Predictors of neonatal mortality: development and validation of prognostic models using prospective data from rural Bangladesh.

Objective: To assess the extent to which maternal histories of newborn danger signs independently or combined with birth weight and/or gestational age (GA) can capture and/or predict postsecond day (age>48 hours) neonatal death. Methods: Data from a cluster-randomised trial conducted in rural Bangladesh were split into development and validation sets. The prompted recall of danger signs and birth weight measurements were collected within 48 hours postchildbirth. Maternally recalled danger signs included cyanosis (any part of the infant's body was blue at birth), non-cephalic presentation (part other than head came out first at birth), lethargy (weak or no arm/leg movement and/or cry at birth), trouble suckling (infant unable to suckle/feed normally in the 2 days after birth or before death, collected 1-month postpartum or from verbal autopsy). Last menstrual period was collected at maternal enrolment early in pregnancy. Singleton newborns surviving 2 days past childbirth were eligible for analysis. Prognostic multivariable models were developed and internally validated. Results: Recalling ≥1 sign of lethargy, cyanosis, non-cephalic presentation or trouble suckling identified postsecond day neonatal death with 65.3% sensitivity, 60.8% specificity, 2.1% positive predictive value (PPV) and 99.3% negative predictive value (NPV) in the development set. Requiring either lethargy or weight <2.5 kg identified 89.1% of deaths (at 39.7% specificity, 1.9% PPV and 99.6% NPV) while lethargy or preterm birth (<37 weeks) captured 81.0% of deaths (at 53.6% specificity, 2.3% PPV and 99.5% NPV). A simplified model (birth weight, GA, lethargy, cyanosis, non-cephalic presentation and trouble suckling) predicted death with good discrimination (validation area under the receiver-operator characteristic curve (AUC) 0.80, 95% CI 0.73 to 0.87). A further simplified model (GA, non-cephalic presentation, lethargy, trouble suckling) predicted death with moderate discrimination (validation AUC 0.74, 95% CI 0.66 to 0.81). Conclusion: Maternally recalled danger signs, coupled to either birth weight or GA, can predict and capture postsecond day neonatal death with high discrimination and sensitivity.

Translational profiling of stress-induced neuroplasticity in the CA3 pyramidal neurons of BDNF Val66Met mice.

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.

Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations.

The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.

BDNF at the synapse: why location matters.

Neurotrophic factors, a family of secreted proteins that support the growth, survival and differentiation of neurons, have been intensively studied for decades due to the powerful and diverse effects on neuronal physiology, as well as their therapeutic potential. Such efforts have led to a detailed understanding on the molecular mechanisms of neurotrophic factor signaling. One member, brain-derived neurotrophic factor (BDNF) has drawn much attention due to its pleiotropic roles in the central nervous system and implications in various brain disorders. In addition, recent advances linking the rapid-acting antidepressant, ketamine, to BDNF translation and BDNF-dependent signaling, has re-emphasized the importance of understanding the precise details of BDNF biology at the synapse. Although substantial knowledge related to the genetic, epigenetic, cell biological and biochemical aspects of BDNF biology has now been established, certain aspects related to the precise localization and release of BDNF at the synapse have remained obscure. A recent series of genetic and cell biological studies have shed light on the question-the site of BDNF release at the synapse. In this Perspectives article, these new insights will be placed in the context of previously unresolved issues related to BDNF biology, as well as how BDNF may function as a downstream mediator of newer pharmacological agents currently under investigation for treating psychiatric disorders.

Developmental regulation of fear learning and anxiety behavior by endocannabinoids.

