Electromechanical coupling of the Kv1.1 voltage-gated K+ channel is fine-tuned by the simplest amino acid residue in the S4-S5 linker.

Investigating the Shaker-related K+ channel Kv1.1, the dysfunction of which is responsible for episodic ataxia 1 (EA1), at the functional and molecular level provides valuable understandings on normal channel dynamics, structural correlates underlying voltage-gating, and disease-causing mechanisms. Most studies focused on apparently functional amino acid residues composing voltage-gated K+ channels, neglecting the simplest ones. Glycine at position 311 of Kv1.1 is highly conserved both evolutionarily and within the Kv channel superfamily, is located in a region functionally relevant (the S4-S5 linker), and results in overt disease when mutated (p.G311D). By mutating the G311 residue to aspartate, we show here that the channel voltage-gating, activation, deactivation, inactivation, and window currents are markedly affected. In silico, modeling shows this glycine residue is strategically placed at one end of the linker helix which must be free to both bend and move past other portions of the protein during the channel's opening and closing. This is befitting of a glycine residue as its small neutral side chain allows for movement unhindered by interaction with any other amino acid. Results presented reveal the crucial importance of a distinct glycine residue, within the S4-S5 linker, in the voltage-dependent electromechanical coupling that control channel gating.

Transiently proliferating perivascular microglia harbor M1 type and precede cerebrovascular changes in a chronic hypertension model.

BACKGROUND: Microglia play crucial roles in the maintenance of brain homeostasis. Activated microglia show a biphasic influence, promoting beneficial repair and causing harmful damage via M2 and M1 microglia, respectively. It is well-known that microglia are initially activated to the M2 state and subsequently switch to the M1 state, called M2-to-M1 class switching in acute ischemic models. However, the activation process of microglia in chronic and sporadic hypertension remains poorly understood. We aimed to clarify the process using a chronic hypertension model, the deoxycorticosterone acetate (DOCA)-salt-treated Wistar rats. METHODS: After unilateral nephrectomy, the rats were randomly divided into DOCA-salt, placebo, and control groups. DOCA-salt rats received a weekly subcutaneous injection of DOCA (40 mg/kg) and were continuously provided with 1% NaCl in drinking water. Placebo rats received a weekly subcutaneous injection of vehicle and were provided with tap water. Control rats received no administration of DOCA or NaCl. To investigate the temporal expression profiles of M1- and M2-specific markers for microglia, the animals were subjected to the immunohistochemical and biochemical studies after 2, 3, or 4 weeks DOCA-salt treatment. RESULTS: Hypertension occurred after 2 weeks of DOCA and salt administration, when round-shaped microglia with slightly shortened processes were observed juxtaposed to the vessels, although the histopathological findings were normal. After 3 weeks of DOCA and salt administration, M1-state perivascular and parenchyma microglia significantly increased, when local histopathological findings began to be observed but cerebrovascular destruction did not occur. On the other hand, M2-state microglia were never observed around the vessels at this period. Interestingly, prior to M1 activation, about 55% of perivascular microglia transiently expressed Ki-67, one of the cell proliferation markers. CONCLUSIONS: We concluded that the resting perivascular microglia directly switched to the pro-inflammatory M1 state via a transient proliferative state in DOCA-salt rats. Our results suggest that the activation machinery of microglia in chronic hypertension differs from acute ischemic models. Proliferative microglia are possible initial key players in the development of hypertension-induced cerebral vessel damage. Fine-tuning of microglia proliferation and activation could constitute an innovative therapeutic strategy to prevent its development.

Gestational stress in mouse dams negatively affects gestation and postpartum hippocampal BDNF and P11 protein levels.

Stress during pregnancy increases the risk to develop psychological disorders such as depression during pregnancy or in the postpartum period. According to the neurotrophin hypothesis of depression, the pathophysiology of depression is caused by reduced neurotrophic activity in the brain. However, most studies only focus on the molecular changes happening to the offspring upon gestational stress. To gain insight into the potential molecular changes happening in the stressed dams, C57Bl6/J mice were stressed during their first week of gestation. At 28 days postpartum, the hippocampus and nucleus accumbens core of the dams, two brain regions heavily implicated in depression, were evaluated using immunohistochemistry to detect changes in the neurotrophin system. Gestational stress decreased the weight of the dams, increased the chance for spontaneous abortion and increased the weight of offspring. Litter size, survival rates and sex distribution were not altered as a consequence of gestational stress. Hippocampal brain-derived neurotrophic factor (BDNF) decreased following exposure to stress during pregnancy. Hippocampal protein levels of p75NTR, a low-affinity receptor for BDNF which can induce apoptosis, were increased following exposure to stress. Protein levels of p11, of which the expression is regulated by BDNF, were decreased in the hippocampus. No changes were found for TrkB immunostaining or apoptosis. Taken together, this shows that stress during pregnancy negatively affects the neurotrophin system in the hippocampus of the dams, thereby reducing hippocampal plasticity. These data confirm that gestational stress has a negative impact on pregnancy.

The influence of anesthetics on substantia nigra tyrosine hydroxylase expression and tau phosphorylation in the hypoxic-ischemic near-term lamb.

BackgroundGeneral anesthetics could protect key neurotransmitter systems, such as the dopaminergic system, from hypoxic-ischemic encephalopathy (HIE) by limiting excessive glutamatergic neurotransmission. However, anesthetics may adversely affect inflammation and tau phosphorylation.MethodsA near-term sheep model of HIE by umbilical cord occlusion (UCO) under anesthesia was used. The effect of propofol and isoflurane on the dopaminergic neurotransmitter phenotype in the substantia nigra (SN) was studied using tyrosine hydroxylase immunohistochemistry. The overall microglial response and tau phosphorylation were also measured in the SN, surrounding the midbrain gray matter structures and the hippocampal white matter.ResultsThe isoflurane-treated UCO group had fewer tyrosine hydroxylase-expressing neurons in the SN at 8 h after the insult than the propofol-treated UCO or sham-operated groups (P<0.05). The microglial response was unchanged in the SN region. In the thalamus and the hippocampal stratum moleculare layer, the propofol-treated UCO group had a lower microglial response than the corresponding sham-operated group. Both UCO and the use of anesthetics additively increased tau phosphorylation in the SN region, thalamus, and hippocampus.ConclusionThe choice of anesthetics is important for an emergency C-section. Propofol could potentially protect the dopaminergic neurotransmitter phenotype within the SN at the cost of a widespread increase in tau phosphorylation.

The phosphodiesterase type 2 inhibitor BAY 60-7550 reverses functional impairments induced by brain ischemia by decreasing hippocampal neurodegeneration and enhancing hippocampal neuronal plasticity.

Cognitive and affective impairments are the most characterized consequences following cerebral ischemia. BAY 60-7550, a selective phosphodiesterase type 2 inhibitor (PDE2-I), presents memory-enhancing and anxiolytic-like properties. The behavioral effects of BAY 60-7550 have been associated with its ability to prevent hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) thereby interfering with neuronal plasticity. Here, we hypothesize that PDE2-I treatment could promote functional recovery after brain ischemia. Mice C57Bl/6 were submitted to bilateral common carotid artery occlusion (BCCAO), an experimental model of transient brain ischemia, for 20 min. During 21 days after reperfusion, the animals were tested in a battery of behavioral tests including the elevated zero maze (EZM), object location task (OLT) and forced swim test (FST). The effects of BAY 60-7550 were evaluated on neuronal nuclei (NeuN), caspase-9, cAMP response element-binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. BCCAO increased anxiety levels, impaired hippocampus-dependent cognitive function and induced despair-like behavior in mice. Hippocampal neurodegeneration was evidenced by a decrease in NeuN and increase incaspase-9 protein levels in BCCAO mice. Ischemic mice also showed low BDNF protein levels in the hippocampus. Repeated treatment with BAY 60-7550 attenuated the behavioral impairments induced by BCCAO in mice. Concomitantly, BAY 60-7550 enhanced expression of pCREB and BDNF protein levels in the hippocampus of ischemic mice. The present findings suggest that chronic inhibition of PDE2 provides functional recovery in BCCAO mice possibly by augmenting hippocampal neuronal plasticity.

Aminochrome induces dopaminergic neuronal dysfunction: a new animal model for Parkinson's disease.

L-Dopa continues to be the gold drug in Parkinson's disease (PD) treatment from 1967. The failure to translate successful results from preclinical to clinical studies can be explained by the use of preclinical models which do not reflect what happens in the disease since these induce a rapid and extensive degeneration; for example, MPTP induces a severe Parkinsonism in only 3 days in humans contrasting with the slow degeneration and progression of PD. This study presents a new anatomy and develops preclinical model based on aminochrome which induces a slow and progressive dysfunction of dopaminergic neurons. The unilateral injection of aminochrome into rat striatum resulted in (1) contralateral rotation when the animals are stimulated with apomorphine; (2) absence of significant loss of tyrosine hydroxylase-positive neuronal elements both in substantia nigra and striatum; (3) cell shrinkage; (4) significant reduction of dopamine release; (5) significant increase in GABA release; (6) significant decrease in the number of monoaminergic presynaptic vesicles; (7) significant increase of dopamine concentration inside of monoaminergic vesicles; (8) significant increase of damaged mitochondria; (9) significant decrease of ATP level in the striatum (10) significant decrease in basal and maximal mitochondrial respiration. These results suggest that aminochrome induces dysfunction of dopaminergic neurons where the contralateral behavior can be explained by aminochrome-induced ATP decrease required both for anterograde transport of synaptic vesicles and dopamine release. Aminochrome could be implemented as a new model neurotoxin to study Parkinson's disease.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

Characterisation of cell-substrate interactions between Schwann cells and three-dimensional fibrin hydrogels containing orientated nanofibre topographical cues.

The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. In the present investigation, we studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied); however, cell behaviour in the hydrogel was somewhat different to that observed on the two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but, when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres, where they extended long processes that followed the longitudinal orientation of the nanofibres. The process length along nanofibre-containing fibrin hydrogel reached near-maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system.

Global hypoxia-ischemia induced inflammation and structural changes in the preterm ovine gut which were not ameliorated by mesenchymal stem cell treatment.

Perinatal asphyxia, a condition of impaired gas exchange during birth, leads to fetal hypoxia-ischemia (HI) and is associated with postnatal adverse outcomes including intestinal dysmotility and necrotizing enterocolitis (NEC). Evidence from adult animal models of transient, locally-induced intestinal HI has shown that inflammation is essential in HI-induced injury of the gut. Importantly, mesenchymal stem cell (MSC) treatment prevented this HI-induced intestinal damage. We therefore assessed whether fetal global HI induced inflammation, injury and developmental changes in the gut and whether intravenous MSC administration ameliorated these HI-induced adverse intestinal effects. In a preclinical ovine model, fetuses were subjected to umbilical cord occlusion (UCO), with or without MSC treatment, and sacrificed 7 days after UCO. Global HI increased the number of myeloperoxidase positive cells in the mucosa, upregulated mRNA levels of interleukin (IL)-1ß and IL-17 in gut tissue and caused T-cell invasion in the intestinal muscle layer. Intestinal inflammation following global HI was associated with increased Ki67+ cells in the muscularis and subsequent muscle hyperplasia. Global HI caused distortion of glial fibrillary acidic protein immunoreactivity in the enteric glial cells and increased synaptophysin and serotonin expression in the myenteric ganglia. Intravenous MSC treatment did not ameliorate these HI-induced adverse intestinal events. Global HI resulted in intestinal inflammation and enteric nervous system abnormalities which are clinically associated with postnatal complications including feeding intolerance, altered gastrointestinal transit and NEC. The intestinal histopathological changes were not prevented by intravenous MSC treatment directly after HI, indicating that alternative treatment regimens for cell-based therapies should be explored.

Developmental fluoxetine exposure increases behavioral despair and alters epigenetic regulation of the hippocampal BDNF gene in adult female offspring.

A growing number of infants are exposed to selective serotonin reuptake inhibitor (SSRI) medications during the perinatal period. Perinatal exposure to SSRI medications alter neuroplasticity and increase depressive- and anxiety-related behaviors, particularly in male offspring as little work has been done in female offspring to date. The long-term effects of SSRI on development can also differ with previous exposure to prenatal stress, a model of maternal depression. Because of the limited work done on the role of developmental SSRI exposure on neurobehavioral outcomes in female offspring, the aim of the present study was to investigate how developmental fluoxetine exposure affects anxiety and depression-like behavior, as well as the regulation of hippocampal brain-derived neurotrophic factor (BDNF) signaling in the hippocampus of adult female offspring. To do this female Sprague-Dawley rat offspring were exposed to prenatal stress and fluoxetine via the dam, for a total of four groups of female offspring: 1) No Stress+Vehicle, 2) No Stress+Fluoxetine, 3) Prenatal Stress+Vehicle, and 4) Prenatal Stress+Fluoxetine. Primary results show that, in adult female offspring, developmental SSRI exposure significantly increases behavioral despair measures on the forced swim test, decreases hippocampal BDNF exon IV mRNA levels, and increases levels of the repressive histone 3 lysine 27 tri-methylated mark at the corresponding promoter. There was also a significant negative correlation between hippocampal BDNF exon IV mRNA levels and immobility in the forced swim test. No effects of prenatal stress or developmental fluoxetine exposure were seen on tests of anxiety-like behavior. This research provides important evidence for the long-term programming effects of early-life exposure to SSRIs on female offspring, particularily with regard to affect-related behaviors and their underlying molecular mechanisms.

Complement activation by ceramide transporter proteins.

C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b-9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

Effects of prenatal Poly I:C exposure on global histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity in the mouse brain.

The aim of our study was to investigate the brain-specific epigenetic effects on global enzymatic histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity after prenatal exposure to maternal immune challenge by polyinosinic:polycytidylic acid (Poly I:C) at gestational day (GD) 17 in C57BL/6JRccHsd mouse offspring. Pregnant mice were randomly divided into 2 groups, receiving either 5 mg/kg Poly I:C or phosphate buffered saline (PBS) intravenously at GD 17. Subsequently, the effects on whole brain enzymatic HDAC and DNMT activity and the protein levels of various HDAC isoforms were assessed in the offspring. Overall, a significant sex × treatment interaction effect was observed after prenatal exposure to maternal immune challenge by Poly I:C, indicative of increased global HDAC activity particularly in female offspring from mothers injected with Poly I:C when compared to controls. Results on the levels of specific HDAC isoforms suggested that neither differences in the levels of HDAC1, HDAC2, HDAC3, HDAC4 or HDAC6 could explain the increased global HDAC activity observed in female Poly I:C offspring. In conclusion, we show that Poly I:C administration to pregnant mice alters global brain HDAC, but not DNMT activity in adult offspring, whereas it is still unclear which specific HDAC(s) mediate(s) this effect. These results indicate the necessity for further research on the epigenetic effects of Poly I:C.

Prenatal stress and early-life exposure to fluoxetine have enduring effects on anxiety and hippocampal BDNF gene expression in adult male offspring.

With the growing use of selective serotonin reuptake inhibitor medications (SSRIs) for the treatment of depression during the perinatal period, questions have been raised about the longterm impact of these medications on development. We aimed to investigate how developmental SSRI exposure may alter affect-related behaviors and associated molecular processes in offspring using a rodent model of maternal stress and depression. For this purpose, prenatally stressed or non-stressed male offspring were exposed to fluoxetine (5 mg/kg/day) or vehicle, via lactation, until weaning. Primary results show that postnatal fluoxetine exposure differentially altered anxiety-like behavior by increasing anxiety in non-stressed offspring and decreasing anxiety in prenatally stressed offspring. In the hippocampus, developmental fluoxetine exposure decreased BDNF IV and TrkB mRNA expression. Prenatal stress alone also decreased escape behaviors and decreased hippocampal BDNF IV mRNA expression. These data provide important evidence for the long-term programming effects of early-life exposure to SSRIs on brain and behavior.

Effects of subcutaneous LPS injection on gestational length and intrauterine and neonatal mortality in mice.

BACKGROUND: Infection during pregnancy can predispose offspring to develop various psychiatric disorders such as depression in later life. In order to investigate the potential mechanisms underlying these associations, animal models of maternal infection have been employed. As such, lipopolysaccharide (LPS) has been commonly used to mimic a bacterial infection in pregnant mice. OBJECTIVE: The original aim of our study was to investigate the effects of different doses of subcutaneous LPS administration on affective behavior in adult mouse offspring. In the present paper, however, we report that subcutaneous LPS administration has a profound impact on gestational length, litter size, and perinatal mortality in the offspring, even at a relatively low dose. METHODS: Pregnant mice were randomly divided into 3 groups, receiving either a high (2 mg/kg) or a low (0.5 mg/kg) dose of LPS or phosphate-buffered saline by means of subcutaneous injection. Subsequently, the effects on gestational length, litter size, and perinatal mortality in the offspring were assessed. RESULTS: After subcutaneous injection with a high dose of LPS, we observed a significant decrease in gestational length and an increase in neonatal mortality. When the low dose was administered, a tendency towards a reduced litter size was observed, most likely reflecting increased intrauterine mortality in response to prenatal maternal LPS exposure. CONCLUSIONS: We showed that subcutaneous administration of 2 mg/kg LPS to pregnant mice in the last phase of gestation should be avoided because of high offspring mortality rates, whereas subcutaneous injection of 0.5 mg/kg LPS seems to result in reabsorption of the fetuses.

Proteomic investigation of the hippocampus in prenatally stressed mice implicates changes in membrane trafficking, cytoskeletal, and metabolic function.

Prenatal stress influences the development of the fetal brain and so contributes to the risk of the development of psychiatric disorders in later life. The hippocampus is particularly sensitive to prenatal stress, and robust abnormalities have been described in the hippocampus in schizophrenia and depression. The aim of this study was to determine whether prenatal stress is associated with distinct patterns of differential protein expression in the hippocampus using a validated mouse model. We therefore performed a comparative proteomic study assessing female hippocampal samples from 8 prenatally stressed mice and 8 control mice. Differential protein expression was assessed using 2-dimensional difference in gel electrophoresis and subsequent mass spectrometry. The observed changes in a selected group of differentially expressed proteins were confirmed by Western blotting. In comparison to controls, 47 protein spots (38 individual proteins) were found to be differentially expressed in the hippocampus of prenatally stressed mice. Functional grouping of these proteins revealed that prenatal stress influenced the expression of proteins involved in brain development, cytoskeletal composition, stress response, and energy metabolism. Western blotting was utilized to validate the changes in calretinin, hippocalcin, profilin-1 and the signal-transducing adaptor molecule STAM1. Septin-5 could not be validated via Western blotting due to methodological issues. Closer investigation of the validated proteins also pointed to an interesting role for membrane trafficking deficits mediated by prenatal stress. Our findings demonstrate that prenatal stress leads to altered hippocampal protein expression, implicating numerous molecular pathways that may provide new targets for psychotropic drug development.

Fluoxetine dose and administration method differentially affect hippocampal plasticity in adult female rats.

Selective serotonin reuptake inhibitor medications are one of the most common treatments for mood disorders. In humans, these medications are taken orally, usually once per day. Unfortunately, administration of antidepressant medications in rodent models is often through injection, oral gavage, or minipump implant, all relatively stressful procedures. The aim of the present study was to investigate how administration of the commonly used SSRI, fluoxetine, via a wafer cookie, compares to fluoxetine administration using an osmotic minipump, with regards to serum drug levels and hippocampal plasticity. For this experiment, adult female Sprague-Dawley rats were divided over the two administration methods: (1) cookie and (2) osmotic minipump and three fluoxetine treatment doses: 0, 5, or 10 mg/kg/day. Results show that a fluoxetine dose of 5 mg/kg/day, but not 10 mg/kg/day, results in comparable serum levels of fluoxetine and its active metabolite norfluoxetine between the two administration methods. Furthermore, minipump administration of fluoxetine resulted in higher levels of cell proliferation in the granule cell layer (GCL) at a 5 mg dose compared to a 10 mg dose. Synaptophysin expression in the GCL, but not CA3, was significantly lower after fluoxetine treatment, regardless of administration method. These data suggest that the administration method and dose of fluoxetine can differentially affect hippocampal plasticity in the adult female rat.

Diagnostic outcome of pro bono neurogenetic diagnostic service in Sri Lanka: A wealth creation.

The inherited disease community in Sri Lanka has been widely neglected. This article aimed to present accumulated knowledge in establishing a pro bono cost-effective national, island-wide, free-of-charge molecular diagnostic service, suggesting a model for other developing countries. The project provided 637 molecular diagnostic tests and reports free of charge to a nation with limited resources. We pioneered the implementation of mobile clinics and home visits, where the research team acted as barefoot doctors with the concept of the doctor and the researcher at the patient's doorstep. Establishing pro bono, cost-effective molecular diagnostics is feasible in developing countries with limited resources and state funding through the effort of dedicated postgraduate students. This service could provide an accurate molecular diagnosis of Duchenne muscular dystrophy, Huntington's disease, Spinocerebellar ataxia, and Spinal muscular atrophy, a diagnostic yield of 54% (343/637), of which 43% (147/343) of the patients identified as amenable for available gene therapies. Initiated human resource development by double doctoral degree opportunities with international collaborations. Established a neurobiobank and a national registry in Sri Lanka, a rich and unique repository, wealth creation for translational collaborative research and sharing of information in neurological diseases, as well as a lodestar for aspiring initiatives from other developing countries.

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting.

BACKGROUND: The phenotype of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients is determined by the type of DMD gene variation, its location, effect on reading frame, and its size. The primary objective of this investigation was to determine the frequency and distribution of DMD gene variants (deletions/duplications) in Sri Lanka through the utilization of a combined approach involving multiplex polymerase chain reaction (mPCR) followed by Multiplex Ligation Dependent Probe Amplification (MLPA) and compare to the international literature. The current consensus is that MLPA is a labor efficient yet expensive technique for identifying deletions and duplications in the DMD gene. METHODOLOGY: Genetic analysis was performed in a cohort of 236 clinically suspected pediatric and adult myopathy patients in Sri Lanka, using mPCR and MLPA. A comparative analysis was conducted between our findings and literature data. RESULTS: In the entire patient cohort (n = 236), mPCR solely was able to identify deletions in the DMD gene in 131/236 patients (DMD-120, BMD-11). In the same cohort, MLPA confirmed deletions in 149/236 patients [DMD-138, BMD -11]. These findings suggest that mPCR has a detection rate of 95% (131/138) among all patients who received a diagnosis. The distal and proximal deletion hotspots for DMD were exons 45-55 and 6-15. Exon 45-60 identified as a novel in-frame variation hotspot. Exon 45-59 was a hotspot for BMD deletions. Comparisons with the international literature show significant variations observed in deletion and duplication frequencies in DMD gene across different populations. CONCLUSION: DMD gene deletions and duplications are concentrated in exons 45-55 and 2-20 respectively, which match global variation hotspots. Disparities in deletion and duplication frequencies were observed when comparing our data to other Asian and Western populations. Identified a 95% deletion detection rate for mPCR, making it a viable initial molecular diagnostic approach for low-resource countries where MLPA could be used to evaluate negative mPCR cases and cases with ambiguous mutation borders. Our findings may have important implications in the early identification of DMD with limited resources in Sri Lanka and to develop tailored molecular diagnostic algorithms that are regional and population specific and easily implemented in resource limited settings.

Goodpasture antigen-binding protein/ceramide transporter binds to human serum amyloid P-component and is present in brain amyloid plaques.

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease.

Electrical stimulation of the motor cortex enhances progenitor cell migration in the adult rat brain.

Clinical and preclinical investigations suggest that epidural stimulation of the motor cortex (MC) can improve stroke-induced neurological deficits. The mechanisms involved in stimulation-induced recovery are not well understood and might involve neurogenesis-related processes. Here, we addressed the question whether MC stimulation influences processes of migration and differentiation of neuronal progenitor cells in vivo. Epidural stimulation electrodes were implanted at the level of the MC in rats, and electrical current was applied for a period of 1 month. Increased cell proliferation was observed in the subventricular zone (SVZ). We also found evidences for enhanced cell migration toward the source of current, a process known as electrotaxis. Some of these cells expressed the neuronal marker, NeuN. In addition, our results indicate that MC stimulation enhances neuronal activity of the dorsal raphe nucleus, leading to an increase in the expression of 5-hydroxytryptamine in the SVZ. It is known that such an increase can promote formation of new cells in the SVZ. Our findings suggest that epidural MC stimulation influences neurogenesis-related processes in animal models.