhibitory input onto principal cells. Electrophysiological analysis of CA1 PV neurons indicated that gephyrin phosphorylation is required for sexually dimorphic function. Moreover, while male and female WT mice showed no difference in hippocampus-dependent memory tasks, GphnS268A/S270A mice exhibited sex- and task-specific deficits, indicating that gephyrin phosphorylation is differentially required by males and females for convergent cognitive function. In fate mapping experiments, we uncovered that gephyrin phosphorylation at S268 and S270 establishes sex differences in putative PV neuron density during early postnatal development. Furthermore, patch-sequencing of putative PV neurons at postnatal day 4 revealed that gephyrin phosphorylation contributes to sex differences in the transcriptomic profile of developing interneurons. Therefore, these early shifts in male-female interneuron development may drive adult sex differences in PV neuron function and connectivity. Our results identify gephyrin phosphorylation as a new substrate organizing PV neuron development at the anatomical, functional, and transcriptional levels in a sex-dependent manner, thus implicating kinase signaling disruption as a new mechanism contributing to the sex-dependent etiology of brain disorders.

División ganglionar posquirúrgica y su efecto en el recuento ganglionar y la estadificación en pacientes con cáncer gástrico / Postoperative lymph node division and its effect on lymph node count and staging in patients with gastric cancer

Introducción. La gastrectomía y disección ganglionar es el estándar de manejo para los pacientes con cáncer gástrico. Factores como la identificación de ganglios por el patólogo, pueden tener un impacto negativo en la estadificación y el tratamiento. El objetivo de este estudio fue comparar el recuento ganglionar de un espécimen quirúrgico después de una gastrectomía completa (grupo A) y de un espécimen con un fraccionamiento por grupos ganglionares (grupo B). Métodos. Estudio de una base de datos retrospectiva de pacientes sometidos a gastrectomía D2 en el Servicio de Cirugía gastrointestinal de la Liga Contra el Cáncer seccional Risaralda, Pereira, Colombia. Se comparó el recuento ganglionar en especímenes quirúrgicos con y sin división ganglionar por regiones anatómicas previo a su envío a patología. Resultados. De los 94 pacientes intervenidos, 65 pertenecían al grupo A y 29 pacientes al grupo B. El promedio de ganglios fue de 24,4±8,6 y 32,4±14,4 respectivamente (p=0,004). El porcentaje de pacientes con más de 15 y de 25 ganglios fue menor en el grupo A que en el grupo B (27 vs 57, p=0,432 y 19 vs 24, p=0,014). El promedio de pacientes con una relación ganglionar menor 0,2 fue mayor en el grupo B (72,4 % vs 55,4 %, p=0,119). Conclusiones. Los resultados de nuestro estudio mostraron que una división por grupos ganglionares previo a la valoración del espécimen por el servicio de patología incrementa el recuento ganglionar y permite establecer de manera certera el pronóstico de los pacientes, teniendo un impacto positivo en su estadificación, para evitar el sobretratamiento

Introduction. A gastrectomy and lymph node dissection is the standard of management for patients with gastric cancer. Factors such as the identification of nodes by the pathologist can have a negative impact on staging and treatment. The objective of this study was to compare the lymph node count of a surgical specimen after a complete gastrectomy (group A) and of a specimen with lymph node by groups (group B). Methods. Study of a retrospective database of patients undergoing D2 gastrectomy in the Risaralda section of the Liga Contra el Cancer Gastrointestinal surgical service, Pereira, Colombia. The lymph node count was compared in surgical specimens with and without lymph node division by anatomical regions, prior to sending them to pathology. Results. Of the 94 patients who underwent surgery, 65 were from group A and 29 patients were from group B. The average number of nodes was 24.4±8.6 and 32.4±14.4, respectively (p=0.004). The percentage of patients with more than 15 and 25 nodes was lower in group A than in group B (27 vs 57, p=0.432 and 19 vs 24, p=0.014). The average number of patients with a nodal ratio less than 0.2 was higher in group B (72.4% vs 55.4%, p=0.119). Conclusions. The results of our study showed that a division by lymph node groups prior to the evaluation of the specimen by the pathology service increases the lymph node count and allows the prognosis of patients to be accurately established, having a positive impact on their staging, to avoid overtreatment.

