Clinical Exome Sequencing Enables Congenital Sialidosis Type II Diagnosis in Two Siblings Presenting with Unreported Clinical Features from a Rare Homozygous Sequence Variant p.(Tyr370Cys) in NEU1.

Sialidosis is a rare autosomal recessive disease that presents with progressive lysosomal storage of sialylated glycopeptides and oligosaccharides caused by homozygous or compound heterozygous sequence variants in the neuraminidase 1 (NEU1) gene. These sequence variants can lead to sialidosis type I and II; the latter is the most severe and presents prenatally or at early age. However, sialidosis diagnosis is challenging, especially in those health systems with limited resources of developing countries. Consequently, it is necessary to dip into high-throughput molecular diagnostic tools to allow for an accurate diagnosis with better cost-effectiveness and turnaround time. We report a 4-member pedigree segregating an ultrarare missense variant, c.1109A>G; p.Tyr370Cys, in NEU1 as detected by whole-exome sequencing. Two short-lived siblings, who presented with previously unreported clinical features from such a homozygous sequence variant, were diagnosed with sialidosis type II. Additionally, we present a novel molecular model exhibiting the consequences of the variant in the sialidase-1 tridimensional structure. This study allowed us to provide a definitive diagnosis for our patients, increase our understanding of this pathogenic variant, and improve genetic counseling.

Alanine Substitution Inactivates Cross-Reacting Epitopes in Dengue Virus Recombinant Envelope Proteins.

The expansion of the habitat of mosquitoes belonging to the Aedes genus puts nearly half of the world's population at risk of contracting dengue fever, and a significant fraction will develop its serious hemorrhagic complication, which can be fatal if not diagnosed properly and treated in a timely fashion. Although several diagnostic methods have been approved for dengue diagnostics, their applicability is limited in rural areas of developing countries by sample preparation costs and methodological requirements, as well as cross-reactivity among the different serotypes of the Dengue virus and other flavivirus, such as the Zika virus. For these reasons, it is necessary to generate more specific antigens to improve serological methods that could be cheaper and used in field operations. Here, we describe a strategy for the inactivation of cross-reacting epitopes on the surface of the Dengue virus envelope protein through the synthetic generation of recombinant peptide sequences, where key amino acid residues from Dengue virus serotype 1 (DENV-1) and 2 (DENV-2) are substituted by alanine residues. The proteins thus generated are recognized by 88% of sera from Dengue NS1+ patients and show improved serotype specificity because they do not react with the antibodies present in seroconverted, PCR-serotyped DEN-4 infected patients.

Genetic Variability of Chikungunya Virus in Southern Mexico.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes Chikungunya fever. CHIKV entered Mexico through the state of Chiapas in October 2014. To fully understand the Chikungunya fever outbreak that occurred in southern Chiapas during 2015, we evaluated 22 PCR-confirmed CHIKV-positive patients, identified CHIKV genetic variability, reconstructed viral dispersal, and assessed possible viral mutations. Viruses were isolated and E2, 6K, and E1 genes were sequenced. We applied phylogenetic and phylogeographic approaches, modeled mutations, and estimated selective pressure. Different CHIKV strains circulated in Chiapas during summer 2015. Three isolates grouped themselves in a well-supported clade. Estimates show that the outbreak started in Ciudad Hidalgo and posteriorly dispersed towards Tapachula and neighboring municipalities. We found six non-synonymous mutations in our isolates. Two mutations occurred in one isolate and the remaining mutations occurred in single isolates. Mutations E2 T116I and E2 K221R changed the protein surface in contact with the host cell receptors. We could not find positive selected sites in our CHIKV sequences from southern Chiapas. This is the first viral phylogeographic reconstruction in Mexico characterizing the CHIKV outbreak in southern Chiapas.

Priming of Hypothalamic Ghrelin Signaling and Microglia Activation Exacerbate Feeding in Rats' Offspring Following Maternal Overnutrition.

Maternal overnutrition during pregnancy leads to metabolic alterations, including obesity, hyperphagia, and inflammation in the offspring. Nutritional priming of central inflammation and its role in ghrelin sensitivity during fed and fasted states have not been analyzed. The current study aims to identify the effect of maternal programming on microglia activation and ghrelin-induced activation of hypothalamic neurons leading to food intake response. We employed a nutritional programming model exposing female Wistar rats to a cafeteria diet (CAF) from pre-pregnancy to weaning. Food intake in male offspring was determined daily after fasting and subcutaneous injection of ghrelin. Hypothalamic ghrelin sensitivity and microglia activation was evaluated using immunodetection for Iba-1 and c-Fos markers, and Western blot for TBK1 signaling. Release of TNF-alpha, IL-6, and IL-1ß after stimulation with palmitic, oleic, linoleic acid, or C6 ceramide in primary microglia culture were quantified using ELISA. We found that programmed offspring by CAF diet exhibits overfeeding after fasting and peripheral ghrelin administration, which correlates with an increase in the hypothalamic Iba-1 microglia marker and c-Fos cell activation. Additionally, in contrast to oleic, linoleic, or C6 ceramide stimulation in primary microglia culture, stimulation with palmitic acid for 24 h promotes TNF-alpha, IL-6, and IL-1ß release and TBK1 activation. Notably, intracerebroventricular (i.c.v.) palmitic acid or LPS inoculation for five days promotes daily increase in food intake and food consumption after ghrelin administration. Finally, we found that i.c.v. palmitic acid substantially activates hypothalamic Iba-1 microglia marker and c-Fos. Together, our results suggest that maternal nutritional programing primes ghrelin sensitivity and microglia activation, which potentially might mirror hypothalamic administration of the saturated palmitic acid.

Modular organization of a hypocretin gene minimal promoter.

Orexins or hypocretins are neurotransmitters produced by a small population of neurons in the lateral hypothalamus. This family of peptides modulates sleep­wake cycle, arousal and feeding behaviors; however, the mechanisms regulating their expression remain to be fully elucidated. There is an interest in defining the key molecular elements in orexin regulation, as these may serve to identify targets for generating novel therapies for sleep disorders, obesity and addiction. Our previous studies showed that the expression of orexin was decreased in mice carrying null­mutations of the transcription factor early B­cell factor 2 (ebf2) and that the promoter region of the prepro­orexin (Hcrt) gene contained two putative ebf­binding sites, termed olf­1 sites. In the present study, a minimal promoter region of the murine Hcrt gene was identified, which was able to drive the expression of a luciferase reporter gene in the human 293 cell line. Deletion of the olf1­site proximal to the transcription start site of the Hcrt gene increased reporter gene expression, whereas deletion of the distal olf1­like site decreased its expression. The lentiviral transduction of murine transcription factor ebf2 cDNA into 293 cells increased the gene expression driven by this minimal Hcrt­gene promoter and an electrophoretic mobility shift assays demonstrated that the distal olf1­like sequence was a binding site for ebf2.

The anti­dengue virus properties of statins may be associated with alterations in the cellular antiviral profile expression.

Dengue virus (DENV) susceptibility to cholesterol depleting treatments has been previously reported. There are numerous questions regarding how DENV seizes cellular machinery and cholesterol to improve viral production and the effect of cholesterol sequestering agents on the cellular antiviral response. The aim of the present study was to evaluate the mechanisms involved in the negative regulation of DENV replication induced by agents that diminish intracellular cholesterol levels. Cholesterol synthesis was pharmacologically (fluvastatin, atorvastatin, lovastatin, pravastatin and simvastatin treatment) and genetically (HMGCR­RNAi) inhibited, in uninfected and DENV2­infected hepatoma Huh­7 cells. The cholesterol levels, DENV titer and cellular antiviral expression profile were evaluated. A reduction in the DENV titer, measured as plaque forming units, was observed in DENV­infected cells following 48 h treatment with 10 µM fluvastatin, 10 µM atorvastatin, 20 µM lovastatin and 20 µM simvastatin, which achieved 70, 70, 65 and 55% DENV2 inhibition, respectively, compared with the untreated cells. In addition, the cytopathic effect was reduced in the statin­treated DENV­infected cells. Statins simultaneously reduced cholesterol levels at 48 h, with the exception of DENV2 infected cells. Genetic inhibition of cholesterol synthesis was performed using RNA interference for 3­hydroxy­3­methylglutaryl­CoA reductase (HMGCR­siRNA), which indicated a slight reduction in DENV2 titer at 48 h post­infection, however, with no significant reduction in cholesterol levels. In addition, DENV2 infection was observed to augment the intracellular cholesterol levels in all experimental conditions. Comparison between the cellular antiviral response triggered by DENV2 infection, statin treatment and HMGCR­siRNA in infected, uninfected, treated and untreated Huh7 cells, showed different expression profiles for the antiviral genes evaluated. All downregulating cholesterol agents evaluated reduced the expression of genes associated with cellular immune and pro­inflammatory responses. These results indicate that statin-mediated downregulation of DENV2 infectious particles number is independent of cholesterol levels and it is partially mediated by the modulation of the cellular antiviral profile.

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l(-1) insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Synthesization, Characterization, and in Vitro Evaluation of Cytotoxicity of Biomaterials Based on Halloysite Nanotubes.

Halloysite is an aluminosilicate clay that has been widely used for controlled drug delivery, immobilization of enzymes, and for the capture of circulating tumor cells (CTCs). Surface modification of halloysite by organosilanes has been explored to improve their properties. In this study halloysite clay nanotubes (HNTs) were functionalized by two different organosilanes: Trimethoxy(propyl)silane (TMPS), and Triethoxy(octyl)silane (EOS). Untreated and modified samples were characterized by scanning electron microscopy (SEM), X-ray diffractometry (XRD), thermogravimetrical analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). Results showed a strong interaction of organosilanes with the chemical groups present in HNTs. Biocompatibility and cytotoxicity of these nanomaterials were determined using C6 rat glioblastoma cells. Our results indicate that prior to functionalization, HNTs show a high biocompatibility and low cytotoxicity. However, HNTs functionalized with EOS and TMPS showed high cytotoxicity by inducing apoptosis. These results allow the identification of potential applications in biomedical areas for HNTs.

Odor memory stability after reinnervation of the olfactory bulb.

The olfactory system, particularly the olfactory epithelium, presents a unique opportunity to study the regenerative capabilities of the brain, because of its ability to recover after damage. In this study, we ablated olfactory sensory neurons with methimazole and followed the anatomical and functional recovery of circuits expressing genetic markers for I7 and M72 receptors (M72-IRES-tau-LacZ and I7-IRES-tau-GFP). Our results show that 45 days after methimazole-induced lesion, axonal projections to the bulb of M72 and I7 populations are largely reestablished. Furthermore, regenerated glomeruli are re-formed within the same areas as those of control, unexposed mice. This anatomical regeneration correlates with functional recovery of a previously learned odorant-discrimination task, dependent on the cognate ligands for M72 and I7. Following regeneration, mice also recover innate responsiveness to TMT and urine. Our findings show that regeneration of neuronal circuits in the olfactory system can be achieved with remarkable precision and underscore the importance of glomerular organization to evoke memory traces stored in the brain.

Supernumerary formation of olfactory glomeruli induced by chronic odorant exposure: a constructivist expression of neural plasticity.

It is accepted that sensory experience instructs the remodelling of neuronal circuits during postnatal development, after their specification has occurred. The story is less clear with regard to the role of experience during the initial formation of neuronal circuits, whether prenatal or postnatal, since this process is now supposed to be primarily influenced by genetic determinants and spontaneous neuronal firing. Here we evaluated this last issue by examining the effect that postnatal chronic exposure to cognate odorants has on the formation of I7 and M72 glomeruli, iterated olfactory circuits that are formed before and after birth, respectively. We took advantage of double knock-in mice whose I7 and M72 primary afferents express green fluorescent protein and ß-galactosidase, correspondingly. Our results revealed that postnatal odorant chronic exposure led to the formation of permanent supernumerary I7 and M72 glomeruli in a dose and time dependent manner. Glomeruli in exposed mice were formed within the same regions of olfactory bulb and occupy small space volumes compared to the corresponding single circuits in non-exposed mice. We suggest that local reorganization of the primary afferents could participate in the process of formation of supernumerary glomeruli. Overall, our results support that sensory experience indeed instructs the permanent formation of specific glomeruli in the mouse olfactory bulb by means of constructivist processes.

Frizzled 1 and frizzled 2 genes function in palate, ventricular septum and neural tube closure: general implications for tissue fusion processes.

The closure of an open anatomical structure by the directed growth and fusion of two tissue masses is a recurrent theme in mammalian embryology, and this process plays an integral role in the development of the palate, ventricular septum, neural tube, urethra, diaphragm and eye. In mice, targeted mutations of the genes encoding frizzled 1 (Fz1) and frizzled 2 (Fz2) show that these highly homologous integral membrane receptors play an essential and partially redundant role in closure of the palate and ventricular septum, and in the correct positioning of the cardiac outflow tract. When combined with a mutant allele of the planar cell polarity gene Vangl2 (Vangl2(Lp)), Fz1 and/or Fz2 mutations also cause defects in neural tube closure and misorientation of inner ear sensory hair cells. These observations indicate that frizzled signaling is involved in diverse tissue closure processes, defects in which account for some of the most common congenital anomalies in humans.

Stem cell therapy for Parkinson's disease: a road map for a successful future.

Cell transplant therapy for Parkinson's disease (PD) has been in use for over 2 decades as an experimental treatment. Different cell types have been proposed as better therapeutic alternatives. However, source availability and therapeutic value of the transplants as compared to current pharmacological options have precluded the use of this kind of surgery in the majority of PD patients. In this article, we discuss the suitability of different types of stem cells for PD therapy, the requirements that the donor cells should fulfill in order to improve upon current methods, and propose alternatives for evaluating the efficacy of PD cell therapy.

Antidepressant-like effects of nicotine and transcranial magnetic stimulation in the olfactory bulbectomy rat model of depression.

In this study, we compared the depression-like symptoms induced by olfactory bulbectomy (OBX) in the two inbred Wistar and Long Evans rat strains. We also analyzed the self-regulated oral intake of nicotine in these strains and the effect of nicotine on the depression-like symptoms of olfactory bulbectomy. Furthermore, we compared the antidepressant-like effects of nicotine on Wistar rats to those of transcranial magnetic stimulation (TMS), which has emerged as a therapeutic alternative for depression management. Our results show that Wistar rats develop depression-like symptoms, demonstrated by the forced swim test (FST), 4 weeks after OBX. However, in bulbectomized Long Evans rats these symptoms cannot be assessed due to a higher degree of variability of the swimming behavior of this strain. These results suggest that there are some innate differences in susceptibility to stress between these two rat strains. In Wistar rats, voluntary oral nicotine intake (1.2 mg/(kg day) for 14 days) as well as nicotine administered as a single daily i.p. injection (1.5 mg/(kg day) for 14 days) decrease the depression-like symptoms of OBX. Daily transcranial magnetic stimulation (60 Hz and 0.7 mT for 2h/day for 14 days) also decreases depression-like symptoms but is less effective than nicotine. In conclusion, our results support the idea that there are possible innate differences for depression susceptibility and that nicotine and TMS may be useful in the treatment of this syndrome.

Expression of nerve growth factor in human pancreatic beta cells.

Nerve growth factor (NGF) is an important modulator of rat pancreatic beta-cell physiology in vitro. In this study, we analysed the expression of NGF, TrkA and insulin in human pancreatic islets from normal, ductal adenocarcinoma and insulinoma-afflicted samples, using double immunofluorescent labelling and confocal microscopy. We found that in normal human pancreas, insulin and NGF are co-expressed in beta cells. Moreover, similar to previous observations in rat, the high affinity NGF receptor TrkA is also expressed in beta cells. Pancreatic beta cells in normal islets from adenocarcinoma and mucinous cystadenocarcinoma patients also expressed NGF. In 2 out of 15 exocrine tumour samples, NGF was detected also in the tissue surrounding the islets, while 2 out of 13 adenocarcinoma tumours expressed this growth factor. In five insulinoma samples, we observed weaker immunofluorescent labelling of insulin and NGF in the neoplastic tissue, compared to the islets not afflicted by the tumour, which may be a consequence of increased hormone secretion rate. We demonstrate that human beta cells express TrkA and NGF. These findings are consistent with the hypothesis that NGF modulates insulin secretion through a paracrine/autocrine loop, similar to the one observed in cultured rat beta cells.

Nerve growth factor increases sodium channel expression in pancreatic beta cells: implications for insulin secretion.

The importance of nerve growth factor (NGF) modulation of pancreatic beta cells is demonstrated by the fact that these cells secrete and respond to this trophic factor. Among NGF effects on beta cells is an increase in Na+ and Ca2+ current densities. This study investigates the mechanisms involved in the NGF-induced increase in Na+ current and the implications of this effect for insulin secretion. The following results were obtained in single beta cells cultured with NGF for 5-7 days: 1) A steady-state level of mRNA coding for type III sodium channel alpha subunit increased twofold compared with that for control cells. 2) The increase in Na+ current density was blocked either by cycloheximide or by actinomycin D, indicating that it is mediated by protein synthesis and mRNA transcription. 3) NGF treatment strengthened, by nearly fourfold, the beta-cell electrical activity; this effect is partially related to the increased Na+ current, because tetrodotoxin (TTX) decreased the duration of the depolarized plateau level. 4) Single beta cells secreted nearly two times more insulin in response to 5.6 or 15.6 mM glucose. This effect was inhibited by TTX, indicating that the enhanced Na+ current plays an important role. These data suggest that NGF could preserve an adequate expression of sodium channels and that impairment of NGF modulation could lead to deficient insulin secretion and diabetes mellitus.