The developing brain undergoes substantial maturation into adulthood and the development of specific neural structures occurs on differing timelines. Transient imbalances between developmental trajectories of corticolimbic structures, which are known to contribute to regulation over fear learning and anxiety, can leave an individual susceptible to mental illness, particularly anxiety disorders. There is a substantial body of literature indicating that the endocannabinoid (eCB) system critically regulates stress responsivity and emotional behavior throughout the life span, making this system a novel therapeutic target for stress- and anxiety-related disorders. During early life and adolescence, corticolimbic eCB signaling changes dynamically and coincides with different sensitive periods of fear learning, suggesting that eCB signaling underlies age-specific fear learning responses. Moreover, perturbations to these normative fluctuations in corticolimbic eCB signaling, such as stress or cannabinoid exposure, could serve as a neural substrate contributing to alterations to the normative developmental trajectory of neural structures governing emotional behavior and fear learning. In this review, we first introduce the components of the eCB system and discuss clinical and rodent models showing eCB regulation of fear learning and anxiety in adulthood. Next, we highlight distinct fear learning and regulation profiles throughout development and discuss the ontogeny of the eCB system in the central nervous system, and models of pharmacological augmentation of eCB signaling during development in the context of fear learning and anxiety.

Stress effects on BDNF expression: effects of age, sex, and form of stress.

Stress has long been associated with the development of neuropsychiatric and neurological disorders. The effects of stress vary depending upon the age during which the stress is incurred, the duration and severity of the stressor, and can further be influenced by levels of circulating gonadal hormones. To date, the majority of research investigating the link between stress and pathology development has focused on stress hormone secretion, receptor activity, and their impact on neuronal development and functioning in developing and adult male and female rodents. In recent years, work has begun to focus on additional neuromodulatory systems that may be significantly impacted by stress that may explain changes in developmental and sex-based susceptibility to stress. New research targets include molecules that play a role in neuronal development and plasticity. Specifically, stress-induced alterations in growth factors such as neurotrophins, in particular brain-derived neurotrophic factor (BDNF), have been identified as a strong candidate modulating stress-associated pathology. Furthermore, changing expression of BDNF and its receptors over development and in response to circulating gonadal hormones extend the attractiveness of this candidate signaling pathway for understanding differences in susceptibility to stress. This review focuses on what is known with regard to the effects of stress on neurotrophin expression in rodents, and the varied effects of stress on BDNF levels as a function of developmental status and sex.

Validation of screening tools to assess appetite among geriatric patients.

Poor appetite is one of the main contributing factors of poor nutritional status among elderly individuals. Recognizing the importance of assessment of appetite, a cross sectional study was conducted to determine the validity of appetite screening tools namely, the Council on Nutrition Appetite questionnaire (CNAQ) and the simplified nutritional appetite questionnaire (SNAQ) against the appetite, hunger and sensory perception questionnaire (AHSPQ), measures of nutritional status and food intake among geriatric patients at the main general hospital in Malaysia. Nutritional status was assessed using the subjective global assessment (SGA) while food intake was measured using the dietary history questionnaire (DHQ). Anthropometric parameters included weight, height, body mass index (BMI), calf circumference (CC) and mid upper arm circumference (MUAC). A total of 145 subjects aged 60 to 86 years (68.3 ± 5.8 years) with 31.7% men and 68.3% women were recruited from outpatients (35 subjects) and inpatients (110 subjects) of Kuala Lumpur Hospital of Malaysia. As assessed by SGA, most subjects were classified as mild to moderately malnourished (50.4%), followed by normal (38.6%) and severely malnourished (11.0%). A total of 79.3% and 57.2% subjects were classified as having poor appetite according to CNAQ and SNAQ, respectively. CNAQ (80.9%) had a higher sensitivity than SNAQ (69.7%) when validated against nutritional status as assessed using SGA. However, the specificity of SNAQ (62.5%) was higher than CNAQ (23.2%). Positive predictive value for CNAQ and SNAQ were 62.6% and 74.7%, respectively. Cronbach's alpha for CNAQ and SNAQ were 0.546 and 0.578, respectively. History of weight loss over the past one year (Adjusted odds ratio 2.49) (p < 0.01) and thiamine intake less than the recommended nutrient intake (RNI) (Adjusted odds ratio 3.04) (p < 0.05) were risk factors for poor appetite among subjects. In conclusion, malnutrition and poor appetite were prevalent among the geriatric outpatients and inpatients. SNAQ was more reliable and valid as an appetite screening tool among this special group of population. There is a need to regularly include nutritional and appetite assessment for early intervention measures in order to prevent consequences of malnutrition.

