Bi-allelic NRXN1α deletion in microglia derived from iPSC of an autistic patient increases interleukin-6 production and impairs supporting function on neuronal networking.

Autism spectrum disorder (ASD) is a set of heterogeneous neurodevelopmental conditions, with a highly diverse genetic hereditary component, including altered neuronal circuits, that has an impact on communication skills and behaviours of the affected individuals. Beside the recognised role of neuronal alterations, perturbations of microglia and the associated neuroinflammatory processes have emerged as credible contributors to aetiology and physiopathology of ASD. Mutations in NRXN1, a member of the neurexin family of cell-surface receptors that bind neuroligin, have been associated to ASD. NRXN1 is known to be expressed by neurons where it facilitates synaptic contacts, but it has also been identified in glial cells including microglia. Asserting the impact of ASD-related genes on neuronal versus microglia functions has been challenging. Here, we present an ASD subject-derived induced pluripotent stem cells (iPSC)-based in vitro system to characterise the effects of the ASD-associated NRXN1 gene deletion on neurons and microglia, as well as on the ability of microglia to support neuronal circuit formation and function. Using this approach, we demonstrated that NRXN1 deletion, impacting on the expression of the alpha isoform (NRXN1α), in microglia leads to microglial alterations and release of IL6, a pro-inflammatory interleukin associated with ASD. Moreover, microglia bearing the NRXN1α-deletion, lost the ability to support the formation of functional neuronal networks. The use of recombinant IL6 protein on control microglia-neuron co-cultures or neutralizing antibody to IL6 on their NRXN1α-deficient counterparts, supported a direct contribution of IL6 to the observed neuronal phenotype. Altogether, our data suggest that, in addition to neurons, microglia are also negatively affected by NRXN1α-deletion, and this significantly contributes to the observed neuronal circuit aberrations.

The Threat Posed by Environmental Contaminants on Neurodevelopment: What Can We Learn from Neural Stem Cells?

Exposure to chemicals may pose a greater risk to vulnerable groups, including pregnant women, fetuses, and children, that may lead to diseases linked to the toxicants' target organs. Among chemical contaminants, methylmercury (MeHg), present in aquatic food, is one of the most harmful to the developing nervous system depending on time and level of exposure. Moreover, certain man-made PFAS, such as PFOS and PFOA, used in commercial and industrial products including liquid repellants for paper, packaging, textile, leather, and carpets, are developmental neurotoxicants. There is vast knowledge about the detrimental neurotoxic effects induced by high levels of exposure to these chemicals. Less is known about the consequences that low-level exposures may have on neurodevelopment, although an increasing number of studies link neurotoxic chemical exposures to neurodevelopmental disorders. Still, the mechanisms of toxicity are not identified. Here we review in vitro mechanistic studies using neural stem cells (NSCs) from rodents and humans to dissect the cellular and molecular processes changed by exposure to environmentally relevant levels of MeHg or PFOS/PFOA. All studies show that even low concentrations dysregulate critical neurodevelopmental steps supporting the idea that neurotoxic chemicals may play a role in the onset of neurodevelopmental disorders.

Sox2 is required for olfactory pit formation and olfactory neurogenesis through BMP restriction and Hes5 upregulation.

The transcription factor Sox2 is necessary to maintain pluripotency of embryonic stem cells, and to regulate neural development. Neurogenesis in the vertebrate olfactory epithelium persists from embryonic stages through adulthood. The role Sox2 plays for the development of the olfactory epithelium and neurogenesis within has, however, not been determined. Here, by analysing Sox2 conditional knockout mouse embryos and chick embryos deprived of Sox2 in the olfactory epithelium using CRISPR-Cas9, we show that Sox2 activity is crucial for the induction of the neural progenitor gene Hes5 and for subsequent differentiation of the neuronal lineage. Our results also suggest that Sox2 activity promotes the neurogenic domain in the nasal epithelium by restricting Bmp4 expression. The Sox2-deficient olfactory epithelium displays diminished cell cycle progression and proliferation, a dramatic increase in apoptosis and finally olfactory pit atrophy. Moreover, chromatin immunoprecipitation data show that Sox2 directly binds to the Hes5 promoter in both the PNS and CNS. Taken together, our results indicate that Sox2 is essential to establish, maintain and expand the neuronal progenitor pool by suppressing Bmp4 and upregulating Hes5 expression.

Mine sludge waste recycling as bio-stimulant for applications in anaerobic wastewater treatment.

This study examined the applicability of two mine sludge wastes, mine tailing sludge (MTS) and acid mine drainage sludge (AMDS) as iron-rich bio-stimulant for enhancing organic matter degradation in anaerobic process. Batch treatment of domestic sewage having 343 ± 10 mg/L chemical oxygen demand (COD) using MTS and AMDS as additives mixed with septic tank sludge as anaerobic inoculum produced lower start-up time, higher efficiency of COD removal, enhanced biomass retention, and higher acidogenic and methanogenic activity after stabilization. Biostimulation induced by mine sludge waste additives in anaerobic system were observed to have correlation with percentage of iron content in the additives, as well as difference in surface charge between biomass and the additives. Treatment efficiency induced by the two mine sludge waste based additives were similar at 90% confidence limit, however, was found to be higher than lower iron containing additive laterite soil, while lower than higher iron containing synthetic zero valent nano iron as additives used for comparison. The study was supported by scanning electron microscope, atomic force microscope and optical microscope images of sludge granule sand surface charge measurement.

Successful Percutaneous Closure of Gerbode Defect and Right Atrial-Aortic Fistula Following Infective Endocarditis.

We report a case of infective endocarditis with a septal abscess that was complicated with abnormal blood flow from the left ventricle to the right atrium (Gerbode defect) along with abnormal blood flow from the aorta to the right atrium (atrial-aortic fistula). This is the first reported case of successful correction of both defects by a percutaneous approach.

Perfluorooctane sulfonate induces neuronal and oligodendrocytic differentiation in neural stem cells and alters the expression of PPARγ in vitro and in vivo.

Perfluorinated compounds are ubiquitous chemicals of major concern for their potential adverse effects on the human population. We have used primary rat embryonic neural stem cells (NSCs) to study the effects of perfluorooctane sulfonate (PFOS) on the process of NSC spontaneous differentiation. Upon removal of basic fibroblast growth factor, NSCs were exposed to nanomolar concentrations of PFOS for 48 h, and then allowed to differentiate for additional 5 days. Exposure to 25 or 50 nM concentration resulted in a lower number of proliferating cells and a higher number of neurite-bearing TuJ1-positive cells, indicating an increase in neuronal differentiation. Exposure to 50 nM also significantly increased the number of CNPase-positive cells, pointing to facilitation of oligodendrocytic differentiation. PPAR genes have been shown to be involved in PFOS toxicity. By q-PCR we detected an upregulation of PPARγ with no changes in PPARα or PPARδ genes. One of the downstream targets of PPARs, the mitochondrial uncoupling protein 2 (UCP2) was also upregulated. The number of TuJ1- and CNPase-positive cells increased after exposure to PPARγ agonist rosiglitazone (RGZ, 3 µM) and decreased after pre-incubation with the PPARγ antagonist GW9662 (5 µM). RGZ also upregulated the expression of PPARγ and UCP2 genes. Meanwhile GW9662 abolished the UCP2 upregulation and decreased Ca²âº activity induced by PFOS. Interestingly, a significantly higher expression of PPARγ and UCP3 genes was also detected in mouse neonatal brain after prenatal exposure to PFOS. These data suggest that PPARγ plays a role in the alteration of spontaneous differentiation of NSCs induced by nanomolar concentrations of PFOS.

Sex Differences in Long-term Outcome of Prenatal Exposure to Excess Glucocorticoids-Implications for Development of Psychiatric Disorders.

Exposure to prenatal insults, such as excess glucocorticoids (GC), may lead to pathological outcomes, including neuropsychiatric disorders. The aim of the present study was to investigate the long-term effects of in utero exposure to the synthetic GC analog dexamethasone (Dex) in adult female offspring. We monitored spontaneous activity in the home cage under a constant 12 h/12 h light/dark cycle, as well as the changes following a 6-h advance of dark onset (phase shift). For comparison, we re-analysed data previously recorded in males. Dex-exposed females were spontaneously more active, and the activity onset re-entrained slower than in controls. In contrast, Dex-exposed males were less active, and the activity onset re-entrained faster than in controls. Following the phase shift, control females displayed a transient reorganisation of behaviour in light and virtually no change in dark, while Dex-exposed females showed limited variations from baseline in both light and dark, suggesting weaker photic entrainment. Next, we ran bulk RNA-sequencing in the suprachiasmatic nucleus (SCN) of Dex and control females. SPIA pathway analysis of ~ 2300 differentially expressed genes identified significantly downregulated dopamine signalling, and upregulated glutamate and GABA signalling. We selected a set of candidate genes matching the behaviour alterations and found consistent differential regulation for ~ 73% of tested genes in SCN and hippocampus tissue samples. Taken together, our data highlight sex differences in the outcome of prenatal exposure to excess GC in adult mice: in contrast to depression-like behaviour in males, the phenotype in females, defined by behaviour and differential gene expression, is consistent with ADHD models.

Modeling Neurological Disorders in 3D Organoids Using Human-Derived Pluripotent Stem Cells.

Modeling neurological disorders is challenging because they often have both endogenous and exogenous causes. Brain organoids consist of three-dimensional (3D) self-organizing brain tissue which increasingly is being used to model various aspects of brain development and disorders, such as the generation of neurons, neuronal migration, and functional networks. These organoids have been recognized as important in vitro tools to model developmental features of the brain, including neurological disorders, which can provide insights into the molecular mechanisms involved in those disorders. In this review, we describe recent advances in the generation of two-dimensional (2D), 3D, and blood-brain barrier models that were derived from induced pluripotent stem cells (iPSCs) and we discuss their advantages and limitations in modeling diseases, as well as explore the development of a vascularized and functional 3D model of brain processes. This review also examines the applications of brain organoids for modeling major neurodegenerative diseases and neurodevelopmental disorders.

High-rate blackwater anaerobic digestion under septic tank conditions with the amendment of biosolids-derived biochar synthesized at different temperatures.

Septic tanks have been widely used for blackwater treatment in developing countries, while high-rate septic tanks with improved methane recovery are yet to be achieved. This study investigated biosolids-derived biochar (synthesized at 300℃, 425℃, and 550℃) as an additive for developing high-rate septic tanks. The experiments were conducted with anaerobic bioreactors operated with synthetic blackwater under septic tank conditions. All biochar amended reactors demonstrated a steady increase in daily methane production for increasing OLR from 0.08 to 3 g COD/L/d. The control reactor showed significant process disturbances at OLRs ≥ 2 g COD/L/d with an accumulation of volatile fatty acids followed by pH drop. At OLR of 3 g COD/L/d, the daily methane production from biochar amended reactors was ~ 4.3 times higher than the control (300 vs. 70 mL per day). Biochar addition established a robust microbiome consisted of a higher abundance of hydrogenotrophic and acetoclastic methanogens and hydrogen-producing fermentative bacteria.

Significance of different mixing conditions on performance and microbial communities in anaerobic digester amended with granular and powdered activated carbon.

Conductive materials amendment in anaerobic digestion (AD) is a promising strategy for boosting the methanogenesis process. Despite mixing is a critical parameter, the behavior of digesters amended with conductive additives upon different mixing conditions has rarely been investigated. This study investigated continuous mixing, intermittent mixing (10 min in every 12 h), and non-mixing conditions for digesters amended with granular activated carbon (GAC) and powdered activated carbon (PAC). The non-mixed GAC digester provided the highest methane yield (318 ± 28 mL/g COD) from synthetic blackwater, while intermittently mixed GAC and control exhibited similar methane yields (290-294 mL/g COD). For non-mixed systems, microbial richness and diversity increased with GAC and PAC amendment. In contrast, continuous and intermittent mixing increased microbial diversity and richness in control reactors while reduced the same in GAC and PAC amended reactors. Overall, various mixing conditions distinctly changed the degree of enrichment/retention of microbes and consequently influenced methane recovery.

Total artificial heart bridge to transplantation: a 9-year experience with 62 patients.

BACKGROUND: The SynCardia CardioWest total artificial heart (CardioWest TAH) is a biventricular, orthotopic, pneumatic, pulsatile blood pump driven by an external console. For each ventricle, the length of the blood-flow path is shorter and the inflow and outflow valves are larger than in any other bridge-to-transplant device, resulting in greater blood flow at smaller pre-load. Such a device should be optimal for bridging transplant candidates who have biventricular failure and for whom all other therapies have failed. METHODS: From January 1, 1993, to April 1, 2002, we prospectively studied 62 consecutive CardioWest TAH implant recipients to document safety and efficacy in bridge to transplantation. We used multisystem monitoring and multidrug therapy for anti-coagulation in 58 patients starting September 1, 1994. RESULTS: Before implantation, patients were critically ill with biventricular heart failure. Mortality in this group from the time of implantation until transplantation was 23%. Causes of death during device support included multi-organ failure (6), sepsis (3), and valve entrapment (2). Forty-eight patients underwent transplantation (77%). Forty-two survived to hospital discharge (68% of the total, 88% of those undergoing transplantation). Adverse events included bleeding (20%), device malfunction (5%), fit complications (3%), mediastinal infections (5%), visceral embolus (1.6%), and stroke during support (1.6%). The linearized stroke rate was 0.068 events per patient-year. CONCLUSIONS: Sixty-eight percent of critically ill transplant candidates for whom medical therapy failed were bridged to transplantation with the CardioWest TAH and survived long-term. Most deaths that occurred during device support were related to pre-implant problems. Infection and stroke were rare events. Therefore, we recommend the CardioWest TAH as the biventricular bridge-to-transplant device of choice.

Successful management of empyema in a patient with a total artificial heart.

A description of successful management of a patient who developed an empyema as a postoperative complication following the insertion of a CardioWest total artificial heart (TAH) as a bridge to cardiac transplantation is presented. By using traditional methods of management, the patient recovered and went on to transplant.

Inherited effects of low-dose exposure to methylmercury in neural stem cells.

Methylmercury (MeHg) is an environmental contaminant with recognized neurotoxic effects, particularly to the developing nervous system. In the present study, we show that nanomolar concentrations of MeHg can induce long-lasting effects in neural stem cells (NSCs). We investigated short-term direct and long-term inherited effects of exposure to MeHg (2.5 or 5.0 nM) using primary cultures of rat embryonic cortical NSCs. We found that MeHg had no adverse effect on cell viability but reduced NSC proliferation and altered the expression of cell cycle regulators (p16 and p21) and senescence-associated markers. In addition, we demonstrated a decrease in global DNA methylation in the exposed cells, indicating that epigenetic changes may be involved in the mechanisms underlying the MeHg-induced effects. These changes were observed in cells directly exposed to MeHg (parent cells) and in their daughter cells cultured under MeHg-free conditions. In agreement with our in vitro data, a trend was found for decreased cell proliferation in the subgranular zone in the hippocampi of adult mice exposed to low doses of MeHg during the perinatal period. Interestingly, this impaired proliferation had a measurable impact on the total number of neurons in the hippocampal dentate gyrus. Importantly, this effect could be reversed by chronic antidepressant treatment. Our study provides novel evidence for programming effects induced by MeHg in NSCs and supports the idea that developmental exposure to low levels of MeHg may result in long-term consequences predisposing to neurodevelopmental disorders and/or neurodegeneration.

Dickkopf 1 mediates glucocorticoid-induced changes in human neural progenitor cell proliferation and differentiation.

Glucocorticoids (GC) are critical for normal development of the fetal brain, and alterations in their levels can induce neurotoxicity with detrimental consequences. Still, there is little information available on the effects of GC on human neural stem/progenitor cells (hNPC). In the present study, we have investigated the effects of the synthetic GC dexamethasone (Dex) on hNPC grown as neurospheres, with special focus on their proliferation and differentiation capacity and the underlying molecular mechanisms. Immunocytochemical stainings showed that Dex markedly decreases proliferation and neuronal differentiation while promoting glia cell formation. Analysis of pathway-specific genes revealed that Dex induces an upregulation of the Wnt-signaling antagonist DKK1. Moreover, Dex- or DKK1-treated hNPCs showed reduced transcriptional levels of the two canonical Wnt target genes cyclin D1 and inhibitor of DNA binding 2 (ID2). Chromatin immunoprecipitation showed that Dex, via the glucocorticoid receptor, interacts with the DKK1 promotor. Treatment of hNPC with recombinant DKK1 or neutralizing antibodies indicated that DKK1 has a critical role in the Dex-induced inhibition of proliferation and neuronal differentiation with a concomitant increase in glial cells. Taken together, our findings show that GC reduce proliferation and interfere with differentiation of hNPCs via the canonical Wnt-signaling pathway.

Neural stem cells for developmental neurotoxicity studies.

The developing nervous system is particularly susceptible to toxicants, and exposure during development may result in long-lasting neurological impairments. The damage can range from subtle to severe, and it may impose substantial burdens on affected individuals, their families, and society. Given the little information available on developmental neurotoxicity (DNT) and the growing number of chemicals that need to be tested, new testing strategies and approaches are necessary to identify developmental neurotoxic agents with speed, reliability, and respect for animal welfare. So far, there are no validated alternative methods for DNT testing. Recently, neural stem/progenitor cells have been proposed as relevant models for alternative DNT testing. In this chapter, we provide detailed protocols for culturing neural stem cells (NSCs), in vitro experimental models, including primary cultures of rat and human embryonic NSCs, rat and mouse adult NSCs, as well as the mouse NSC line C17.2 that we have implemented and successfully used for neurotoxicity studies.

Treatment of peripartum cardiomyopathy with mechanical assist devices and cardiac transplantation.

BACKGROUND: Peripartum cardiomyopathy is a life threatening illness. If maximal medical therapy fails, patients may then be treated with mechanical circulatory support devices and (or) cardiac transplantation. Our purpose is to demonstrate the long-term efficacy of these surgical interventions. METHODS: A retrospective review of 18 patients diagnosed with peripartum cardiomyopathy from 1994 to 2009 was conducted. RESULTS: Eighteen patients were referred with a median of seven year delay between onset of symptoms and presentation. Eight (44%) had medical therapy with only one death at five years, seven are alive, and two patients are awaiting transplantation. Six patients were implanted with devices. From this group, there were two hospital deaths, one native heart recovery, and three heart transplants with 100% survival. Four patients (21%) were treated with transplantation alone and all survived. Combined device and (or) transplant survival was 80% at one year. CONCLUSIONS: The natural history of this group varied; 38% of the medically treated patients are stable on medical therapy (3 of 8) and 67% of the device patients (4 of 6) are alive. One of six device patients (17%) was successfully bridged to native heart recovery. Mechanical assist devices can be used as a bridge to recovery or as a bridge to cardiac transplantation for the treatment of peripartum cardiomyopathy patients who fail medical management. In addition, cardiac transplantation alone is also a viable treatment option for patients who fail medical management and do not require a mechanical assist device.

Risk factor analysis for bridge to transplantation with the CardioWest total artificial heart.

