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Background Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. Methods Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. Results Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1β and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. Conclusions These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

Accurate estimate of fetal maturity could provide individualized guidance for delivery of complicated pregnancies. However, current methods are invasive, have low accuracy, and are limited to fetal lung maturation. To identify diagnostic gestational biomarkers, we performed transcriptomic profiling of lung and brain, as well as cell-free RNA from amniotic fluid of preterm and term rhesus macaque fetuses. These data identify potentially new and prior-associated gestational age differences in distinct lung and neuronal cell populations when compared with existing single-cell and bulk RNA-Seq data. Comparative analyses found hundreds of genes coincidently induced in lung and amniotic fluid, along with dozens in brain and amniotic fluid. These data enable creation of computational models that accurately predict lung compliance from amniotic fluid and lung transcriptome of preterm fetuses treated with antenatal corticosteroids. Importantly, antenatal steroids induced off-target gene expression changes in the brain, impinging upon synaptic transmission and neuronal and glial maturation, as this could have long-term consequences on brain development. Cell-free RNA in amniotic fluid may provide a substrate of global fetal maturation markers for personalized management of at-risk pregnancies.

Serotonin receptor 4 (5-HT4R) plays an important role in regulating mood, anxiety, and cognition, and drugs that activate this receptor have fast-acting antidepressant (AD)-like effects in preclinical models. However, 5-HT4R is widely expressed throughout the central nervous system (CNS) and periphery, making it difficult to pinpoint the cell types and circuits underlying its effects. Therefore, we generated a Cre-dependent 5-HT4R knockout mouse line to dissect the function of 5-HT4R in specific brain regions and cell types. We show that the loss of functional 5-HT4R specifically from excitatory neurons of hippocampus led to robust AD-like behavioral responses and an elevation in baseline anxiety. 5-HT4R was necessary to maintain the proper excitability of dentate gyrus (DG) granule cells and cell type-specific molecular profiling revealed a dysregulation of genes necessary for normal neural function and plasticity in cells lacking 5-HT4R. These adaptations were accompanied by an increase in the number of immature neurons in ventral, but not dorsal, dentate gyrus, indicating a broad impact of 5-HT4R loss on the local cellular environment. This study is the first to use conditional genetic targeting to demonstrate a direct role for hippocampal 5-HT4R signaling in modulating mood and anxiety. Our findings also underscore the need for cell type-based approaches to elucidate the complex action of neuromodulatory systems on distinct neural circuits.

Neurodegenerative diseases are characterised by progressive damage to the nervous system including the selective loss of vulnerable populations of neurons leading to motor symptoms and cognitive decline. Despite millions of people being affected worldwide, there are still no drugs that block the neurodegenerative process to stop or slow disease progression. Neuronal death in these diseases is often linked to the misfolded proteins that aggregate within the brain (proteinopathies) as a result of disease-related gene mutations or abnormal protein homoeostasis. There are two major degradation pathways to rid a cell of unwanted or misfolded proteins to prevent their accumulation and to maintain the health of a cell: the ubiquitin–proteasome system and the autophagy–lysosomal pathway. Both of these degradative pathways depend on the modification of targets with ubiquitin. Aging is the primary risk factor of most neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. With aging there is a general reduction in proteasomal degradation and autophagy, and a consequent increase of potentially neurotoxic protein aggregates of β-amyloid, tau, α-synuclein, SOD1 and TDP-43. An often over-looked yet major component of these aggregates is ubiquitin, implicating these protein aggregates as either an adaptive response to toxic misfolded proteins or as evidence of dysregulated ubiquitin-mediated degradation driving toxic aggregation. In addition, non-degradative ubiquitin signalling is critical for homoeostatic mechanisms fundamental for neuronal function and survival, including mitochondrial homoeostasis, receptor trafficking and DNA damage responses, whilst also playing a role in inflammatory processes. This review will discuss the current understanding of the role of ubiquitin-dependent processes in the progressive loss of neurons and the emergence of ubiquitin signalling as a target for the development of much needed new drugs to treat neurodegenerative disease.

Most drugs and xenobiotics are metabolized in the liver. Amongst others, different cytochrome P450 (CYP) enzymes catalyze the metabolic conversion of foreign compounds, and various transport proteins are engaged in the excretion of metabolites from the hepatocytes. Inter-species and inter-individual differences in the hepatic levels and activities of drug-metabolizing enzymes and transporters result from genetic as well as from environmental factors, and play a decisive role in determining the pharmacokinetic properties of a compound in a given test system. To allow for a meaningful comparison of results from metabolism studies, it is, therefore, of utmost importance to know about the specific metabolic properties of the test systems, especially about the levels of metabolic enzymes such as the CYPs. Using a targeted proteomics approach, we, therefore, compared the hepatic levels of important CYP enzymes and transporters in different experimental systems in vivo and in vitro, namely Wistar rats, C57/Bl6 mice, mice humanized for the two xeno-sensing receptors PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor), mice with human hepatocyte-repopulated livers, human HepaRG hepatocarcinoma cells, primary human hepatocytes, and human liver biopsies. In addition, the effects of xenobiotic inducers of drug metabolism on CYP enzymes and transporters were analyzed in selected systems. This study for the first time presents a comprehensive overview of similarities and differences in important drug metabolism-related proteins among the different experimental models. Electronic supplementary material The online version of this article (10.1007/s00204-020-02939-4) contains supplementary material, which is available to authorized users.

We review the history of antenatal corticosteroid therapy (ACS) and present recent experimental data to demonstrate that this, one of the pillars of perinatal care, has been inadequately evaluated to minimize fetal exposure to these powerful medications. There have been concerns since 1972 that fetal exposures to ACS convey risk. However, this developmental modulator, with its multiple widespread biologic effects, has not been evaluated for drug choice, dose, or duration of treatment, despite over 30 randomized trials. The treatment used in the United States is two intramuscular doses of a mixture of 6 mg betamethasone phosphate (Beta P) and 6 mg betamethasone acetate (Beta Ac). To optimize outcomes with ACS, the goal should be to minimize fetal drug exposure. We have determined that the minimum exposure needed for fetal lung maturation in sheep, monkeys, and humans (based on published cord blood corticosteroid concentrations) is about 1 ng/ml for a 48-h continuous exposure, far lower than the concentration reached by the current dosing. Because the slowly released Beta Ac results in prolonged fetal exposure, a drug containing Beta Ac is not ideal for ACS use.ImpactUsing sheep and monkey models, we have defined the minimum corticosteroid exposure for a fetal lung maturation.These results should generate new clinical trials of antenatal corticosteroids (ACS) at much lower fetal exposures to ACS, possibly given orally, with fewer risks for the fetus.

Background: The addition of budesonide (Bud) 0.25 mg/kg to surfactant decreased the lung and systemic responses to mechanical ventilation in preterm sheep and the rates and severity of bronchopulmonary dysplasia (BPD) in preterm infants. We hypothesized that lower budesonide concentrations in surfactant will decrease injury while decreasing systemic corticosteroid exposure. Methods: Preterm lambs received either (1) protective tidal volume (V T) ventilation with surfactant from birth or (2) injurious V T ventilation for 15 min and then surfactant treatment. Lambs were further assigned to surfactant mixed with (i) Saline, (ii) Bud 0.25 mg/kg, (iii) Bud 0.1 mg/kg, or (iv) Bud 0.04 mg/kg. All lambs were then ventilated with protective V T for 6 h. Results: Plasma Bud levels were proportional to the dose received and decreased throughout ventilation. In both protective and injurious V T ventilation

Xenobiotica-metabolizing enzyme (XME) induction is a relevant biological/biochemical process vital to understanding the toxicological profile of xenobiotics. Early recognition of XME induction potential of compounds under development is therefore important, yet its determination by traditional XME activity measurements is time consuming and cost intensive. A proof-of-principle study was therefore designed due to the advent of faster and less cost-intensive methods for determination of enzyme protein and transcript levels to determine whether two such methods may substitute for traditional measurement of XME activity determinations. The results of the study show that determination of enzyme protein levels by peptide group-specific immunoaffinity enrichment/MS and/or determination of gene expression by NanoString nCounter may serve as substitutes for traditional evaluation methodology and/or as an early predictor of potential changes in liver enzymes. In this study, changes of XME activity by the known standard XME inducers phenobarbital, beta-naphthoflavone and Aroclor 1254 were demonstrated by these two methods. To investigate the applicability of these methods to demonstrate XME-inducing activity of an unknown, TS was also examined and found to be an XME inducer. More specifically, TS was found to be a phenobarbital-type inducer (likely mediated by CAR rather than PXR as nuclear receptor), but not due to Ah receptor-mediated or antioxidant response element-mediated beta-naphthoflavone-type induction. The results for TS were confirmed via enzymatic activity measurements. The results of the present study demonstrate the potential applicability of NanoString nCounter mRNA quantitation and peptide group-specific immunoaffinity enrichment/MS protein quantitation for predicting compounds under development to be inducers of liver XME activity.

Antenatal steroid (ANS) therapy is the standard care for women at imminent risk of preterm labor. Despite extensive and long-standing use, 40%–50% of babies exposed antenatally to steroids do not derive benefit; remaining undelivered 7 days or more after ANS treatment is associated with a lack of treatment benefit and increased risk of harm. We used a pregnant sheep model to evaluate the impact of continuous versus pulsed ANS treatments on fetal lung maturation at an extended, 8-day treatment to delivery interval. Continuous low-dose ANS treatments for more than 72 h in duration improved fetal lung maturation at 8 days after treatment initiation. If fetal ANS exposure was interrupted, the beneficial ANS effect was lost. Truncated treatments, including that simulating the current clinical treatment regimen, did not improve lung function. Variable fetal lung maturation was correlated to the amount of saturated phosphatidylcholine present in the lung fluid. These data demonstrate that 1) the durability of ANS therapy may be enhanced by employing an extended, low-dose treatment regimen by reducing total dose and 2) interrupting the continuity of fetal exposure by allowing it to fall below a minimal threshold was associated with comparably poor functional maturation of the preterm ovine lung.

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

Extracellular vesicles (EVs) have garnered increasing interest as delivery vehicles for multiple classes of therapeutics based on their role as mediators in an important, natural intercellular communication system. We recently described a platform to allow the design, production and in vivo study of human EVs with specific properties (drug or tropism modifiers). This article seeks to compare and expand upon historical biodistribution and kinetic data by comparing systemically and compartmentally administered labeled engineered EVs using in vivo and ex vivo techniques.EVs were surface-labeled to high radiochemical purity and specific activity withOver time, systemic administration of engineered EVs distributed preferentially to the liver and spleen (Intravenous, IV), gastrointestinal tract and lymph nodes (Intraperitoneal, IP) and local/regional lymph nodes (Subcutaneous, SC). Immunostaining of dissected organs displaying PET signal revealed co-localization of an EV marker (PTGFRN) with a subset of macrophage markers (CD206, F4/80, IBA1). Compartmental dosing into NHP cerebrospinal fluid (CSF) resulted in a heterogenous distribution of labeled EVs depending upon whether the route was intrathecal (ITH), intracisterna magna (ICM) or intracerebroventricular (ICV), compared to the homogeneous distribution observed in rodents. Thus anatomically, ITH administration in NHP revealed meningeal distribution along the neuraxis to the base of the skull. In contrast ICM and ICV dosing resulted in meningeal distribution around the skull and to the cervical and thoracic spinal column. Further characterization using IHC shows uptake in a subset of meningeal macrophages.The present studies provide a comprehensive assessment of the fate of robustly and reproducibly labeled engineered EVs across several mammalian species. The in vivo distribution was observed to be both spatially and temporally dependent upon the route of administration providing insight into potential targeting opportunities for engineered EVs carrying a therapeutic payload.

Pemphigoid diseases are autoimmune chronic inflammatory skin diseases, which are characterized by blistering of the skin and/or mucous membranes, and circulating and tissue-bound autoantibodies. The well-established pathomechanisms comprise autoantibodies targeting various structural proteins located at the dermal-epidermal junction, leading to complement factor binding and activation. Several effector cells are thus attracted and activated, which in turn inflict characteristic tissue damage and subepidermal blistering. Moreover, the detection of linear complement deposits in the skin is a diagnostic hallmark of all pemphigoid diseases. However, recent studies showed that blistering might also occur independently of complement. This review reassesses the importance of complement in pemphigoid diseases based on current research by contrasting and contextualizing data from in vitro, murine and human studies.

In many vertebrates, courtship occurs through the performance of elaborate behavioral displays that are as spectacular as they are complex. The question of how sexual selection acts upon these animals’ neuromuscular systems to transform a repertoire of pre-existing movements into such remarkable (if not unusual) display routines has received relatively little research attention. This is a surprising gap in knowledge, given that unraveling this extraordinary process is central to understanding the evolution of behavioral diversity and its neural control. In many vertebrates, courtship displays often push the limits of neuromuscular performance, and often in a ritualized manner. These displays can range from songs that require rapid switching between two independently controlled ‘voice boxes’ to precisely choreographed acrobatics. Here, we propose a framework for thinking about how the brain might not only control these displays, but also shape their evolution. Our framework focuses specifically on a major midbrain area, which we view as a likely important node in the orchestration of the complex neural control of behavior used in the courtship process. This area is the periaqueductal grey (PAG), as studies suggest that it is both necessary and sufficient for the production of many instinctive survival behaviors, including courtship vocalizations. Thus, we speculate about why the PAG, as well as its key inputs, might serve as targets of sexual selection for display behavior. In doing so, we attempt to combine core ideas about the neural control of behavior with principles of display evolution. Our intent is to spur research in this area and bring together neurobiologists and behavioral ecologists to more fully understand the role that the brain might play in behavioral innovation and diversification.

Dendritic cells (DCs) activated via TLR ligation experience metabolic reprogramming, in which the cells are heavily dependent on glucose and glycolysis for the synthesis of molecular building blocks essential for maturation, cytokine production, and the ability to stimulate T cells. Although the TLR‐driven metabolic reprogramming events are well documented, fungal‐mediated metabolic regulation via C‐type lectin receptors such as Dectin‐1 and Dectin‐2 is not clearly understood. Here, we show that activation of DCs with fungal‐associated β‐glucan ligands induces acute glycolytic reprogramming that supports the production of IL‐1β and its secretion subsequent to NOD‐, LRR‐ and pyrin domain‐containing protein 3 (NLRP3) inflammasome activation. This acute glycolytic induction in response to β‐glucan ligands requires spleen tyrosine kinase signaling in a TLR‐independent manner, suggesting now that different classes of innate immune receptors functionally induce conserved metabolic responses to support immune cell activation. These studies provide new insight into the complexities of metabolic regulation of DCs immune effector function regarding cellular activation associated with protection against fungal microbes., Fungal‐associated Beta‐glucan ligands induce acute glycolytic reprogramming in DCs in a Syk‐dependent TLR‐independent manner that contributes to IL‐1beta production.

The sphenopalatine ganglion (SPG) is a vasoactive mediator of the anterior intracranial circulation in mammals. SPG stimulation has been demonstrated to alter blood-brain barrier (BBB) permeability, although this phenomenon is not well characterized.To determine the effect of SPG stimulation on the BBB using rat models.Extravasation of fluorescent tracer 70 kDa FITC-dextran into rat brain specimens was measured across a range of stimulation parameters to assess BBB permeability. Tight junction (TJ) morphology was compared by assessing differences in the staining of proteins occludin and ZO-1 and analyzing ultrastructural changes on transmission electron microscopy (TEM) between stimulated and unstimulated specimens.SPG stimulation at 10 Hz maximally increased BBB permeability, exhibiting a 6-fold increase in fluorescent traceruptake (1.66% vs 0.28%, p 0.0001). This effect was reversed 4-hours after stimulation (0.36% uptake, p = 0.99). High-frequency stimulation at 20 Hz and 200 Hz did not increase tracer extravasation, (0.26% and 0.28% uptake, p = 0.999 and p = 0.998, respectively). Stimulation was associated a significant decrease in the colocalization of occludin and ZO-1 with endothelial markers in stimulated brains compared to control (74.6% vs. 39.7% and 67.2% vs. 60.4% colocalization, respectively, p 0.0001), and ultrastructural changes in TJ morphology associated with increased BBB permeability were observed on TEM.This study is the first to show a reversible, frequency-dependent increase in BBB permeability with SPG stimulation and introduces a putative mechanism of action through TJ disruption. Bypassing the BBB with SPG stimulation could enable new paradigms in delivering therapeutics to the CNS. Further study of this technology is needed.

Background The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. Methods Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. Results Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p

In mood disorders, psychomotor and sensory abnormalities are prevalent, disabling, and intertwined with emotional and cognitive symptoms. Corticostriatal neurons in motor and somatosensory cortex are implicated in these symptoms, yet mechanisms of their vulnerability are unknown. Here, we demonstrate that S100a10 corticostriatal neurons exhibit distinct serotonin responses and have increased excitability, compared with S100a10-negative neurons. We reveal that prolonged social isolation disrupts the specific serotonin response which gets restored by chronic antidepressant treatment. We identify cell-type-specific transcriptional signatures in S100a10 neurons that contribute to serotonin responses and strongly associate with psychomotor and somatosensory function. Our studies provide a strong framework to understand the pathogenesis and create new avenues for the treatment of mood disorders.

Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, noninvasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T-cell–specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor-bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody–treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and nonresponders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor-draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy. Significance: These findings reveal differences in T-cell activation between responders and nonresponders early into anti-PD-1 treatment, which may impact many facets of immuno-oncology, including patient selection, management, and development of novel combinatorial approaches.

Songbirds hold a prominent role in the fields of neurobiology, evolution, and social behavior. Many of these fields have assumed that females lacked the ability to produce song and have therefore treated song as a male-specific behavior. Consequently, much of our understanding regarding the evolution and neural control of song behavior has been driven by these assumptions. Here we review literature from diverse fields to provide a broader perspective of the role of females in vocal communication and courtship. Recent evidence indicates that song evolved in both males and females and instances of female song are still common. The specialized neural circuit known as the "song system," which is necessary for singing in males, is also present in females, including those that do not sing, implying broader functions that include evaluating male song and controlling courtship behavior. In addition to having flexible, individualized preferences, females actively shape their social network through their interactions with males, females, and juveniles. We suggest that by developing more accurate hypotheses concerning the role of females we may better understand the evolution and neural mechanisms of song production and courtship behavior.

Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of nonruminant PAPs for use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, because the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here, we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides, and LC-MS/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of six ring trial samples obtained in former PCR and microscopy proficiency tests. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.

Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development.Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed.DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT.This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH.There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.

Hyperoxia-induced lung injury plays a key role in the development of bronchopulmonary dysplasia (BPD), characterized by inflammatory injury and impaired lung development in preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, currently it is uncertain how lung injury contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that regulate intercellular and inter-organ communications. Gasdermin D (GSDMD) has emerged as a key executor of inflammasome-mediated cell death and inflammation. In this study, we utilized a neonatal rat model of BPD to assess if hyperoxia stimulates lung release of circulating EVs and if these EVs induce lung and brain injury. We found that hyperoxia-exposed rats had elevated numbers of plasma-derived EVs compared to rats maintained in room air. These EVs also had increased cargos of surfactant protein C, a marker of type II alveolar epithelial cells (AEC), and the active (p30) form of GSDMD. When these EVs were adoptively transferred into normal newborn rats via intravenous injection, they were taken up both by lung and brain tissues. Moreover, EVs from hyperoxic animals induced not only the pathological hallmarks of BPD, but also brain inflammatory injury in recipient rats, as well as inducing cell death in cultured pulmonary vascular endothelial cells and neural stem cells (NSC). Similarly, hyperoxia-exposed cultured AEC-like cells released EVs that also contained increased GSDMD-p30 and these EVs induced pyroptotic cell death in NSC. Overall, these data indicate that hyperoxia-activated circulating EVs mediate a lung to brain crosstalk resulting in brain injury and suggest a mechanism that links lung injury and neurodevelopmental impairment in BPD infants.