Celastrol inhibits aminoglycoside-induced ototoxicity via heat shock protein 32.

Hearing loss is often caused by death of the mechanosensory hair cells of the inner ear. Hair cells are susceptible to death caused by aging, noise trauma, and ototoxic drugs, including the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Ototoxic drugs result in permanent hearing loss for over 500,000 Americans annually. We showed previously that induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. In order to begin to translate these findings into a clinical therapy aimed at inhibiting ototoxic drug-induced hearing loss, we have now examined a pharmacological HSP inducer, celastrol. Celastrol induced upregulation of HSPs in utricles, and it provided significant protection against aminoglycoside-induced hair cell death in vitro and in vivo. Moreover, celastrol inhibited hearing loss in mice receiving systemic aminoglycoside treatment. Our data indicate that the major heat shock transcription factor HSF-1 is not required for celastrol-mediated protection. HSP32 (also called heme oxygenase-1, HO-1) is the primary mediator of the protective effect of celastrol. HSP32/HO-1 inhibits pro-apoptotic c-Jun N-terminal kinase (JNK) activation and hair cell death. Taken together, our data indicate that celastrol inhibits aminoglycoside ototoxicity via HSP32/HO-1 induction.

Effect of brain-derived neurotrophic factor haploinsufficiency on stress-induced remodeling of hippocampal neurons.

Chronic restraint stress (CRS) induces the remodeling (i.e., retraction and simplification) of the apical dendrites of hippocampal CA3 pyramidal neurons in rats, suggesting that intrahippocampal connectivity can be affected by a prolonged stressful challenge. Since the structural maintenance of neuronal dendritic arborizations and synaptic connectivity requires neurotrophic support, we investigated the potential role of brain derived neurotrophic factor (BDNF), a neurotrophin enriched in the hippocampus and released from neurons in an activity-dependent manner, as a mediator of the stress-induced dendritic remodeling. The analysis of Golgi-impregnated hippocampal sections revealed that wild type (WT) C57BL/6 male mice showed a similar CA3 apical dendritic remodeling in response to three weeks of CRS to that previously described for rats. Haploinsufficient BDNF mice (BDNF(±) ) did not show such remodeling, but, even without CRS, they presented shorter and simplified CA3 apical dendritic arbors, like those observed in stressed WT mice. Furthermore, unstressed BDNF(±) mice showed a significant decrease in total hippocampal volume. The dendritic arborization of CA1 pyramidal neurons was not affected by CRS or genotype. However, only in WT mice, CRS induced changes in the density of dendritic spine shape subtypes in both CA1 and CA3 apical dendrites. These results suggest a complex role of BDNF in maintaining the dendritic and spine morphology of hippocampal neurons and the associated volume of the hippocampal formation. The inability of CRS to modify the dendritic structure of CA3 pyramidal neurons in BDNF(±) mice suggests an indirect, perhaps permissive, role of BDNF in mediating hippocampal dendritic remodeling.

Dendritic trafficking of BDNF mRNA is mediated by translin and blocked by the G196A (Val66Met) mutation.

Alternatively spliced brain-derived neurotrophic factor (BDNF) transcripts are targeted to distinct cellular compartments in neurons but the mechanisms underlying this sorting are unknown. Although only some BDNF isoforms are targeted to dendrites, we have found that the coding region common to all BDNF transcripts contains a constitutively active dendritic targeting signal and that this signal is suppressed in transcripts containing exons 1 or 4, which are restricted to the cell soma and proximal dendrites. This dendritic targeting signal is mediated by translin, an RNA-binding protein implicated in RNA trafficking, and is disrupted by the G196A mutation associated with memory deficits and psychiatric disorders. Molecular modeling and mutational studies indicate that the G196A mutation blocks dendritic targeting of BDNF mRNA by disrupting its interaction with translin. These findings implicate abnormal dendritic trafficking of BDNF mRNA in the pathophysiology of neuropsychiatric disorders linked to the G196A mutation.

