Discovery and characterization of sgRNA-sequence-independent DNA cleavage from CRISPR/Cas9 in mouse embryos.

The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and their affection in embryo development. To study these events, sgRNAs with different off-target rates were designed and compared after micro-injected into mouse zygotes, and γH2AX was used for DNA cleavage sites analysis by immunostaining and CUT&Tag. Although the low off-target sgRNA were usually selected for production gene editing animals, γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group than in the control group. Further, the result of CUT&Tag sequencing analysis showed more double-strand breaks (DSBs) related sequences were detected in low off-target sgRNA-injected group than control and the distribution of DSB related sequences had no chromosome specificity. Gene Ontology (GO) annotation analysis of the DSB related sequences showed that these sequences were mainly concentrated at genes associated with some important biological processes, molecular functions, and cell components. In a conclusion, there are many sgRNA-sequence-independent DSBs in early mouse embryos when the Cas9 system is used for gene editing and the DSB related sequence could be detected and characterized in the genome. These results and method should also be considered in using or optimizing the Cas9 system.

Integrative Omics Reveals Rapidly Evolving Regulatory Sequences Driving Primate Brain Evolution.

Although the continual expansion of the brain during primate evolution accounts for our enhanced cognitive capabilities, the drivers of brain evolution have scarcely been explored in these ancestral nodes. Here, we performed large-scale comparative genomic, transcriptomic, and epigenomic analyses to investigate the evolutionary alterations acquired by brain genes and provide comprehensive listings of innovatory genetic elements along the evolutionary path from ancestral primates to human. The regulatory sequences associated with brain-expressed genes experienced rapid change, particularly in the ancestor of the Simiiformes. Extensive comparisons of single-cell and bulk transcriptomic data between primate and nonprimate brains revealed that these regulatory sequences may drive the high expression of certain genes in primate brains. Employing in utero electroporation into mouse embryonic cortex, we show that the primate-specific brain-biased gene BMP7 was recruited, probably in the ancestor of the Simiiformes, to regulate neuronal proliferation in the primate ventricular zone. Our study provides a comprehensive listing of genes and regulatory changes along the brain evolution lineage of ancestral primates leading to human. These data should be invaluable for future functional studies that will deepen our understanding not only of the genetic basis of human brain evolution but also of inherited disease.

Molecular mechanisms underlying human spatial cognitive ability revealed with neurotransmitter and transcriptomic mapping.

Mental rotation, one of the cores of spatial cognitive abilities, is closely associated with spatial processing and general intelligence. Although the brain underpinnings of mental rotation have been reported, the cellular and molecular mechanisms remain unexplored. Here, we used magnetic resonance imaging, a whole-brain spatial distribution atlas of 19 neurotransmitter receptors, transcriptomic data from Allen Human Brain Atlas, and mental rotation performances of 356 healthy individuals to identify the genetic/molecular foundation of mental rotation. We found significant associations of mental rotation performance with gray matter volume and fractional amplitude of low-frequency fluctuations in primary visual cortex, fusiform gyrus, primary sensory-motor cortex, and default mode network. Gray matter volume and fractional amplitude of low-frequency fluctuations in these brain areas also exhibited significant sex differences. Importantly, spatial correlation analyses were conducted between the spatial patterns of gray matter volume or fractional amplitude of low-frequency fluctuations with mental rotation and the spatial distribution patterns of neurotransmitter receptors and transcriptomic data, and identified the related genes and neurotransmitter receptors associated with mental rotation. These identified genes are localized on the X chromosome and are mainly involved in trans-synaptic signaling, transmembrane transport, and hormone response. Our findings provide initial evidence for the neural and molecular mechanisms underlying spatial cognitive ability.

Neural Stem Cells Transplanted into Rhesus Monkey Cortical Traumatic Brain Injury Can Survive and Differentiate into Neurons.

Cortical traumatic brain injury (TBI) is a major cause of cognitive impairment accompanied by motor and behavioral deficits, and there is no effective treatment strategy in the clinic. Cell transplantation is a promising therapeutic strategy, and it is necessary to verify the survival and differentiation of cells after transplantation in large animal models like rhesus monkeys. In this study, we transplanted neural stem cells (NSCs) and simultaneously injected basic fibroblast growth factor/epidermal growth factor (bFGF/EGF) into the cortex (visual and sensory cortices) of rhesus monkeys with superficial TBI. The results showed that the transplanted NSCs did not enter the cerebrospinal fluid (CSF) and were confined to the transplantation site for at least one year. The transplanted NSCs differentiated into mature neurons that formed synaptic connections with host neurons, but glial scar formation between the graft and the host tissue did not occur. This study is the first to explore the repairing effect of transplanting NSCs into the superficial cerebral cortex of rhesus monkeys after TBI, and the results show the ability of NSCs to survive long-term and differentiate into neurons, demonstrating the potential of NSC transplantation for cortical TBI.

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera.

Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO4 treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

EGF/bFGF promotes survival, migration and differentiation into neurons of GFP-labeled rhesus monkey neural stem cells xenografted into the rat brain.