BACKGROUND: Safety and efficacy studies of various mechanical circulatory support devices are important, but may not be strictly comparable. Lacking prospective randomized studies for different devices, we believe that comparison of risk factor analyses may give the surgeon a tool more powerful than current studies for matching a patient with an appropriate device. In this paper, we report risk factor profiles for bridge to transplantation with the CardioWest total artificial heart and summarize reports for other devices. METHODS: A multiinstitutional risk factor analysis of the CardioWest total artificial heart, as a bridge to transplantation in 81 patients, was conducted. Univariate analyses were performed on 43 preimplantation prognostic factors. From this group, eight factors were chosen for multivariate analysis. Our results were compared with all recent risk factor analyses for other devices. RESULTS: Independent predictors for death at three intervals by multivariate analysis were as follows: "implant to transplant": history of smoking (odds ratio, 34); "implant to 30 days after transplant": history of smoking (odds ratio, 10.00), prothrombin time greater than 16 seconds (odds ratio, 4.76); and "implant to 1 year after transplant": prothrombin time greater than 16 seconds (odds ratio, 3.85). The major difference between this experience and multiple reported experiences with left ventricular assist devices is that for left ventricular assist devices, but not for the temporary CardioWest total artificial heart, right heart failure, high central venous pressure, and being on a ventilator (with or without sepsis) were independent predictors of mortality. CONCLUSIONS: Risk factors for bridge to transplantation with the CardioWest total artificial heart are different from those reported for left ventricular assist devices. Recognition of these risk factor differences may facilitate appropriate device selection.

Pediatric bridge to heart transplantation: application of the Berlin Heart, Medos and Thoratec ventricular assist devices.

BACKGROUND: Bridge to transplantation (BTT) is an accepted option when a donor heart is not available. Extensive clinical study has been done with BTT in the adult population, but comparatively fewer data are available in the pediatric population with regard to pulsatile devices. METHODS: Ten pediatric patients are presented, all of whom underwent BTT or recovery with pneumatic paracorporeal systems. The Berlin Heart bi-ventricular assist device (BVAD) was utilized in 1 patient, the Medos VAD in 4 patients (1 left ventricular assist device [LVAD], 3 BVADs) and the Thoratec VAD in 5 patients (3 BVADs, 2 LVADs). The pediatric population consisted of 3 females and 7 males. Mean age of the population was 7.4 years, weight 25 kg and body surface area (BSA) 0.88 m(2). Etiology for heart failure consisted of 4 viral, 3 congenital and 3 idiopathic cardiomyopathies. Before implant, all patients had evidence of progressive cardiac failure despite inotropic support, and 2 patients had been on extracorporeal membrane oxygenation (ECMO). Mean duration on the device was 34.3 days (8 to 107 days). RESULTS: Two patients suffered stroke and recovered without sequelae. Two patients died of ischemic stroke and 1 of sepsis. Seven patients survived (6 transplanted and 1 weaned) for a survival rate of 70% compared with survival for ECMO as BTT, which was 40% to 50%. All survivors had complications related to bleeding, thromboembolic events and infections. CONCLUSIONS: The Thoratec VAD can be placed in small patients with large hearts that can accommodate the available cannulas. The Berlin Heart and the Medos VAD have a selection of ventricles with small stroke volumes. All 3 systems can be used successfully in the pediatric population as BTT with better survival than with ECMO.

CardioWest total artificial heart in a moribund adolescent with left ventricular thrombi.

Bridge to transplant is a well-known strategy to enable patients with congestive heart failure to live until transplant. A 15-year-old boy with Beckers' muscular dystrophy and cardiomyopathy was accepted for heart transplantation. He suffered a cardiac arrest and was placed on extracorporeal membrane oxygenator. A paracorporeal biventricular assist device and a total artificial heart were considered for bridge to transplant. A CardioWest total artificial heart was chosen because of the patient's size. Multiple left ventricular thrombi were identified at the time of the ventriculectomy. The patient did well with the total artificial heart was transplanted and discharged home. The unknown presence of significant left ventricular thrombi raises the question of outcome with a paracorporeal ventricular assist device.