Brain-derived neurotrophic factor as a model system for examining gene by environment interactions across development.

There has been a dramatic rise in gene x environment studies of human behavior over the past decade that have moved the field beyond simple nature versus nurture debates. These studies offer promise in accounting for more variability in behavioral and biological phenotypes than studies that focus on genetic or experiential factors alone. They also provide clues into mechanisms of modifying genetic risk or resilience in neurodevelopmental disorders. Yet, it is rare that these studies consider how these interactions change over the course of development. In this paper, we describe research that focuses on the impact of a polymorphism in a brain-derived neurotrophic factor (BDNF) gene, known to be involved in learning and development. Specifically we present findings that assess the effects of genotypic and environmental loadings on neuroanatomic and behavioral phenotypes across development. The findings illustrate the use of a genetic mouse model that mimics the human polymorphism, to constrain the interpretation of gene-environment interactions across development in humans.

Emotional incontinence--the other poststroke phenomenon.

Emotional incontinence is a disorder of emotional control following brain damage. It refers to the heightened tendency to cry or less commonly laugh, out of proportion to the underlying mood. Recognition of this phenomenon is often lacking as it is confused with other related sequelae of brain damage such as depression. This is a case report of an elderly female exhibiting poststroke emotional incontinence.

Limit on the electron neutrino magnetic moment from the kuo-sheng reactor neutrino experiment.

A search of neutrino magnetic moment was carried out at the Kuo-Sheng Nuclear Power Station at a distance of 28 m from the 2.9 GW reactor core. With a high purity germanium detector of mass 1.06 kg surrounded by scintillating NaI(Tl) and CsI(Tl) crystals as anti-Compton detectors, a detection threshold of 5 keV and a background level of 1 kg(-1) keV(-1) day(-1) at 12-60 keV were achieved. Based on 4712 and 1250 h of reactor ON and OFF data, respectively, the limit on the neutrino magnetic moment of mu(nu;(e))<1.3x10(-10)mu(B) at 90% confidence level was derived. An indirect bound of the nu;(e) radiative lifetime of m(3)(nu)tau(nu)>2.8x10(18) eV(3) s can be inferred.

A capsule review of recent studies on the application of mass spectrometry in the analysis of Chinese medicinal herbs.

Chinese herbal medicine is gaining increasing popularity worldwide as an alternative approach to the development of pharmaceuticals in therapeutic applications. Chemical characterization and compositional analysis of Chinese medicines provide the necessary scientific basis for the discovery and development of new drugs of natural origin. Applications of mass spectrometry in the analysis of Chinese herbal medicines have been growing rapidly in recent years owing to the rapid technical advances and increasing availability of the instrumentation. This paper reviews the current status of how different mass spectrometric techniques are being used to support research studies of Chinese medicines. The focus is on crude herbal medicines and their derived products. The review is not meant to be exhaustive, but rather to provide a general overview of the various research activities in this rapidly expanding field. In the discussion of specific herbs, the emphasis is placed on ginseng and Danshen, two of the herbs for which active experimental work is on-going in the authors' laboratories. Other selected herbs will be discussed only briefly, aiming primarily to illustrate the current status of research in the area.

HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1alpha is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction thereby promotes the rapid degradation of HIF-1alpha. Under hypoxic conditions, HIF-1alpha is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1alpha, and that this regulation depends on a posttranslational modification of HIF-1alpha. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1alpha in this region, as well as MS and in vitro binding assays, we now provide evidence that this modification, which occurs under normoxic conditions, is hydroxylation of Pro-564 of HIF-1alpha. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.

The uniqueness of being a neurotrophin receptor.

Neurotrophins rely on Trk tyrosine kinase and p75 receptors for signal transduction. Recently, other roles for these receptors have been identified. Many questions have been raised about the mechanism by which these receptors mediate diverse cellular functions. Studies indicate a great deal of neurotrophin signaling specificity may stem from ligand-receptor selectivity and intracellular protein recruitment.