This chapter on therapeutic drift with antenatal steroids will make the case that this pilar of treatment to improve the outcomes of preterm infants, despite multiple Randomized Control Trials (RCTs) and meta-analysis, has multiple gaps in solid clinical data to support any expanded use of Antenatal Corticosteroids (ACS). A basic problem is that agents used for ACS have never been evaluated to minimize fetal exposures. Based on the premise that all drug exposure to the fetus should be minimized and only used when necessary, ACS is a potent developmental modulator that has never been evaluated to minimize the dose and duration of fetal exposure. The use of ACS is expanding to late preterm infants where the benefit is modest, to elective C-sections, and periviable fetuses, with minimal RCT data of long-term benefit. Relevant animal experiments demonstrate that much lower doses will induce lung maturation in sheep and primates. Another area of drift in the use of ACS is based on the assumption that the old RCT data accurately predict the magnitude of benefit when ACS is used today with entirely different OB and neonatal care strategies to improve outcomes. We do not have data that demonstrate the effectiveness of ACS in very low resource environments, where most of the preterm mortality occurs. The final concern is the risk of ACS to the infant and child. Short-term risks are minimal but dysmaturation effects of ACS on multiple organ systems (lung, heart, brain, and kidney) may result in disease presentation in later life.

Respiratory complicˆations are the major cause of morbidity and mortality among preterm infants, which is partially prevented by the administration of antenatal corticosteroids (ACS). Most very preterm infants are exposed to chorioamnionitis, but short- and long-term effects of ACS treatment in this setting are not well defined. In low-resource settings, ACS increased neonatal mortality by perhaps increasing infection. We report that treatment with low-dose ACS in the setting of inflammation induced by intraamniotic lipopolysaccharide (LPS) in rhesus macaques improves lung compliance and increases surfactant production relative to either exposure alone. RNA sequencing shows that these changes are mediated by suppression of proliferation and induction of mesenchymal cellular death via TP53. The combined exposure results in a mature-like transcriptomic profile with inhibition of extracellular matrix development by suppression of collagen genes COL1A1, COL1A2, and COL3A1 and regulators of lung development FGF9 and FGF10. ACS and inflammation also suppressed signature genes associated with proliferative mesenchymal progenitors similar to the term gestation lung. Treatment with ACS in the setting of inflammation may result in early respiratory advantage to preterm infants, but this advantage may come at a risk of abnormal extracellular matrix development, which may be associated with increased risk of chronic lung disease.

The family of human muscarinic acetylcholine receptors (MRs) is characterized by a high sequence homology among the five subtypes (M

Norepinephrine (NE) function is often implicated in regulating arousal levels. Recent theory suggests that the noradrenergic system also regulates the optimization of behavior with respect to reward maximization by controlling a switch between exploration and exploitation of the specific actions that yield greatest utility. We show in the songbird that NE can act directly on a cortical motor area and cause a switch between exploratory and exploitative behavior.

Background Antenatal corticosteroid therapy is a standard of care for women at imminent risk of preterm labor. However, the optimal (maximum benefit and minimal risk of side effects) antenatal corticosteroid dosing strategy remains unclear. Although conveying overall benefit when given to the right patient at the right time, antenatal corticosteroid treatment efficacy is highly variable and is not risk-free. Building on earlier findings, we hypothesized that when administered in combination with slow-release betamethasone acetate, betamethasone phosphate and the high maternal-fetal betamethasone concentrations it generates are redundant for fetal lung maturation. Objective Using an established sheep model of prematurity and postnatal ventilation of the preterm lamb, we aimed to compare the pharmacodynamic effects of low-dosage treatment with betamethasone acetate only against a standard dosage of betamethasone phosphate and betamethasone acetate as recommended by the American College of Obstetricians and Gynecologists for women at risk of imminent preterm delivery between 24 0/7 and 35 6/7 weeks’ gestation. Study Design Ewes carrying a single fetus at 122±1 days’ gestation (term=150 days) were randomized to receive either (1) maternal intramuscular injections of sterile saline (the saline negative control group, n=12), (2) 2 maternal intramuscular injections of 0.25 mg/kg betamethasone phosphate+betamethasone acetate administered at 24-hour dosing intervals (the betamethasone phosphate+betamethasone acetate group, n=12); or (3) 2 maternal intramuscular injections of 0.125 mg/kg betamethasone acetate administered at 24-hour dosing intervals (the betamethasone acetate group, n=11). The fetuses were surgically delivered 48 hours after treatment initiation and ventilated for 30 minutes to determine functional lung maturation. The fetuses were euthanized after ventilation, and the lungs were collected for analysis using quantitative polymerase chain reaction and Western blot assays. Fetal plasma adrenocorticotropic hormone levels were measured in the cord blood samples taken at delivery. Results Preterm lambs were defined as either antenatal corticosteroid treatment responders or nonresponders using an arbitrary cutoff, being a PaCO2 level at 30 minutes of ventilation being more extreme than 2 standard deviations from the mean value of the normally distributed saline control group values. Compared with the animals in the saline control group, the animals in the antenatal corticosteroid treatment groups showed significantly improved lung physiological responses (blood gas and ventilation data) and had a biochemical signature (messenger RNA and surfactant protein assays) consistent with functional maturation. However, the betamethasone acetate group had a significantly higher treatment response rate than the betamethasone phosphate+betamethasone acetate group. These physiological results were strongly correlated to the amount of surfactant protein A. Birthweight was lower in the betamethasone phosphate+betamethasone acetate group and the fetal hypothalamic-pituitary-adrenal axis was suppressed to a greater extent in the betamethasone phosphate+betamethasone acetate group. Conclusion Low-dosage antenatal corticosteroid therapy solely employing betamethasone acetate was sufficient for fetal lung maturation. The elevated maternal-fetal betamethasone concentrations associated with the coadministration of betamethasone phosphate did not in addition improve lung maturation but were associated with greater fetal hypothalamic-pituitary-adrenal axis suppression, a lower antenatal corticosteroid treatment response rate, and lower birthweight—outcomes not desirable in a clinical setting. These data warranted a clinical investigation of sustained low-dosage antenatal corticosteroid treatments that avoid high maternal-fetal betamethasone exposures.

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), lead to the selective degeneration of discrete cell types in the CNS despite the ubiquitous expression of many genes linked to disease. Therapeutic advancement depends on understanding the unique cellular adaptations that underlie pathology of vulnerable cells in the context of disease-causing mutations. Here, we employ bacTRAP molecular profiling to elucidate cell type-specific molecular responses of cortical upper motor neurons in a preclinical ALS model. Using two bacTRAP mouse lines that label distinct vulnerable or resilient projection neuron populations in motor cortex, we show that the regulation of oxidative phosphorylation (Oxphos) pathways is a common response in both cell types. However, differences in the baseline expression of genes involved in Stem and the handling of reactive oxygen species likely lead to the selective degeneration of the vulnerable cells. These results provide a framework to identify cell-type-specific processes in neurodegenerative disease.

With the reintroduction of nonruminant processed animal proteins (PAPs) for use in aquaculture in 2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date, light microscopy and polymerase chain reaction are the official methods for proving the absence of illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species differentiation are needed. To address this issue, we developed and validated an 8-plex mass spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-macroglobulin peptides using a group-specific antibody, and a subsequent analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry for species identification and quantification. This workflow can be used to quantitatively determine the species origin in future feed authentication studies.

Males and females use distinct brain circuits to cope with similar challenges. Using RNA sequencing of ribosome-bound mRNA from hippocampal CA3 neurons, we found remarkable sex differences and discovered that female mice displayed greater gene expression activation after acute stress than males. Stress-sensitive BDNF Val66Met mice of both sexes show a pre-stressed translational phenotype in which the same genes that are activated without applied stress are also induced in wild-type mice by an acute stressor. Behaviourally, only heterozygous BDNF Val66Met females exhibit spatial memory impairment, regardless of acute stress. Interestingly, this effect is not observed in ovariectomized heterozygous BDNF Val66Met females, suggesting that circulating ovarian hormones induce cognitive impairment in Met carriers. Cognitive deficits are not observed in males of either genotype. Thus, in a brain region not normally associated with sex differences, this work sheds light on ways that genes, environment and sex interact to affect the transcriptome’s response to a stressor., Animals’ response to acute stress is known to be influenced by sex and genetics. Here the authors performed RNA-seq on actively translated mRNAs in hippocampal CA3 neurons in mice, and document the effects of sex and genotype (i.e., BDNF Val66Met) on acute stress-induced gene expression.

Summary: Translational profiling methodologies enable the systematic characterization of cell types in complex tissues, such as the mammalian brain, where neuronal isolation is exceptionally difficult. Here, we report a versatile strategy for profiling CNS cell types in a spatiotemporally restricted fashion by engineering a Cre-dependent adeno-associated virus expressing an EGFP-tagged ribosomal protein (AAV-FLEX-EGFPL10a) to access translating mRNAs by translating ribosome affinity purification (TRAP). We demonstrate the utility of this AAV to target a variety of genetically and anatomically defined neural populations expressing Cre recombinase and illustrate the ability of this viral TRAP (vTRAP) approach to recapitulate the molecular profiles obtained by bacTRAP in corticothalamic neurons across multiple serotypes. Furthermore, spatially restricting adeno-associated virus (AAV) injections enabled the elucidation of regional differences in gene expression within this cell type. Altogether, these results establish the broad applicability of the vTRAP strategy for the molecular dissection of any CNS or peripheral cell type that can be engineered to express Cre. : Nectow et al. describe vTRAP, a technology to purify translating mRNAs from genetically defined cell types in a spatiotemporally restricted fashion. Multiplexing vTRAP with other technologies offers a comprehensive strategy to interrogate the precise role of individual, cell-type-specific genes in neural circuit function. Keywords: AAV, molecular profiling, TRAP, MCH, corticothalamic, Ntsr1, RNA-seq

The δ opioid receptor (δR) is a promising alternate target for pain management because δR agonists show decreased abuse potential compared with current opioid analgesics that target the μ opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacyin vivo, requiring the use of high doses that often cause adverse effects, such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacyin vivoby limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface bothin vitroandin vivo. PTEN inhibitionin vivoincreases the percentage of TG neurons expressing δR on the surface and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonistsin vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENTOpioid analgesics, such as morphine, which target the μ opioid receptor (μR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. Although the δ opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular localization of δR in neurons is a key factor that contributes to low agonist efficacy, and presents a proof of mechanism that relocating δR improves efficacy.

BACKGROUND: Dexamethasone-phosphate (Dex-PO4) and the combination betamethasone-phosphate (Beta-PO4) + betamethasone-acetate (Beta-Ac) are the most used antenatal corticosteroids to promote fetal lung maturation. We compared fetal lung maturation induced by Beta-Ac+Beta-PO4, Dex-PO4, or Beta-PO4 alone.METHODS: Pregnant ewes received two intramuscular doses 24 h apart of 0.25 mg/kg/dose of Beta-Ac+Beta-PO4, Dex-PO4 or Beta-PO4; or 2 doses of 0.125 mg/kg/dose of Beta-PO4 at 6, 12, or 24h intervals. Fetuses were delivered 48 h after the first dose and ventilated for 30 min. We assessed ventilatory variables, vital signs, and blood gas. After ventilation pressure-volume curves were measured and lungs were sampled for analysis.RESULTS: All treatments improved lung compliance and ventilation efficiency. Only Beta-Ac + Beta-PO4 required lower positive inspiratory pressure compared with control. Beta-Ac + Beta-PO4, and Beta-PO4, alone, but not Dex-PO4, increased the mRNA of surfactant proteins compared with control. Low-dose Beta-PO4 did not increase mRNA of surfactant proteins. There were no differences among Beta-PO4, treatment intervals.CONCLUSION: Beta-Ac + Beta-PO4 given as two doses 24 h apart was more effective in promoting fetal lung maturation than Dex-PO4 or Beta-PO4 alone, consistent with a prolonged exposure provided by the Beta-Ac + Beta-PO4. These results support the clinical use of combined Beta-Ac + Beta-PO4, preparations over phosphate corticosteroids alone for fetal lung maturation.

Supplemental oxygen (O2) therapy in preterm infants impairs lung development, but the impact of O2 on long‐term systemic vascular structure and function has not been well‐explored. The present study tested the hypothesis that neonatal O2 therapy induces long‐term structural and functional alterations in the systemic vasculature, resulting in vascular stiffness observed in children and young adults born preterm. Newborn Sprague‐Dawley rats were exposed to normoxia (21% O2) or hyperoxia (85% O2) for 1 and 3 weeks. A subgroup exposed to 3 weeks hyperoxia was recovered in normoxia for an additional 3 weeks. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound and pressure myography. Aorta remodeling was assessed by collagen deposition and expression. Left ventricular (LV) function was assessed by echocardiography. We found that neonatal hyperoxia exposure increased vascular stiffness at 3 weeks, which persisted after normoxic recovery at 6 weeks of age. These findings were accompanied by increased PWV, aortic remodeling, and altered LV function as evidenced by decreased ejection fraction, cardiac output, and stroke volume. Importantly, these functional changes were associated with increased collagen deposition in the aorta. Together, these findings demonstrate that neonatal hyperoxia induces early and sustained biomechanical alterations in the systemic vasculature and impairs LV function. Early identification of preterm infants who are at risk of developing systemic vascular dysfunction will be crucial in developing targeted prevention strategies that may improve the long‐term cardiovascular outcomes in this vulnerable population., Prolonged neonatal oxygen exposure causes persistent aortic structural and biomechanical alterations, which was even more pronounced after recovery in normoxia. This was accompanied by a marked impairment in left ventricular function. These findings have important implications as it suggests that premature babies exposed to oxygen may be predisposed to vascular dysfunction, leading to increased risk of cardiovascular disease in later life. As this affected population begins to age, there will be a critical need to identify the underlying pathophysiological mechanisms for these vascular morbidities in preterm survivors.

Background Administration of antenatal steroids is standard of care for women assessed to be at imminent risk of preterm delivery. There is a marked variation in antenatal steroid dosing strategy, selection for treatment criteria, and agent choice worldwide. This, combined with very limited optimization of antenatal steroid use per se, means that treatment efficacy is highly variable, and the rate of respiratory distress syndrome is decreased to perhaps as low as 40%. In some cases, antenatal steroid use is associated with limited benefit and potential harm. Objective We hypothesized that individual differences in maternofetal steroid exposure would contribute to observed variability in antenatal steroid treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between maternofetal steroid exposure and antenatal steroid treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. Study Design Ewes carrying a single fetus underwent surgery to catheterize a fetal and maternal jugular vein at 119 days’ gestation. Animals recovered for 24 hours before being randomized to either (1) a single maternal intramuscular injection of 2 mL saline (negative control group, n=10) or (2) a single maternal intramuscular injection of 0.25 mg/kg betamethasone phosphate plus acetate (antenatal steroid group, n=20). Serial maternal and fetal plasma samples were collected from each animal after 48 hours before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. Results One animal from the control group and one animal from the antenatal steroid group did not complete their treatment protocol and were removed from analyses. Animals in the antenatal steroid group were divided into a responder subgroup (n=12/19) and a nonresponder subgroup (n=7/19) using a cutoff of partial pressure of arterial CO2 at 30-minute ventilation within 2 standard deviations of the mean value from saline-treated negative control group animals. Although antenatal steroid improved fetal lung maturation in the undivided antenatal steroid group and in the responder subgroup both physiologically (blood gas– and ventilation-related data) and biochemically (messenger ribonucleic acid expression related to fetal lung maturation), these values did not improve relative to saline-treated control group animals in the antenatal steroid nonresponder subgroup. No differences in betamethasone distribution, clearance, or protein binding were identified between the antenatal steroid responder and nonresponder subgroups. Conclusion This study correlated individual maternofetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation that was not significantly different to saline-treated control group animals. These nonresponsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of antenatal steroid therapy is not solely determined by maternofetal drug levels and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid signaling.

Clostridium perfringens epsilon toxin (ETX) is responsible for causing the economically devastating disease, enterotoxaemia, in livestock. It is well accepted that ETX causes blood brain barrier (BBB) permeability, however the mechanisms involved in this process are not well understood. Using in vivo and in vitro methods, we determined that ETX causes BBB permeability in mice by increasing caveolae-dependent transcytosis in brain endothelial cells. When mice are intravenously injected with ETX, robust ETX binding is observed in the microvasculature of the central nervous system (CNS) with limited to no binding observed in the vasculature of peripheral organs, indicating that ETX specifically targets CNS endothelial cells. ETX binding to CNS microvasculature is dependent on MAL expression, as ETX binding to CNS microvasculature of MAL-deficient mice was not detected. ETX treatment also induces extravasation of molecular tracers including 376Da fluorescein salt, 60kDA serum albumin, 70kDa dextran, and 155kDA IgG. Importantly, ETX-induced BBB permeability requires expression of both MAL and caveolin-1, as mice deficient in MAL or caveolin-1 did not exhibit ETX-induced BBB permeability. Examination of primary murine brain endothelial cells revealed an increase in caveolae in ETX-treated cells, resulting in dynamin and lipid raft-dependent vacuolation without cell death. ETX-treatment also results in a rapid loss of EEA1 positive early endosomes and accumulation of large, RAB7-positive late endosomes and multivesicular bodies. Based on these results, we hypothesize that ETX binds to MAL on the apical surface of brain endothelial cells, causing recruitment of caveolin-1, triggering caveolae formation and internalization. Internalized caveolae fuse with early endosomes which traffic to late endosomes and multivesicular bodies. We believe that these multivesicular bodies fuse basally, releasing their contents into the brain parenchyma., Author summary Clostridium perfringens epsilon toxin (ETX) is an extremely lethal bacterial toxin known to cause a devastating disease in livestock animals and may be a possible cause of multiple sclerosis in humans. ETX is well known to cause disruption of the blood-brain barrier (BBB), a critical structure necessary for proper brain function. Deterioration of this barrier allows entry of toxic blood-borne material to enter the brain. Although ETX-induced BBB dysfunction is well accepted, how this happens is unknown. Here, we demonstrate that ETX causes BBB permeability by inducing formation of cell-surface invaginations called caveolae in endothelial cells, the cells that line blood vessels. Importantly, only endothelial cells from the brain and other central nervous system organs appear to be a target of ETX, as the toxin only binds to blood vessels in these organs and not blood vessels from other organs. These ETX-induced caveolae fuse with other caveolae and specialized intracellular vesicles called endosomes. We predict that these endosomes engulf blood-borne material during their internalization, allowing material to travel from the blood, through the cell, and into brain tissue. We also show that expression of the protein MAL and caveolin-1 is necessary for ETX-induced BBB permeability.

Mechanical ventilation from birth with normal tidal volumes (VT) causes lung injury and systemic responses in preterm sheep. The addition of budesonide to surfactant therapy decreases these injury markers. Budesonide and surfactant will decrease the injury from injurious VT ventilation in preterm sheep. Lambs at 126 ± 1 day gestational age were ventilated from birth with either: 1) Normal VT [surfactant 200 mg/kg before ventilation, positive end expiratory pressure (PEEP) 5 cmH2O, VT 8 mL/kg] or 2) Injury VT (high pressure, 100% oxygen, no PEEP) for 15 min, then further randomized to surfactant + saline or surfactant + 0.25 mg/kg budesonide with Normal VT for 6 h. Lung function and lung, liver, and brain tissues were evaluated for indicators of injury. Injury VT + saline caused significant injury and systemic responses, and Injury VT + budesonide improved lung physiology. Budesonide decreased lung inflammation and decreased pro-inflammatory cytokine mRNA in the lung, liver, and brain to levels similar to Normal VT + saline. Budesonide was present in plasma within 15 min of treatment in both ventilation groups, and less than 5% of the budesonide remained in the lung at 6 h. mRNA sequencing of liver and periventricular white matter demonstrated multiple pathways altered by both Injury VT and budesonide and the combination exposure. In lambs receiving Injury VT, the addition of budesonide to surfactant improved lung physiology and decreased pro-inflammatory cytokine responses in the lung, liver, and brain to levels similar to lambs receiving Normal VT.

Antenatal corticosteroids (ACS) are standard of care for women at risk of preterm delivery, although choice of drug, dose or route have not been systematically evaluated. Further, ACS are infrequently used in low resource environments where most of the mortality from prematurity occurs. We report proof of principle experiments to test betamethasone-phosphate (Beta-P) or dexamethasone-phosphate (Dex-P) given orally in comparison to the clinical treatment with the intramuscular combination drug beta-phosphate plus beta-acetate in a Rhesus Macaque model. First, we performed pharmacokinetic studies in non-pregnant monkeys to compare blood levels of the steroids using oral dosing with Beta-P, Dex-P and an effective maternal intramuscular dose of the beta-acetate component of the clinical treatment. We then evaluated maternal and fetal blood steroid levels with limited fetal sampling under ultrasound guidance in pregnant macaques. We found that oral Beta is more slowly cleared from plasma than oral Dex. The blood levels of both drugs were lower in maternal plasma of pregnant than in non-pregnant macaques. Using the pharmacokinetic data, we treated groups of 6-8 pregnant monkeys with oral Beta-P, oral Dex-P, or the maternal intramuscular clinical treatment and saline controls and measured pressure-volume curves to assess corticosteroid effects on lung maturation at 5d. Oral Beta-P improved the pressure-volume curves similarly to the clinical treatment. Oral Dex-P gave more variable and nonsignificant responses. We then compared gene expression in the fetal lung, liver and hippocampus between oral Beta-P and the clinical treatment by RNA-sequencing. The transcriptomes were largely similar with small gene expression differences in the lung and liver, and no differences in the hippocampus between the groups. As proof of principle, ACS therapy can be effective using inexpensive and widely available oral drugs. Clinical dosing strategies must carefully consider the pharmacokinetics of oral Beta-P or Dex-P to minimize fetal exposure while achieving the desired treatment responses.

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca(2+). SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission.

Antenatal corticosteroids (ANS) are the major intervention to decrease respiratory distress syndrome and mortality from premature birth and are standard of care. The use of ANS is expanding to include new indications and gestational ages, although the recommended dosing was never optimized. The most widely used treatment is two intramuscular doses of a 1:1 mixture of betamethasone-phosphate (Beta-P) and betamethasone-acetate (Beta-Ac) – the clinical drug. We tested in a primate model the efficacy of the slow release Beta-Ac alone for enhancing fetal lung maturation and to reduce fetal corticosteroid exposure and potential toxic effects. Pregnant rhesus macaques at 127 days of gestation (80% of term) were treated with either the clinical drug (0.25 mg/kg) or Beta-Ac (0.125 mg/kg). Beta-Ac alone increased lung compliance and surfactant concentration in the fetal lung equivalently to the clinical drug. By transcriptome analyses the early suppression of genes associated with immune responses and developmental pathways were less affected by Beta-Ac than the clinical drug. Promoter and regulatory analysis prediction identified differentially expressed genes targeted by the glucocorticoid receptor in the lung. At 5 days the clinical drug suppressed genes associated with neuronal development and differentiation in the fetal hippocampus compared to control, while low dose Beta-Ac alone did not. A low dose ANS treatment with Beta-Ac should be assessed for efficacy in human trials.

Treatment with antenatal corticosteroids (ACS) is standard of care for women at risk of preterm birth between 24 and 34 weeks' gestation. ACS are increasingly used for other indications, including threated or indicated late preterm birth, elective cesarean, and in at-risk pregnancies for periviable gestations. However, the various drugs and doses being used worldwide have not been rigorously evaluated to optimize clinical responses and to minimize potential risks. Translational research in animal models indicate that a constant, low concentration fetal exposure to ACS is sufficient for lung maturation, resulting in lower fetal exposures. Clinical trials to develop dosing strategies with inexpensive and widely available drugs could promote greater use in low resource environments.

Starting from the β-adrenoceptor agonist isoprenaline and beta-blocker carvedilol, we designed and synthesized three different chemotypes of agonist/antagonist hybrids. Investigations of ligand-mediated receptor activation using bioluminescence resonance energy transfer biosensors revealed a predominant effect of the aromatic head group on the intrinsic activity of our ligands, as ligands with a carvedilol head group were devoid of agonistic activity. Ligands composed of a catechol head group and an antagonist-like oxypropylene spacer possess significant intrinsic activity for the activation of Gαs, while they only show weak or even no β-arrestin-2 recruitment at both β1- and β2-AR. Molecular dynamics simulations suggest that the difference in G protein efficacy and β-arrestin recruitment of the hybrid ( S)-22, the full agonist epinephrine, and the β2-selective, G protein-biased partial agonist salmeterol depends on specific hydrogen bonding between Ser5.46 and Asn6.55, and the aromatic head group of the ligands.

The use of antenatal corticosteroids (ACS) in low-resource environments is sporadic. Further, drug choice, dose, and route of ACS are not optimized. We report the pharmacokinetics and pharmacodynamics of oral dosing of ACS using a preterm sheep model.We measured pharmacokinetics of oral betamethasone-phosphate (Beta-P) and dexamethasone-phosphate (Dex-P) using catheterized pregnant sheep. We compared fetal lung maturation responses of oral Beta-P and Dex-P to the standard treatment with 2 doses of the i.m. mixture of Beta-P and betamethasone-acetate at 2, 5, and 7 days after initiation of ACS.Oral Dex-P had lower bioavailability than Beta-P, giving a lower maximum maternal and fetal concentration. A single oral dose of 0.33 mg/kg of Beta-P was equivalent to the standard clinical treatment assessed at 2 days; 2 doses of 0.16 mg/kg of oral Beta-P were equivalent to the standard clinical treatment at 7 days as assessed by lung mechanics and gas exchange after preterm delivery and ventilation. In contrast, oral Dex-P was ineffective because of its decreased bioavailability.Using a sheep model, we demonstrate the use of pharmacokinetics to develop oral dosing strategies for ACS. Oral dosing is feasible and may facilitate access to ACS in low-resource environments.

Mechanical ventilation with normal tidal volumes (VT) causes lung and systemic inflammation in preterm sheep. Mechanical ventilation is associated with bronchopulmonary dysplasia (BPD) in preterm infants, and the addition of budesonide to surfactant decreases BPD in clinical trials. Budesonide with surfactant will decrease the lung injury from mechanical ventilation for 24 h in preterm sheep. Lambs at 126 ± 1 day gestational age were delivered and randomized to either: 1) surfactant (200 mg/kg) or 2) surfactant mixed with budesonide (0.25 mg/kg) before mechanical ventilation with VT of 7–8 ml/kg for 2, 6, or 24 h ( n = 6 or 7/group). Lung physiology and budesonide levels in the plasma and the lung were measured. Lung tissue, bronchoalveolar lavage fluid (BALF), liver, and brain tissues were evaluated for indicators of injury. High initial budesonide plasma levels of 170 ng/ml decreased to 3 ng/ml at 24 h. Lung tissue budesonide levels were less than 1% of initial dose by 24 h. Although physiological variables were generally similar, budesonide-exposed lambs required lower mean airway pressures, had higher hyperoxia responses, and had more stable blood pressures. Budesonide decreased proinflammatory mRNA in the lung, liver, and brain. Budesonide also decreased total protein and proinflammatory cytokines in BALF, and decreased inducible nitric oxide synthase activation at 24 h. In ventilated preterm lambs, most of the budesonide left the lung within 24 h. The addition of budesonide to surfactant improved physiology, decreased markers of lung injury, and decreased systemic responses in liver and brain.

One of the great unanswered biological questions is the absolute necessity of the polyunsaturated lipid docosahexaenoic acid (DHA; 22:6n-3) in retinal and neural tissues. Everything from the simple eye spot of dinoflagellates to cephalopods to every class of vertebrates uses DHA, yet it is abundant only in cold water marine food chains. Docosapentaenoic acids (DPAs; 22:5n-6 and especially 22:5n-3) are fairly plentiful in food chains yet cannot substitute for DHA. About 600 million years ago, multi-cellular, air breathing systems evolved rapidly and 32 phyla came into existence in a short geological time span; the "Cambrian Explosion". Eukaryotic intracellular detail requires cell membranes, which are constructed of complex lipids, and proteins. Proteins and nucleic acids would have been abundant during the first 2.5-5 billion years of anaerobic life but lipids, especially unsaturated fatty acids, would not. We hypothesize lipid biology was a key driver of the Cambrian Explosion, because it alone provides for compartmentalization and specialization within cells DHA has six methylene interrupted double bonds providing controlled electron flow at precise energy levels; this is essential for visual acuity and truthful execution of the neural pathways which make up our recollections, information processing and consciousness. The last double bond is critical for the evolution and function of the photoreceptor and neuronal and synaptic signaling systems. It completes a quantum mechanical device for the regulation of current flow with absolute signal precision based on electron tunneling (ET). DHA's methylene interruption distance is6 Å, making ET transfer between the π-orbitals feasible throughout the molecule. The possibility fails if one double bond is removed and replaced by a saturated bond as in the DPAs. The molecular biophysical foundation of neural signaling can also include the discrete pattern of paired spin states that arise in the DHA double bond and methylene regions. The complexity depends upon the number of C13 and H1 molecular sites in which spin states are coupled. Electron wave harmonics with entanglement and cohesion provide a mechanism for learning and memory, and power cognition and complex human brain functions.

Molecular imaging using near-infrared (NIR) fluorescence is useful for intraoperative imaging and real-time margin identification. Directly conjugated IR dyes possess useful properties for in vivo imaging, but conjugation often substantially alters the circulation dynamics of targeting moieties. We developed and characterized a new tumor-targeting probe, affiFAP, which consists of a protein that specifically binds EGFR (affibody) and a fluorogen activating protein (FAP). This compact molecular recognition reagent can reversibly bind and activate fluorescence of otherwise nonfluorescent dyes and allows tumor visualization with low nonspecific tissue staining. We demonstrate molecular pre-targeting of affiFAPs and subsequent systemic or topical application of fluorogenic dye to achieve high contrast, fast clearance, and good tissue penetration that may be used in clinical settings to molecularly define tumor margins.

Genetic susceptibility and environmental factors (such as stress) can interact to affect the likelihood of developing a mood disorder. Stress-induced changes in the hippocampus have been implicated in mood disorders, and mutations in several genes have now been associated with increased risk, such as brain-derived neurotrophic factor (BDNF). The hippocampus has important anatomical subdivisions, and pyramidal neurons of the vulnerable CA3 region show significant remodeling after chronic stress, but the mechanisms underlying their unique plasticity remain unknown. This study characterizes stress-induced changes in the in vivo translating mRNA of this cell population using a CA3-specific enhanced green fluorescent protein (EGFP) reporter fused to the L10a large ribosomal subunit (EGFPL10a). RNA-sequencing after isolation of polysome-bound mRNAs allows for cell-type-specific, genome-wide characterization of translational changes after stress. The data demonstrate that acute and chronic stress produce unique translational profiles and that the stress history of the animal can alter future reactivity of CA3 neurons. CA3-specific EGFPL10a mice were then crossed to the stress-susceptible BDNF Val66Met mouse line to characterize how a known genetic susceptibility alters both baseline translational profiles and the reactivity of CA3 neurons to stress. Not only do Met allele carriers exhibit distinct levels of baseline translation in genes implicated in ion channel function and cytoskeletal regulation, but they also activate a stress response profile that is highly dissimilar from wild-type mice. Closer examination of genes implicated in the mechanisms of neuroplasticity, such as the NMDA and AMPA subunits and the BDNF pathway, reveal how wild-type mice upregulate many of these genes in response to stress, but Met allele carriers fail to do so. These profiles provide a roadmap of stress-induced changes in a genetically homogenous population of hippocampal neurons and illustrate the profound effects of gene-environment interactions on the translational profile of these cells.

Background Although Ureaplasma species are the most common organisms associated with prematurity, their effects on the maternal and fetal immune system remain poorly characterized. Methods Rhesus macaque dams at approximately 80% gestation were injected intra-amniotically with 107 colony-forming units of Ureaplasma parvum or saline (control). Fetuses were delivered surgically 3 or 7 days later. We performed comprehensive assessments of inflammation and immune effects in multiple fetal and maternal tissues. Results Although U. parvum grew well in amniotic fluid, there was minimal chorioamnionitis. U. parvum colonized the fetal lung, but fetal systemic microbial invasion was limited. Fetal lung inflammation was mild, with elevations in CXCL8, tumor necrosis factor (TNF) α, and CCL2 levels in alveolar washes at day 7. Inflammation was not detected in the fetal brain. Significantly, U. parvum decreased regulatory T cells (Tregs) and activated interferon γ production in these Tregs in the fetus. It was detected in uterine tissue by day 7 and induced mild inflammation and increased expression of connexin 43, a gap junction protein involved with labor. Conclusions U. parvum colonized the amniotic fluid and caused uterine inflammation, but without overt chorioamnionitis. It caused mild fetal lung inflammation but had a more profound effect on the fetal immune system, decreasing Tregs and polarizing them toward a T-helper 1 phenotype.

Two 28-day feeding studies were performed in male rats to investigate combination effects of azole fungicides in a broad dose range. Following separate administration of cyproconazole, epoxiconazole, prochloraz, propiconazole, and tebuconazole at five dose levels, the first three compounds were selected to be administered in two different mixtures at three dose levels including very low doses. Here we present the data obtained by clinical observations, pathology, histopathology, clinical chemistry and haematology. The liver was the common main target organ of all compounds and their mixtures. In addition, epoxiconazole exhibited an effect on the adrenals. Furthermore, food consumption and efficiency and body weight (gain) were affected. Adverse effects of the combinations were observed at dose levels at which the individual substances caused similar effects. No evidence of adverse effects was found at dose levels below the previously established NOAELs. Our findings indicate that the concept of dose additivity appears sufficiently protective for risk assessment of the fungicides examined. Besides toxicological testing, tissue residues of the azole compounds in liver, testis and kidney were determined revealing remarkable differences following administration of the single substances and of the mixtures.