The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

CD8+ T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion1. Although it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the ß1-adrenergic receptor ADRB1. We identify that exhausted CD8+ T cells increase ADRB1 expression and that exposure of ADRB1+ T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8+ T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of ß1-adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, ß-blockers and ICB synergize to boost CD8+ T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients2,3, and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

Commissural dentate granule cell projections and their rapid formation in the adult brain.

Dentate granule cells (GCs) have been characterized as unilaterally projecting neurons within each hippocampus. Here, we describe a unique class, the commissural GCs, which atypically project to the contralateral hippocampus in mice. Although commissural GCs are rare in the healthy brain, their number and contralateral axon density rapidly increase in a rodent model of temporal lobe epilepsies. In this model, commissural GC axon growth appears together with the well-studied hippocampal mossy fiber sprouting and may be important for the pathomechanisms of epilepsy. Our results augment the current view on hippocampal GC diversity and demonstrate powerful activation of a commissural wiring program in the adult brain.

Pcdh11x controls target specification of mossy fiber sprouting.

Circuit formation is a defining characteristic of the developing brain. However, multiple lines of evidence suggest that circuit formation can also take place in adults, the mechanisms of which remain poorly understood. Here, we investigated the epilepsy-associated mossy fiber (MF) sprouting in the adult hippocampus and asked which cell surface molecules define its target specificity. Using single-cell RNAseq data, we found lack and expression of Pcdh11x in non-sprouting and sprouting neurons respectively. Subsequently, we used CRISPR/Cas9 genome editing to disrupt the Pcdh11x gene and characterized its consequences on sprouting. Although MF sprouting still developed, its target specificity was altered. New synapses were frequently formed on granule cell somata in addition to dendrites. Our findings shed light onto a key molecular determinant of target specificity in MF sprouting and contribute to understanding the molecular mechanism of adult brain rewiring.

Neonatal Anoxia Impacts Rats' Maternal Behavior and Offspring Development due to Ultrasonic Vocalization's Impairment.

Hypoxic-ischemic encephalopathy is a severe clinical condition, among others, affecting the brain after offspring exposure to neonatal anoxia, which causes persistent sensorimotor and cognitive deficits. During peripartum, maternal behaviors are crucial for the healthy development of the offspring. In rats, the vocalization of newborns, around 40 kHz, corresponds to separation calls that encourage their mothers to retrieve them. Alterations in this pattern affect the maternal behavior addressed to the offspring. This study aimed to evaluate the maternal behavior of primiparous rats whose offspring were exposed to neonatal anoxia in P2 (postpartum day) during the lactation period, to assess mother-pup interactions through the pups' vocalization from P3 to P18. It also intends to quantify eventual neuronal alterations in the mothers' medial preoptic area after the last weaning (P21) through FOS protein expression. Anoxia offspring were found to reduce maternal behaviors toward them, increased frequency of separation calls in the male anoxia group, and reduced vocalization rate in the female anoxia group compared to their respective controls. Body weight gain reduction of males' and females' anoxia was observed. We concluded that anoxia exerts deleterious effects on the vocalization patterns of the pups, with sex differences that alter maternal behavior toward them. Impaired USV makes an additional negative impact on the already noxious effects of neonatal anoxia. Understanding those phenomena applies/contributes to guiding procedures and strategies to mitigate the deleterious outcomes and orient research concerning the complexity of neonatal anoxia events and the influence of maternal care quality concerning the pups, which should also be considered sex differences.

Adolescence is a sensitive period for prefrontal microglia to act on cognitive development.

The prefrontal cortex (PFC) is a cortical brain region that regulates various cognitive functions. One distinctive feature of the PFC is its protracted adolescent maturation, which is necessary for acquiring mature cognitive abilities in adulthood. Here, we show that microglia, the brain's resident immune cells, contribute to this maturational process. We find that transient and cell-specific deficiency of prefrontal microglia in adolescence is sufficient to induce an adult emergence of PFC-associated impairments in cognitive functions, dendritic complexity, and synaptic structures. While prefrontal microglia deficiency in adolescence also altered the excitatory-inhibitory balance in adult prefrontal circuits, there were no cognitive sequelae when prefrontal microglia were depleted in adulthood. Thus, our findings identify adolescence as a sensitive period for prefrontal microglia to act on cognitive development.

Recurrent rewiring of the adult hippocampal mossy fiber system by a single transcriptional regulator, Id2.

Circuit formation in the central nervous system has been historically studied during development, after which cell-autonomous and nonautonomous wiring factors inactivate. In principle, balanced reactivation of such factors could enable further wiring in adults, but their relative contributions may be circuit dependent and are largely unknown. Here, we investigated hippocampal mossy fiber sprouting to gain insight into wiring mechanisms in mature circuits. We found that sole ectopic expression of Id2 in granule cells is capable of driving mossy fiber sprouting in healthy adult mouse and rat. Mice with the new mossy fiber circuit solved spatial problems equally well as controls but appeared to rely on local rather than global spatial cues. Our results demonstrate reprogrammed connectivity in mature neurons by one defined factor and an assembly of a new synaptic circuit in adult brain.

Metabolic regulation of T cells in the tumor microenvironment by nutrient availability and diet.

Recent advances in immunotherapies such as immune checkpoint blockade (ICB) and chimeric antigen receptor T cells (CAR-T) for the treatment of cancer have generated excitement over their ability to yield durable, and potentially curative, responses in a multitude of cancers. These findings have established that the immune system is capable of eliminating tumors and led us to a better, albeit still incomplete, understanding of the mechanisms by which tumors interact with and evade destruction by the immune system. Given the central role of T cells in immunotherapy, elucidating the cell intrinsic and extrinsic factors that govern T cell function in tumors will facilitate the development of immunotherapies that establish durable responses in a greater number of patients. One such factor is metabolism, a set of fundamental cellular processes that not only sustains cell survival and proliferation, but also serves as a means for cells to interpret their local environment. Nutrient sensing is critical for T cells that must infiltrate into a metabolically challenging tumor microenvironment and expand under these harsh conditions to eliminate cancerous cells. Here we introduce T cell exhaustion with respect to cellular metabolism, followed by a discussion of nutrient availability at the tumor and organismal level in relation to T cell metabolism and function to provide rationale for the study and targeting of metabolism in anti-tumor immune responses.

Neonatal anoxia increases nociceptive response in rats: Sex differences and lumbar spinal cord and insula alterations.

Neonatal anoxia is a well-known world health problem that results in neurodevelopmental deficits, such as sensory alterations that are observed in patients with cerebral palsy and autism disorder, for which oxygen deprivation is a risk factor. Nociceptive response, as part of the sensory system, has been reported as altered in these patients. To determine whether neonatal oxygen deprivation alters nociceptive sensitivity and promotes medium- and long-term inflammatory feedback in the central nervous system, Wistar rats of around 30 h old were submitted to anoxia (100% nitrogen flux for 25 min) and evaluated on PND23 (postpartum day) and PND90. The nociceptive response was assessed by mechanical, thermal, and tactile tests in the early postnatal and adulthood periods. The lumbar spinal cord (SC, L4-L6) motor neurons (MNs) and the posterior insular cortex neurons were counted and compared with their respective controls after anoxia. In addition, we evaluated the possible effect of anoxia on the expression of astrocytes in the SC at adulthood. The results showed increased nociceptive responses in both males and females submitted to anoxia, although these responses were different according to the nociceptive stimulus. A decrease in MNs in adult anoxiated females and an upregulation of GFAP expression in the SC were observed. In the insular cortex, a decrease in the number of cells of anoxiated males was observed in the neonatal period. Our findings suggest that oxygen-deprived nervous systems in rats may affect their response at the sensorimotor pathways and respective controlling centers with sex differences, which were related to the used stimulus.

Inhibition of Fusarium oxysporum f. sp. nicotianae Growth by Phenylpropanoid Pathway Intermediates.

Fusarium wilt in tobacco caused by the fungus Fusarium oxysporum f. sp. nicotianae is a disease­management challenge worldwide, as there are few effective and environmentally benign chemical agents for its control. This challenge results in substantial losses in both the quality and yield of tobacco products. Based on an in vitro analysis of the effects of different phenylpropanoid intermediates, we found that the early intermediates trans­cinnamic acid and para­coumaric acid effectively inhibit the mycelial growth of F. oxysporum f. sp. nicotianae strain FW316F, whereas the downstream intermediates quercetin and caffeic acid exhibit no fungicidal properties. Therefore, our in vitro screen suggests that trans­cinnamic acid and para­coumaric acid are promising chemical agents and natural lead compounds for the suppression of F. oxysporum f. sp. nicotianae growth.

Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications.

Post-translational modifications (PTMs) are one of the most important regulatory mechanisms in cells, and they play key roles in cell signaling both in health and disease. PTM catalyzing enzymes have become significant drug targets, and therefore, tremendous interest has been focused on the development of broad-scale assays to monitor several different PTMs with a single detection platform. Most of the current methodologies suffer from low throughput or rely on antibody recognition, increasing the assay costs, and decreasing the multifunctionality of the assay. Thus, we have developed a sensitive time-resolved Förster resonance energy transfer (TR-FRET) detection method for PTMs of cysteine residues using a single-peptide approach performed in a 384-well format. In the developed assay, the enzyme-specific biotinylated substrate peptide is post-translationally modified at the cysteine residue, preventing the subsequent thiol coupling with a reactive AlexaFluor 680 acceptor dye. In the absence of enzymatic activity, increase in the TR-FRET signal between the biotin-bound Eu(III)-labeled streptavidin donor and the cysteine-coupled AlexaFluor 680 acceptor dye is observed. We demonstrate the detection concept with cysteine modifying S-nitrosylation and ADP-ribosylation reactions using a chemical nitric oxide donor S-nitrosoglutathione and enzymatic ADP-ribosyltransferase PtxS1-subunit of pertussis toxin, respectively. As a proof of concept, three peptide substrates derived from the small GTPase K-Ras and the inhibitory α-subunit of the heterotrimeric G-protein Gαi showed expected functionality in both chemical and enzymatic assays. Measurements yielded signal-to-background ratios of 28.7, 33.0, and 8.7 between the modified and the nonmodified substrates for the three peptides in the S-nitrosylation assay, 5.8 in the NAD+ hydrolysis assay, and 6.8 in the enzymatic ADP-ribosyltransferase inhibitor dose-response assay. The developed antibody-free assay for cysteine-modifying enzymes provides a detection platform with low nanomolar peptide substrate consumption, and the assay is potentially applicable to investigate various cysteine-modifying enzymes in a high throughput compatible format.

Neonatal anoxia impairs long-term energy metabolism and somatic development of Wistar rats.

BACKGROUND: Neonatal anoxia may cause neurological injuries, behavioral alterations and changes in somatic growth. Somatic developmental changes suggest a possible effect of anoxia on energy metabolism and/or feeding behavior. Short-term effects of oxygen deficit on energy homeostasis have been described. In contrast, just a few studies report long-term effects. This study investigated the effects of neonatal anoxia on energy metabolism and somatic development at adulthood of males and females Wistar rats. METHOD: Male (m) and female (f) rats were exposed, on postnatal day 2 (P2), to either 25-min of Anoxia or Control treatment. At P34 part of the subjects of each group was fasted for 18 h, refeed for 1 h and then perfused 30 min later, at P35; the remaining subjects were submitted to these treatments at P94 and perfused at P95. Therefore, there were 8 groups: AmP35, AmP95, AfP35, AfP95, CmP35, CmP95, CfP35 and CfP95. For subjects perfused at P95, body weight and food intake were recorded up to P90. For subjects perfused at P35 and P95, glycemia, leptin and insulin were assessed after fasting and refeed. After perfusion the encephalon and pancreas were collected for Fos immunohistochemistry and Hematoxylin-Eosin stain analyses. RESULTS: Even though neonatal anoxia did not interfere with regular food intake, it reduced body weight gain along growing in both male and female subjects as compared to the corresponding controls. At P35 neonatal anoxia decreased post-prandial glycemia and increased insulin. While at P95 neonatal anoxia altered the pancreatic histomorphology and increased post-fasting weight loss, decreasing leptin, insulin and glycemia secretion, as well Fos immunoreactivity (IR) in ARC. CONCLUSION: Neonatal anoxia impairs long-term energy metabolism and somatic development in Wistar rats, with differences related to sex and age.

Thermal Dissociation Assay for Time-Resolved Fluorescence Detection of Protein Post-Translational Modifications.

Post-translational modifications (PTMs) of proteins provide an important mechanism for cell signal transduction control. Impaired PTM control is a key feature in multiple different disease states, and thus the enzyme-controlling PTMs have drawn attention as highly promising drug targets. Due to the importance of PTMs, various methods to monitor PTM enzyme activity have been developed, but universal high-throughput screening (HTS), a compatible method for different PTMs, remains elusive. Here, we present a homogeneous single-label thermal dissociation assay for the detection of enzymatic PTM removal. The developed method allows the use of micromolar concentration of substrate peptide, which is expected to be beneficial when monitoring enzymes with low activity and peptide binding affinity. We prove the thermal dissociation concept functionality using peptides for dephosphorylation, deacetylation, and demethylation and demonstrate the HTS-compatible flash isothermal method for PTM enzyme activity monitoring. Using specific inhibitors, we detected literature-comparable IC50 values and Z' factors from 0.61 to 0.72, proving the HTS compatibility of the thermal peptide-break technology.

Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides.

We have developed a rapid and sensitive universal peptide-based time-resolved luminescence assay for detection of enzymatic post-translational modifications (PTMs). PTMs play essential roles in intracellular signaling and cell regulation, thus providing functional protein diversity in cell. Due this, impaired PTM patterns have been linked to multiple disease states. Clear link between PTMs and pathological conditions have also driven assay development further, but still today most of the methodologies are based on single-specificity or group-specific PTM-recognition. We have previously introduced leuzine-zipper based peptide-break technology as a viable option for universal PTM detection. Here, we introduce peptide-break technology utilizing single-label homogeneous quenching resonance energy transfer (QRET) and charge-based peptide-peptide interaction. We demonstrate the functionality of the new assay concept in phosphorylation, deacetylation, and citrullination. In a comparable study between previously introduced leucine-zipper and the novel charge-based approach, we found equal PTM detection performance and sensitivity, but the peptide design for new targets is simplified with the charged peptides. The new concept allows the use of short <20 amino acid peptides without limitations rising from the leucine-zipper coiled-coil structure. Introduced methodology enables wash-free PTM detection in a 384-well plate format, using low nanomolar enzyme concentrations. Potentially, the peptide-break technique using charged peptides may be applicable for natural peptide sequences directly obtained from the target protein.

Toward universal protein post-translational modification detection in high throughput format.

Post-translational modification (PTM) of proteins plays essential regulatory roles in a variety of pathological conditions. Reliable and practical assays are required to accelerate the discovery of inhibitors and activators for PTM related diseases. Today, methodologies are based on specific or group-specific PTM recognition of e.g. phosphate for kinase activity without extending to other type of PTMs. Here we have established a universal time-resolved luminescence assay on a peptide-break platform for the direct detection of wide variety of PTMs. The developed assay is based on the leucine zipper concept wherein a europium-chelate labeled detection peptide and a non-labeled peptide substrate form a highly luminescent dimer. As an active PTM enzyme at sub or low nanomolar concentration modifies the substrate peptide, the luminescent signal of the detached detection peptide is quenched in the presence of soluble quenchers. The functionality of this universal assay technique has been demonstrated for the monitoring of phosphorylation, dephosphorylation, deacetylation, and citrullination with high applicability also to other PTMs in a high throughput format.

Efeito do estresse pré-natal na regulação da inflamação alérgica pulmonar no modelo murino de asma experimental

Devido ao seu rápido crescimento, o feto é particularmente vulnerável a insultos e modificações no millieu hormonal. Este fato sugere que situações adversas experimentadas pela mãe grávida podem alterar o desenvolvimento e a saúde da prole, explicado principalmente pela permeabilidade da barreira placentária a diversos hormônios e substâncias. O objetivo deste trabalho foi estudar o efeito do estresse pré-natal na regulação da inflamação alérgica pulmonar, empregando o modelo murino de asma experimental. Para este propósito foram utilizadas camundongas virgens da linhagem Swiss, com 50 dias de idade. Foi empregado o modelo de choque nas patas para promover o estresse pré-natal e o modelo do "metrô de Nova Efeito do estresse pré-natal na regulação da inflamação alérgica pulmonar no modelo murino de asma experimental 65 Iorque" para o estresse pós-natal. As fêmeas foram distribuídas em 4 grupos experimentais: CC: fêmeas não estressadas; CE: fêmeas estressadas pós-natalmente aos 60 dias de idade (PND60), EC: fêmeas nascidas de mães estressadas entre o dia 15 (GD15) e 18 de gestação (GD18); EE: fêmeas nascidas de mães estressadas entre o GD15 e GD18 e estressadas pós-natalmente aos PND60. A indução da inflamação alérgica pulmonar foi realizada através da sensibilização dos animais com solução de ovalbumina (OVA) 0,1 mg.Kg-1 sc para avaliação do leucograma, lavado broncoalveolar (BAL), celularidade hematopoiética medular e neuroquímica. Os experimentos foram realizados 24h após a última sessão de nebulização. O número de células do BAL foi significantemente maior nos animais do grupo EE, em relação àqueles dos grupos CC (P0.05) para os linfócitos, neutrófilos, eosinófilos e monócitos; porém, observou-se diferenças significativas (P<0.05) entre o número de bastonetes dos grupos, sendo maior nos animais do grupo CC em relação àqueles do grupo EC. O número de células hematopoiéticas da medula óssea foi significantemente (P<0.05) menor nos animais do grupo EE, em relação àqueles do grupo CC. No córtex pré-frontal, há diferenças significantes na relação Ácido Homovanílico/ Dopamina (HVA/DA) (P<0.05), sendo maior nos animais do grupo EC, em relação àqueles do grupo CE. Em conclusão, o estresse pré-natal levou a modulação de células do sistema imune (SI) dos neonatos, evidenciado após a exposição a estresse agudo pós-natal, amplificando a resposta alérgica pulmonar. Sugere-se que a maior susceptibilidade dos animais do grupo EE seja consequência de alterações induzidas pelo estresse pré-natal no eixo hipotálamo-pituitária-adrenal (HPA).

Debido a su rápido crecimiento el feto es particularmente vulnerable a los cambios en el ambiente hormonal. Esto sugiere que situaciones adversas de la madre durante la gestación pueden alterar el desarrollo y la salud de la descendencia, principalmente debido a la permeabilidad de la barrera placentaria a diversas hormonas y sustancias. El objetivo del presente trabajo fue estudiar el efecto del estrés prenatal sobre la regulación de la inflamación alérgica pulmonar, empleando el modelo murino de asma experimental. Para este propósito fueron utilizadas ratonas vírgenes de linaje suizo de 50 d de edad. Fue empleado el modelo de descargas eléctricas en las patas (Footshock) para inducir el estrés prenatal y el modelo de estrés denominado "metro de Nueva York" para el estrés posnatal. Las hembras fueron divididas en 4 grupos experimentales: CC: hembras no estresadas; CE: hembras estresadas posnatalmente a los 60 d de edad (PND60); EC: hembras nacidas de madres estresadas entre el día 15 (GD15) y 18 de gestación (GD18); EE: hembras nacidas de madres estresadas entre el GD15 y GD18 y estresadas posnatalmente al PND60. La inducción de la inflamación alérgica pulmonar fue realizada a través de la sensibilización de los animales con solución de ovoalbúmina (OVA) 0,1 mg.Kg-1 sc. para posteriormente evaluar leucograma, lavado broncoalveolar (BAL), celularidad hematopoyética medular y neuroquímica. Los experimentos fueron realizados 24 horas después de la última sesión de nebulización. El número de células del BAL fue significativamente mayor en los animales del grupo EE, en comparación con los del grupo CC (P0.05) para los linfocitos, neutrófilos, eosinófilos y monocitos; sin embargo, se observaron diferencias significativas (P<0.05) entre los grupos en el número de bastonetes, siendo mayor en los animales del grupo CC en relación al grupo EC. El número de células hematopoyéticas de la médula ósea fue significativamente menor (P<0.05) en los animales del Grupo EE, en comparación con los del grupo de CC. En la corteza prefrontal, hubo diferencias significativas en la relación Ácido Homovanílico/ Dopamina (HVA/DA) (P<0.05), siendo mayor en los animales del grupo EC, en comparación con los del grupo CE. En conclusión, el estrés prenatal produjo modulación de las células del sistema inmune (SI) de los neonatos, evidenciado después de la exposición a un estrés agudo posnatal, por la amplificación de la respuesta alérgica pulmonar. Se sugiere que la mayor susceptibilidad de los animales del grupo EE sea resultado de los cambios inducidos por el estrés prenatal en el eje hipotálamo-pituitaria-adrenal (HPA).

Due to the rapid growth of the fetus it is particularly vulnerable to insults and changes in hormonal milieu. Therefore, is suggested that adverse situations experienced by the pregnant mother can alter the development and health of offspring, mainly due to the permeability of the placental barrier to various hormones and substances. The aim of the present investigation was to study the effects of prenatal stress in the regulation of pulmonary allergic inflammation, employing the murine model of experimental asthma. For this purpose, were used virgin female mice, Swiss lineage, of 50 days old. The models used were foot shock to induce prenatally stress, and "New York subway" stress to induce postnatally stress. Females were divided into 4 groups: CC group: not stressed females; CE group: postnatally stressed females (PND60); EC: females born from stressed mothers (GD15 to GD18); EE Group: females born from stressed mothers (GD15 to GD18) (footshock) and postnatally stressed (PND60). The induction of allergic pulmonary inflammation was done through sensitization of animals with 0,1 mg.Kg-1 sc of ovalbumin (OVA) solution, to further evaluate leukogram, bronchoalveolar lavage (BAL) hematopoietic marrow cellularity and neurochemistry. The experiments were performed 24 hours after the last session of nebulization. The number of BAL cells was significantly higher in EE group animals compared with the CC group (P0.05) for lymphocytes, neutrophils, eosinophils and monocytes; however, there were significant differences (P<0.05) observed in the number of rods cells between groups, being higher in animals the CC group compared to EC group. The number of hematopoietic cells of the bone marrow was significantly lower (P<0.05) in animals of Group EE, compared with CC group. In the prefrontal cortex, there were significant differences in homovanillic acid /dopamine (HVA/DA) (P<0.05) rate, being higher in the EC group, compared to EC group. In conclusion, prenatal stress modulated the immune system (SI) cells of neonates, evidenced after exposure to a post-natal acute stress by amplification of pulmonary allergic response. It is suggested that the increased susceptibility of animals EE group is a result of changes induced by prenatal stress on hypothalamus pituitary-adrenal (HPA) axis.

Correction: Validation of Novel Biomarkers for Prostate Cancer Progression by the Combination of Bioinformatics, Clinical and Functional Studies.

[This corrects the article DOI: 10.1371/journal.pone.0155901.].

Validation of Novel Biomarkers for Prostate Cancer Progression by the Combination of Bioinformatics, Clinical and Functional Studies.

The identification and validation of biomarkers for clinical applications remains an important issue for improving diagnostics and therapy in many diseases, including prostate cancer. Gene expression profiles are routinely applied to identify diagnostic and predictive biomarkers or novel targets for cancer. However, only few predictive markers identified in silico have also been validated for clinical, functional or mechanistic relevance in disease progression. In this study, we have used a broad, bioinformatics-based approach to identify such biomarkers across a spectrum of progression stages, including normal and tumor-adjacent, premalignant, primary and late stage lesions. Bioinformatics data mining combined with clinical validation of biomarkers by sensitive, quantitative reverse-transcription PCR (qRT-PCR), followed by functional evaluation of candidate genes in disease-relevant processes, such as cancer cell proliferation, motility and invasion. From 300 initial candidates, eight genes were selected for validation by several layers of data mining and filtering. For clinical validation, differential mRNA expression of selected genes was measured by qRT-PCR in 197 clinical prostate tissue samples including normal prostate, compared against histologically benign and cancerous tissues. Based on the qRT-PCR results, significantly different mRNA expression was confirmed in normal prostate versus malignant PCa samples (for all eight genes), but also in cancer-adjacent tissues, even in the absence of detectable cancer cells, thus pointing to the possibility of pronounced field effects in prostate lesions. For the validation of the functional properties of these genes, and to demonstrate their putative relevance for disease-relevant processes, siRNA knock-down studies were performed in both 2D and 3D organotypic cell culture models. Silencing of three genes (DLX1, PLA2G7 and RHOU) in the prostate cancer cell lines PC3 and VCaP by siRNA resulted in marked growth arrest and cytotoxicity, particularly in 3D organotypic cell culture conditions. In addition, silencing of PLA2G7, RHOU, ACSM1, LAMB1 and CACNA1D also resulted in reduced tumor cell invasion in PC3 organoid cultures. For PLA2G7 and RHOU, the effects of siRNA silencing on proliferation and cell-motility could also be confirmed in 2D monolayer cultures. In conclusion, DLX1 and RHOU showed the strongest potential as useful clinical biomarkers for PCa diagnosis, further validated by their functional roles in PCa progression. These candidates may be useful for more reliable identification of relapses or therapy failures prior to the recurrence local or distant metastases.