I kappa B kinase is critical for TNF-alpha-induced VCAM1 gene expression in renal tubular epithelial cells.

The expression of VCAM1 is up-regulated in renal proximal tubular epithelial cells (TEC) in a variety of inflammatory renal diseases, a prominent example of which is acute renal allograft rejection. VCAM1 may play an important role in these diseases because it binds to the integrins very late Ag-4 and alpha(4)beta(7) on lymphocytes and monocytes, thereby providing a potential mechanism to recruit these leukocytes to sites of inflammation. The molecular mechanisms underlying VCAM1 regulation in renal TEC are essentially unknown. We now report that VCAM1 mRNA is dramatically up-regulated in C1, a cell line derived from renal TEC, on exposure to TNF-alpha. Two NF-kappaB binding sites in the VCAM1 promoter are critical for the TNF-alpha-induced VCAM1 transcriptional up-regulation, and both sites bind to p65-p50 NF-kappaB complexes. TNF-alpha induces activation of inhibitor of NF-kappaB (IkappaB) kinase-beta (IKK-beta), a protein kinase that phosphorylates the NF-kappaB inhibitor IkappaB, and thereby targets the latter for degradation via the ubiquitin-proteasome pathway. Moreover, dominant negative versions of IKK inhibit TNF-alpha activation of a VCAM1 promoter reporter. We conclude that the IKK/NF-kappaB pathway is critical in the TNF-alpha-induced up-regulation of VCAM1 mRNA in renal TEC.

Dynamic, site-specific interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor protein.

Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that plays a critical role in regulating genes involved in erythropoiesis and angiogenesis. Recent evidence indicates that the von Hippel-Lindau tumor suppressor protein (VHL) is part of a ubiquitin ligase complex that promotes the degradation of HIF-1alpha under normoxic conditions. Under hypoxic conditions, HIF-1alpha is markedly stabilized. A critical issue in understanding the hypoxic response is the identification of hypoxia-regulated steps. We show here that hypoxia and cobalt treatment modulate the capacity of a HIF-1alpha fragment comprising residues 531-652 to coimmunoprecipitate with VHL. Hypoxia and cobalt both significantly diminish the interaction, and furthermore, normoxia treatment after hypoxia rapidly normalizes it. This HIF-1alpha fragment confers hypoxia and cobalt inducibility on a heterologous protein. Significantly, contained within this fragment is a short 27-residue sequence that behaves identically in all respects noted above. Finally, evidence is provided to show that cobalt and hypoxia both induce a posttranslational modification (or loss of one) in HIF-1alpha that affects its binding to VHL. We propose that dynamic, site-specific interaction of HIF-1alpha with VHL provides one mechanism by which HIF-1alpha can be regulated.

Activation of Trk neurotrophin receptors in the absence of neurotrophins.

Neurotrophins regulate neuronal cell survival and synaptic plasticity through activation of Trk receptor tyrosine kinases. Binding of neurotrophins to Trk receptors results in receptor autophosphorylation and downstream phosphorylation cascades. Here, we describe an approach to use small molecule agonists to transactivate Trk neurotrophin receptors. Activation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons was observed after treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors. These effects were reproduced by using the adenosine agonist CGS 21680 and were counteracted with the antagonist ZM 241385, indicating that this transactivation event by adenosine involves adenosine 2A receptors. The increase in Trk activity could be inhibited by the use of the Src family-specific inhibitor, PP1, or K252a, an inhibitor of Trk receptors. In contrast to other G protein-coupled receptor transactivation events, adenosine used Trk receptor signaling with a longer time course. Moreover, adenosine activated phosphatidylinositol 3-kinase/Akt through a Trk-dependent mechanism that resulted in increased cell survival after nerve growth factor or brain-derived neurotrophic factor withdrawal. Therefore, adenosine acting through the A(2A) receptors exerts a trophic effect through the engagement of Trk receptors. These results provide an explanation for neuroprotective actions of adenosine through a unique signaling mechanism and raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases.