Failure Of Neural Tube Closure Research Articles

Pin1 Downregulation Is Involved in Excess Retinoic Acid-Induced Failure of Neural Tube Closure.

Neural tube defects (NTDs), which are caused by impaired embryonic neural tube closure, are one of the most serious and common birth defects. Peptidyl-prolyl cis/trans isomerase 1 (Pin1) is a prolyl isomerase that uniquely regulates cell signaling by manipulating protein conformation following phosphorylation, although its involvement in neuronal development remains unknown. In this study, we explored the involvement of Pin1 in NTDs and its potential mechanisms both in vitro and in vivo. The levels of Pin1 expression were reduced in NTD models induced by all-trans retinoic acid (Atra). Pin1 plays a significant role in regulating the apoptosis, proliferation, differentiation, and migration of neurons. Moreover, Pin1 knockdown significantly was found to exacerbate oxidative stress (OS) and endoplasmic reticulum stress (ERs) in neuronal cells. Further studies showed that the Notch1-Nrf2 signaling pathway may participate in Pin1 regulation of NTDs, as evidenced by the inhibition and overexpression of the Notch1-Nrf2 pathway. In addition, immunofluorescence (IF), co-immunoprecipitation (Co-IP), and GST pull-down experiments also showed that Pin1 interacts directly with Notch1 and Nrf2. Thus, our study suggested that the knocking down of Pin1 promotes NTD progression by inhibiting the activation of the Notch1-Nrf2 signaling pathway, and it is possible that this effect is achieved by disrupting the interaction of Pin1 with Notch1 and Nrf2, affecting their proteostasis. Our research identified that the regulation of Pin1 by retinoic acid (RA) and its involvement in the development of NTDs through the Notch1-Nrf2 axis could enhance our comprehension of the mechanism behind RA-induced brain abnormalities.

A CASE REPORT OF MYELOMENIGOCELE WITH ARNOLD CHIARI MALFORMATIN II-HIGHLIGHTING THE IMPORTANCE OF ANEMIA MUKTH BHARATH PROGRAMME

Introduction:Neural tube defects are common complex congenital malformations resulting from failure of neural tube closure during embryogenesis, which usually completes by 28 days post conception. Myelomeningocele is almost always associated with Arnold Chiari Malformation II (CM II)and vice versa. Aims &Objectives : As most of the neural tube defects can be prevented by pre-conceptional folate supplements, we would like to high light the importance of proper implementation and utilization of Anemia Mukhth Bharath Programme. Background: Neural tube defects (NTDs) are caused by a combination of multiple genes and multiple environmental factors like folic acid deficiency, insulin dependent diabetes, obesity and certain medications which interfere with folate metabolism.One of the beneficiaries under the Anemia Mukth Bharath programme are women in the reproductive age group in the pre-conception period and up to the first trimester of the pregnancy. They are advised to have400 mcg of Folic Acid tablets, daily, to reduce the incidence of neural tube defects in the fetus. Case report :We present a case of Chiari malformation associated with megaloblastic anemia with an attempt to highlight the importance of proper utilization of national program for prevention of iron and folate deficiency in women of reproductive age group. Clinical relevance : Neural tube defects are largely preventable anomalies by simple and effective management of folate deficiency preconceptionally. Conclusion :Proper utilization of the Anemia Mukth Bharath program which is available to the beneficiaries at their doorstep can not only prevent maternal anemia but also prevent fetal loss due to NTD.

Identification and functional analysis of rare HECTD1 missense variants in human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased extracellular heat shock protein 90 (eHSP90) secretion to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with NTDs in humans.

Up-regulation of miR-10a-5p expression inhibits the proliferation and differentiation of neural stem cells by targeting Chl1.

Neural tube defects (NTDs) are characterized by the failure of neural tube closure during embryogenesis and are considered the most common and severe central nervous system anomalies during early development. Recent microRNA (miRNA) expression profiling studies have revealed that the dysregulation of several miRNAs plays an important role in retinoic acid (RA)-induced NTDs. However, the molecular functions of these miRNAs in NTDs remain largely unidentified. Here, we show that miR-10a-5p is significantly upregulated in RA-induced NTDs and results in reduced cell growth due to cell cycle arrest and dysregulation of cell differentiation. Moreover, the cell adhesion molecule L1-like ( Chl1) is identified as a direct target of miR-10a-5p in neural stem cells (NSCs) in vitro, and its expression is reduced in RA-induced NTDs. siRNA-mediated knockdown of intracellular Chl1 affects cell proliferation and differentiation similar to those of miR-10a-5p overexpression, which further leads to the inhibition of the expressions of downstream ERK1/2 MAPK signaling pathway proteins. These cellular responses are abrogated by either increased expression of the direct target of miR-10a-5p ( Chl1) or an ERK agonist such as honokiol. Overall, our study demonstrates that miR-10a-5p plays a major role in the process of NSC growth and differentiation by directly targeting Chl1, which in turn induces the downregulation of the ERK1/2 cascade, suggesting that miR-10a-5p and Chl1 are critical for NTD formation in the development of embryos.

Downregulation of extraembryonic tension controls body axis formation in avian embryos

Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that the chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. Experimental relaxation of the VM early in development impairs blastoderm expansion, while maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, failure of neural tube closure, and axis rupture. Biochemical and structural analysis shows that VM weakening is associated with the reduction of outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized potential cause of body axis defects through mis-regulation of extraembryonic tissue tension.

Microdeletions on chromosomes 2 and 20 are associated with equine early pregnancy loss

Copy number variants (CNVs) are chromosomal microdeletions and microduplications greater than 1000 bp and can confer disease risk, if they impact gene dosage. Specific pathogenic CNVs have been described as associated with miscarriage and stillbirth in women but are unexplored in equine pregnancy. This study aimed to identify and characterize pathogenic CNVs associated with equine early embryonic loss (EEL) and early fetal losses collectively referred to as early pregnancy loss (EPL). Conceptuses were submitted to the laboratory by the attending veterinarians following clinical confirmation of pregnancy failure between gestation days 15 and 65. DNA was isolated from the allantochorion (ALC) (n=52) and fetus (n=13) of EPL pregnancies. As a control, DNA was isolated from allantochorion and fetuses of manually terminated clinically normal pregnancies (CNP n=20), and peripheral blood mononuclear cells (PBMCs) were isolated from adult horses (n=371). DNA was hybridized to 96 sample Axiom Equine 670K Arrays. DNAcopy was used to identify autosomal CNVs >1000 bp in length after excluding samples with <95% call rate, MAPD <0.35, log2 intensity between -0.2 and +0.2, and a significance of p>0.05. Two microdeletions were found in at least two EPL conceptuses that were absent in all control samples. A review of the literature confirmed both microdeletions were unreported to date (n=3040 individuals). In this study, PCR confirmed the deletions(del2:91608493-91610830 and del20:30652320-30656312). Bioinformatic analysis of del2:91608493-91610830 using ENSEMBL revealed that the 2.3 kb deletion region contained part of exon1 and intron1 of the mastermind like transcriptional coactivator 3 (MAML3) gene, a co-activator of Notch signaling known to play a key role in early development. This deletion in MAML3 was identified in the ALC of two EPLs lost at 42 and 64 days of gestation. Del20:30652320-30656312 was identified in seven EPLs, including both the fetal and ALC genome of two EPLs and only the fetal genome of an additional five EPLs. Bioinformatic analysis revealed the deletion in ECA20 encompassed a 4 kb region that included exon1 and partial intron1 of both transcripts for tubulin beta class 1 (TUBB), which is essential to neurogenesis. Five of the seven EPLs failed at day 35-40, and three of the fetuses had abnormalities of the central nervous system, including one failure of neural tube closure. Whilst causality can't be confirmed from these experiments, the rarity of microdeletions in viable horses, and the biological plausibility of the deleted gene impacting early development is supportive of a role for both microdeletions in lethality of the conceptus in the first two months of pregnancy. Further experiments are required to determine if the deletions are acquired through the germline.

Prenatal cadmium exposure impairs neural tube closure via inducing excessive apoptosis in neuroepithelium

Birth defects have become a public health concern. The hazardous environmental factors exposure to embryos could increase the risk of birth defects. Cadmium, a toxic environmental factor, can cross the placental barrier during pregnancy. Pregnant woman may be subjected to cadmium before taking precautionary protective actions. However, the link between birth defects and cadmium remains obscure. Cadmium exposure can induce excessive apoptosis in neuroepithelium during embryonic development progresses. Cadmium exposure activated the p53 via enhancing the adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) and reactive oxygen species’ (ROS) level. And cadmium decreases the level of Paired box 3 (Pax3) and murine double minute 2 (Mdm2), disrupting the process of p53 ubiquitylation. And p53 accumulation induced excessive apoptosis in neuroepithelium during embryonic development progresses. Excessive apoptosis led to the failure of neural tube closure. The study emphasizes that environmental materials may increase the health risk for embryos. Cadmium caused the failure of neural tube closure during early embryotic day. Pregnant women may be exposed by cadmium before taking precautionary protective actions, because of cadmium concentration-containing foods and environmental tobacco smoking. This suggests that prenatal cadmium exposure is a threatening risk factor for birth defects.

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

First person – Sarah Escuin and Saba Raza-Knight

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models &amp; Mechanisms, helping researchers promote themselves alongside their papers. Sarah Escuin and Saba Raza-Knight are co-first authors on ‘ Dual mechanism underlying failure of neural tube closure in the Zic2 mutant mouse’, published in DMM. Sarah conducted the research described in this article while a research associate in the lab of Prof. Andrew Copp at University College London, London, UK, investigating the mechanisms of neurulation and identifying the causes underlying neural tube defects. Saba conducted the research described in this article while an MB PhD student in the lab of Prof. Andrew Copp and Prof. Nicholas Greene at University College London, London, UK. She is now a specialty trainee in neurosurgery at Salford Royal Hospital, Salford, UK, and an honorary clinical lecturer at the University of Manchester, Manchester, UK, investigating neurovascular disorders, cancer biology and genetics, and hydrocephalus, with an active role in medical education.

Dual mechanism underlying failure of neural tube closure in the Zic2 mutant mouse.

Understanding the molecular mechanisms that lead to birth defects is an important step towards improved primary prevention. Mouse embryos homozygous for the Kumba (Ku) mutant allele of Zic2 develop severe spina bifida with complete lack of dorsolateral hinge points (DLHPs) in the neuroepithelium. Bone morphogenetic protein (BMP) signalling is over-activated in Zic2Ku/Ku embryos, and the BMP inhibitor dorsomorphin partially rescues neural tube closure in cultured embryos. RhoA signalling is also over-activated, with accumulation of actomyosin in the Zic2Ku/Ku neuroepithelium, and the myosin inhibitor Blebbistatin partially normalises neural tube closure. However, dorsomorphin and Blebbistatin differ in their effects at tissue and cellular levels: DLHP formation is rescued by dorsomorphin but not Blebbistatin, whereas abnormal accumulation of actomyosin is rescued by Blebbistatin but not dorsomorphin. These findings suggest a dual mechanism of spina bifida origin in Zic2Ku/Ku embryos: BMP-dependent formation of DLHPs is faulty, together with RhoA-dependent F-actin accumulation in the neuroepithelium. Hence, we identify a multi-pathway origin of spina bifida in a mammalian system that may provide a developmental basis for understanding the corresponding multifactorial human defects.

Neural Tube Defects and Folate Deficiency: Is DNA Repair Defective?

Neural tube defects (NTDs) are complex congenital malformations resulting from failure of neural tube closure during embryogenesis, which is affected by the interaction of genetic and environmental factors. It is well known that folate deficiency increases the incidence of NTDs; however, the underlying mechanism remains unclear. Folate deficiency not only causes DNA hypomethylation, but also blocks the synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) and increases uracil misincorporation, resulting in genomic instabilities such as base mismatch, DNA breakage, and even chromosome aberration. DNA repair pathways are essential for ensuring normal DNA synthesis, genomic stability and integrity during embryonic neural development. Genomic instability or lack of DNA repair has been implicated in risk of development of NTDs. Here, we reviewed the relationship between folate deficiency, DNA repair pathways and NTDs so as to reveal the role and significance of DNA repair system in the pathogenesis of NTDs and better understand the pathogenesis of NTDs.

Somitic mesoderm morphogenesis is necessary for neural tube closure during Xenopus development.

Neural tube closure is a fundamental process during vertebrate embryogenesis, which leads to the formation of the central nervous system. Defective neural tube closure leads to neural tube defects which are some of the most common human birth defects. While the intrinsic morphogenetic events shaping the neuroepithelium have been studied extensively, how tissues mechanically coupled with the neural plate influence neural tube closure remains poorly understood. Here, using Xenopus laevis embryos, live imaging in combination with loss of function experiments and morphometric analysis of fixed samples we explore the reciprocal mechanical communication between the neural plate and the somitic mesoderm and its impact on tissue morphogenesis. We show that although somitic mesoderm convergent extension occurs independently from neural plate morphogenesis neural tube closure depends on somitic mesoderm morphogenesis. Specifically, impaired somitic mesoderm remodelling results in defective apical constriction within the neuroepithelium and failure of neural tube closure. Last, our data reveal that mild abnormalities in somitic mesoderm and neural plate morphogenesis have a synergistic effect during neurulation, leading to severe neural tube closure defects. Overall, our data reveal that defective morphogenesis of tissues mechanically coupled with the neural plate can not only drastically exacerbate mild neural tube defects that may arise from abnormalities within the neural tissue but can also elicit neural tube defects even when the neural plate is itself free of inherent defects.

Pathogenesis of neural tube defects: The regulation and disruption of cellular processes underlying neural tube closure.

Neural tube closure (NTC) is crucial for proper development of the brain and spinal cord and requires precise morphogenesis from a sheet of cells to an intact three-dimensional structure. NTC is dependent on successful regulation of hundreds of genes, a myriad of signaling pathways, concentration gradients, and is influenced by epigenetic and environmental cues. Failure of NTC is termed a neural tube defect (NTD) and is a leading class of congenital defects in the United States and worldwide. Though NTDs are all defined as incomplete closure of the neural tube, the pathogenesis of an NTD determines the type, severity, positioning, and accompanying phenotypes. In this review, we survey pathogenesis of NTDs relating to disruption of cellular processes arising from genetic mutations, altered epigenetic regulation, and environmental influences by micronutrients and maternal condition. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Stem Cells and Development.

Inflammation in embryology: A review of neuroinflammation in spina bifida

The occurrence of neuroinflammation after the failure of neural tube closure, resulting in spina bifida aperta, is well established but whether or not neuroinflammation contributes to damage to the neuroepithelium prior to and during closure is not known. Neuroinflammation may occur at different time periods after perturbation to the developing spinal cord. Evidence suggests that early neuroinflammation is detrimental, whereas the later chronic phase of neuroinflammation may have useful roles. The role of neuroinflammation in neural tube defects is complex. It is important to make the distinction of whether neuroinflammation is important for neuroprotection or detrimental to the neural tissue. This may directly be influenced by the location, magnitude and duration of the insult, as well as the expression of neurotrophic or neurotoxic molecules. The current understanding remains that the chronic damage to the developing spinal cord is likely due to the chemical and mechanical damage of the exposed neural tissue owing to the aggressive intrauterine environment, described as the “two-hit mechanism”. Astrogliosis in the exposed spinal cord has been described in animal models of spina bifida after the failure of closure during embryonic life. Still, its association with neuroinflammatory processes is poorly understood. In this review, we will discuss the current understanding of neuroinflammation in neural tube defects, specifically spina bifida, and highlight inflammation-targeted strategies that may potentially be used to treat this pathophysiological condition.

Biodegradation of poly(l-lactic acid) and poly(\u03b5-caprolactone) patches by human amniotic fluid in an in-vitro simulated fetal environment

Open spina bifida or myelomeningocele (MMC) is a devastating neurologic congenital defect characterized by primary failure of neural tube closure of the spinal column during the embryologic period. Cerebrospinal fluid leak caused by the MMC spinal defect in the developing fetus can result in a constellation of encephalic anomalies that include hindbrain herniation and hydrocephalus. The exposure of extruded spinal cord to amniotic fluid also poses a significant risk for inducing partial or complete paralysis of the body parts beneath the spinal aperture by progressive spinal cord damage in-utero. A randomized trial demonstrated that prenatal repair by fetal surgery, sometimes using patches, to cover the exposed spinal cord with a watertight barrier is effective in reducing the postnatal neurologic morbidity as evidenced by decreased incidence and severity of postnatal hydrocephalus and the reduced need for ventricular-peritoneal shunting. Currently, the use of inert or collagen-based patches are associated with high costs and inadequate structural properties. Specifically, the inert patches do not degrade after implantation, causing the need for a post-natal removal surgery associated with trauma for the newborn. Our present study is aimed towards in-vitro degradation studies of a newly designed patch, which potentially can serve as a superior alternative to existing patches for MMC repair. This novel patch was fabricated by blending poly(l-lactic acid) and poly(ε-caprolactone). The 16-week degradation study in amniotic fluid was focused on tracking changes in crystallinity and mechanical properties. An additional set of designed patches was exposed to phosphate-buffered saline (PBS), as a time-paired control. Crystallinity studies indicate the progress of hydrolytic degradation of the patch in both media, with a preference to bulk erosion in phosphate buffered saline and surface erosion in amniotic fluid. Mechanical testing results establish that patch integrity is not compromised up to 16 weeks of exposure either to body fluids analog (PBS) or to amniotic fluid.

Nuclear factor I-C disrupts cellular homeostasis between autophagy and apoptosis via miR-200b-Ambra1 in neural tube defects

Impaired autophagy and excessive apoptosis disrupt cellular homeostasis and contribute to neural tube defects (NTDs), which are a group of fatal and disabling birth defects caused by the failure of neural tube closure during early embryonic development. However, the regulatory mechanisms underlying NTDs and outcomes remain elusive. Here, we report the role of the transcription factor nuclear factor I-C (NFIC) in maintaining cellular homeostasis in NTDs. We demonstrated that abnormally elevated levels of NFIC in a mouse model of NTDs can interact with the miR-200b promoter, leading to the activation of the transcription of miR-200b, which plays a critical role in NTD formation, as reported in our previous study. Furthermore, miR-200b represses autophagy and triggers apoptosis by directly targeting the autophagy-related gene Ambra1 (Autophagy/Beclin1 regulator 1). Notably, miR-200b inhibitors mitigate the unexpected effects of NFIC on autophagy and apoptosis. Collectively, these results indicate that the NFIC-miR-200b-Ambra1 axis, which integrates transcription- and epigenome-regulated miRNAs and an autophagy regulator, disrupts cellular homeostasis during the closure of the neural tube, and may provide new insight into NTD pathogenesis.

Back to the Beginning: An Algorithmic Approach to Neonatal Myelomeningocele Repair.

Myelomeningoceles are formed by prenatal failure of neural tube closure and can cause hydrocephalus, motor abnormalities, and developmental delay. Although small defects are amenable to primary closure, larger defects often require complex reconstruction. Our goal was to identify factors associated with postoperative soft tissue complications and develop a systematic approach for myelomeningocele closure. A retrospective review was performed at the Children's Memorial Hermann Hospital from January 2013 to January 2019. Patients were identified using International Classification of Diseases, Ninth Revision/Tenth Revision , codes for myelomeningocele. Cohorts were stratified by reconstruction type and defect location. Primary outcomes were incidence of complications including cerebrospinal fluid leak, superficial and deep infection, and wound dehiscence. In addition, we developed an algorithm to standardize closure approach for patients with myelomeningoceles. A total of 172 patients with myelomeningocele were identified with 73 patients undergoing postnatal repair. Overall, 72% of defects were >5 cm. Defects were in the lumbar (9%), sacral (8%), and junctional (83%) regions. Overall, 30.1% patients underwent lumbar myofascial repair with 39.7% requiring fasciocutaneous flaps. Larger defects (>5 cm) were more likely to be closed with complex fasciocutaneous flaps (82.8% vs 66.0%, P = 0.11). No significant differences were observed in complication rates. In this series, patients with larger myelomeningoceles appear to benefit from complex flap closure. We propose a 5-layer closure for patients with myelomeningocele including the routine use of a myofascial layer. Cutaneous closure technique should be tailored based on specific defect characteristics as outlined in our algorithm. This approach streamlines myelomeningocele repair while optimizing outcomes and decreasing downstream complications.

Mycotoxin Fumonisin B1 Interferes Sphingolipid Metabolisms and Neural Tube Closure during Early Embryogenesis in Brown Tsaiya Ducks.

Fumonisin B1 (FB1) is among the most common contaminants produced by Fusarium spp. fungus from corns and animal feeds. Although FB1 has been known to cause physical or functional defects of embryos in humans and several animal species such as Syrian hamsters, rabbits, and rodents, little is known about the precise toxicity to the embryos and the underlying mechanisms have not been fully addressed. The present study aimed to investigate its developmental toxicity and potential mechanisms of action on sphingolipid metabolism in Brown Tsaiya Ducks (BTDs) embryos. We examined the effect of various FB1 dosages (0, 10, 20 and 40 µg/embryo) on BTD embryogenesis 72 h post-incubation. The sphingomyelin content of duck embryos decreased (p < 0.05) in the highest FB1-treated group (40 µg). Failure of neural tube closure was observed in treated embryos and the expression levels of a neurulation-related gene, sonic hedgehog (Shh) was abnormally decreased. The sphingolipid metabolism-related genes including N-acylsphingosine amidohydrolase 1 (ASAH1), and ceramide synthase 6 (CERS6) expressions were altered in the treated embryos compared to those in the control embryos. Apparently, FB1 have interfered sphingolipid metabolisms by inhibiting the functions of ceramide synthase and folate transporters. In conclusion, FB1-caused developmental retardation and abnormalities, such as neural tube defects in Brown Tsaiya Duck embryos, as well as are partly mediated by the disruption of sphingolipid metabolisms.

Closure of large lumbosacral defect using a combined method of bilateral bipedicle flap with lateral releasing incision and Integra® dermal regeneration template

BACKGROUND: Myelomeningocele is one of the most complex congenital malformations of the central nervous system. It is one of the most common types of spina bifida which involves a failure of neural tube closure. Reconstruction surgery for myelomeningocele had always been challenging for plastic and neurosurgeons.&#x0D; CLINICAL CASE: We report a case of a new-born with lumbosacral myelomeningocele who received treatment in the Hospital Universiti Sains Malaysia. The myelomeningocele was repaired by the neurosurgery team and subsequently, the child was left with huge lumbosacral skin defect. The large defect was successfully covered by using a combined method of bilateral bipedicle flap with lateral releasing incision and remaining lumbosacral and secondary defect resurfaced using Integra dermal regeneration template (DRT). We used ACTICOAT interfaced negative pressure wound therapy (NPWT) as our main dressing in preparing the wound bed for autologous epidermal graft. The result of our closure technique provides tension free closure.&#x0D; DISCUSSION: We incorporated bilateral bipedicle fasciocutaneous flap technique together with DRT for closure of the lumbosacral defect. The bilateral bipedicle flap with lateral releasing incision served to reduce tension on the skin at bilateral lumbar region. The DRT downsized the lumbosacral defect and NPWT dressing provided an optimal sterile environment in giving time for neodermis generation. The remaining secondary defect were also resurfaced utilizing DRT and autologous skin grafting.&#x0D; CONCLUSIONS: The outcome of surgery demonstrated that the combined use of bilateral bipedicle fasciocutaneous flap with lateral releasing incision and DRT with delayed skin grafting is safe, effective and provide long term stable and supple scar for large, exposed dura defect.

CRISPR/Cas9-mediated gene editing on Sox2ot promoter leads to its truncated expression and does not influence neural tube closure and embryonic development in mice

Sox2 overlapping transcript (Sox2ot) is a long non-coding RNA (lncRNA), which harbors one of the major regulators of pluripotency, the Sox2 gene, in its intronic region. Sox2ot is primarily expressed in the developing neuroepithelium. However, its role in neural tube closure and embryonic development remains unclear. To investigate if Sox2ot is required for neural tube closure and embryonic development, Sox2ot promoter was deleted by CRISPR-Cas9 genome editing technology to prevent Sox2ot gene expression in mice. We designed 9 guide RNAs to specifically target the Sox2ot promoter and 3 gRNAs induced gene editing on the promoter of the Sox2ot gene in cells transfected with Cas9 mRNA and gRNAs. Then, these gRNAs and Cas9 mRNA were injected into mouse zygotes and implanted into pseudopregnant mice. A Sox2ot promoter-deleted mouse line was identified with complete deletion of promoter as well as deletion of exon 1 and exon 2. Sox2ot transcript was truncated with a lack of exon 1 and exon 2 in Sox2ot promoter-deleted mice. Furthermore, neural tube closure and embryonic development were checked at E9.5, E10.5, E14.5, E17.5 and after-birth (P2) and we did not find any failure of neural tube closure and aberrant embryonic development in Sox2ot promoter-deleted mice. Thus, our study demonstrated that CRISPR-Cas9 gene editing in Sox2ot promoter leads to its truncated expression and does not influence neural tube closure and embryonic development.