Stem cell replacement therapy is considered a promising treatment for diseases of the central nervous system. Improving the ratio of surviving transplanted cells and the efficiency of differentiation into functional neuronal cells are the most important issues related to research on neuroregenerative medicine. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have been reported to promote the proliferation and differentiation of neural stem cells (NSCs) in vitro, but whether they have the same effect in vivo is unclear. In this study, NSCs derived from rhesus monkey embryonic stem cells (ESCs) were resuspended in medium with or without EGF/bFGF and xenotransplanted into the rat striatum. No behavioral abnormalities or teratoma formation were observed in the recipient engrafted rats. GFP-labeled cells exhibited a higher survival rate and longer migration in the EGF/bFGF group than control group at 2 months after transplantation. Moreover, the percentages of Tuj1+ neurons and Map2+ neurons in the EGF/bFGF group were significantly higher than those in the control group, while the percentages of astrocytes and oligodendrocytes were significantly lower in the EGF/bGFG group than control group. These findings indicated that EGF/bFGF can promote protrusion of nerve fibers and the survival and neuronal differentiation of transplanted NSCs in the recipient brain, suggesting that EGF/bFGF has a potential application for stem cell therapy.

White Matter Structural and Network Topological Changes Underlying the Behavioral Phenotype of MECP2 Mutant Monkeys.

To explore the brain structural basis underlying the behavioral abnormalities associated with Rett syndrome (RTT), we carried out detailed longitudinal noninvasive magnetic resonance imaging analyses of RTT monkey models created by gene-editing, from weaning, through adolescence, till sexual maturation. Here, we report abnormal developmental dynamics of brain white matter (WM) microstructures and network topological organizations via diffusion tensor imaging. Specifically, disrupted WM microstructural integrity was observed at 9 months, but recovered thereafter, whereas WM network topological properties showed persistent abnormal dynamics from 9 to 37 months. Changes in the WM microstructure and WM network topology were correlated well with RTT-associated behavioral abnormalities including sleep latency, environmental exploration, and conflict encounters. Deleterious and protracted early WM myelination process likely lead to abnormal synaptic pruning, resulting in poor functional segregations. Together, this study provides initial evidence for changes in WM microstructure and network topological organization, which may underlie the neuro-patho-etilogy of RTT.

Comparison of quintrione and quinclorac on mechanism of action.

Quintrione is a new post-emergence herbicide developed for use in rice; however, the mechanism of action remains unclear. We determined the phytotoxicity of quintrione, and the contributions of hormone levels and lipid peroxidation to phytotoxicity, by comparing them to those induced by quinclorac. We also investigated 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity and carotenoid content following treatment with quintrione by comparing them to those induced by quinclorac and mesotrione. We found that quintrione and quinclorac both inhibited the growth of Echinochloa crusgalli var. zelayensis, but that quinclorac was a little more effective. At 24 h, quintrione and quinclorac significantly increased ethylene production and the contents of abscisic acid (ABA) and indole acetic acid (IAA) compared with the control. No significant differences were observed between quintrione and quinclorac on the three plant hormones. Quintrione and quinclorac also induced the formation of malondialdehyde (MDA), which is associated with lipid peroxidation, with no significant difference between them. Carotenoid content was reduced in E. crusgalli var. zelayensis following treatments with quintrione, quinclorac, and mesotrione. At 120 h, carotenoid contents were significantly higher following the quintrione and quinclorac treatments, in comparison with mesotrione treatment. There were no significant differences between quintrione and quinclorac in the inhibition of HPPD activity, and the effects of both were significantly less than the effect of mesotrione. In summary, E. crusgalli var. zelayensis was susceptible to both quintrione and quinclorac. The mechanism of action of quintrione, like that of quinclorac, was related to levels of plant hormones and lipid peroxidation; however, quintrione was a poor inhibitor of HPPD activity compared to mesotrione.

Recent Advances in Polymer Flooding in China.

Polymer flooding is drawing lots of attention because of the technical maturity in some reservoirs. The first commercial polymer flooding in China was performed in the Daqing oilfield and is one of the largest applications in the world. Some laboratory tests from Daqing researchers in China showed that the viscoelasticity of high molecular weight polymers plays a significant role in increasing displacement efficiency. Hence, encouraged by the conventional field applications and new findings on the viscoelasticity effect of polymers on residual oil saturation (ROS), some high-concentration high-molecular-weight (HCHMW) polymer-flooding field tests have been conducted. Although some field tests were well-documented, subsequent progress was seldom reported. It was recently reported that HCHMW has a limited application in Daqing, which does not agree with observations from laboratory core flooding and early field tests. However, the cause of this discrepancy is unclear. Thus, a systematic summary of polymer-flooding mechanisms and field tests in China is necessary. This paper explained why HCHMW is not widely used when considering new understandings of polymer-flooding mechanisms. Different opinions on the viscoelasticity effect of polymers on ROS reduction were critically reviewed. Other mechanisms of polymer flooding, such as wettability change and gravity stability effect, were discussed with regard to widely reported laboratory tests, which were explained in terms of the viscoelasticity effects of polymers on ROS. Recent findings from Chinese field tests were also summarized. Salt-resistance polymers (SRPs) with good economic performance using produced water to prepare polymer solutions were very economically and environmentally promising. Notable progress in SRP flooding and new amphiphilic polymer field tests in China were summarized, and lessons learned were given. Formation blockage, represented by high injection pressure and produced productivity ability, was reported in several oil fields due to misunderstanding of polymers' injectivity. Although the influence of viscoelastic polymers on reservoir conditions is unknown, the injection of very viscous polymers to displace medium-to-high viscosity oils is not recommended. This is especially important for old wells that could cause damage. This paper clarified misleading notions on polymer-flooding implementations based on theory and practices in China.

Annotation and cluster analysis of spatiotemporal- and sex-related lncRNA expression in rhesus macaque brain.

Long noncoding RNAs (lncRNAs) mediate important epigenetic regulation in a wide range of biological processes and diseases. We applied comprehensive analyses of RNA-seq and CAGE-seq (cap analysis of gene expression and sequencing) to characterize the dynamic changes in lncRNA expression in rhesus macaque (Macaca mulatta) brain in four representative age groups. We identified 18 anatomically diverse lncRNA modules and 14 mRNA modules representing spatial, age, and sex specificities. Spatiotemporal- and sex-biased changes in lncRNA expression were generally higher than those observed in mRNA expression. A negative correlation between lncRNA and mRNA expression in cerebral cortex was observed and functionally validated. Our findings offer a fresh insight into spatial-, age-, and sex-biased changes in lncRNA expression in macaque brain and suggest that the changes represent a previously unappreciated regulatory system that potentially contributes to brain development and aging.

Mutant alpha-synuclein causes age-dependent neuropathology in monkey brain.

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD.

A new method for piercing the tentorium cerebelli for implanting fragile electrodes into the brain stem in the rhesus monkey (Macaca mulatta).

Recent developments in neuron recording techniques include the invention of some fragile electrodes. The fragility of these electrodes impedes their successful use in deep brain recordings because it is difficult to penetrate the electrodes through the dura mater, especially the tentorium cerebelli (TC) enclosing the cerebellum and brain stem. This paper reports a new method to pierce the TC for inserting fragile electrodes into the inferior colliculus of rhesus monkeys. Briefly, a unique tool kit, consisting of needles with sharp tips, a guide tube and an "impactor," was used in a multistep protocol to pierce the TC. The impactor provided a brief force that quickly thrusts the needles through the meninges without causing significant damage to the brain tissue under the TC. Using this novel approach, tetrodes were successfully implanted into the inferior colliculus of a rhesus monkey and neuronal discharge signals were recorded. This method, which is simple, convenient and economical, allows neurophysiologists to study the electrophysiological characteristics of deep brain structures under the TC with advanced, albeit fragile, electrodes.

The Impact of Microglia on Neurodevelopment and Brain Function in Autism.

Microglia, as one of the main types of glial cells in the central nervous system (CNS), are widely distributed throughout the brain and spinal cord. The normal number and function of microglia are very important for maintaining homeostasis in the CNS. In recent years, scientists have paid widespread attention to the role of microglia in the CNS. Autism spectrum disorder (ASD) is a highly heterogeneous neurodevelopmental disorder, and patients with ASD have severe deficits in behavior, social skills, and communication. Most previous studies on ASD have focused on neuronal pathological changes, such as increased cell proliferation, accelerated neuronal differentiation, impaired synaptic development, and reduced neuronal spontaneous and synchronous activity. Currently, more and more research has found that microglia, as immune cells, can promote neurogenesis and synaptic pruning to maintain CNS homeostasis. They can usually reduce unnecessary synaptic connections early in life. Some researchers have proposed that many pathological phenotypes of ASD may be caused by microglial abnormalities. Based on this, we summarize recent research on microglia in ASD, focusing on the function of microglia and neurodevelopmental abnormalities. We aim to clarify the essential factors influenced by microglia in ASD and explore the possibility of microglia-related pathways as potential research targets for ASD.

Cell reprogramming therapy for Parkinson's disease.

Parkinson's disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson's disease. The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson's disease, which could substantially alleviate the symptoms of Parkinson's disease in clinical practice. However, ethical issues and tumor formation were limitations of its clinical application. Induced pluripotent stem cells can be acquired without sacrificing human embryos, which eliminates the huge ethical barriers of human stem cell therapy. Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons, without the need for intermediate proliferation states, thus avoiding issues of immune rejection and tumor formation. Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson's disease. However, there are also ethical concerns and the risk of tumor formation that need to be addressed. This review highlights the current application status of cell reprogramming in the treatment of Parkinson's disease, focusing on the use of induced pluripotent stem cells in cell replacement therapy, including preclinical animal models and progress in clinical research. The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson's disease, as well as the controversy surrounding in vivo reprogramming. These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson's disease.

Competition between homogeneous and heterogeneous crystallization of CaCO3 during water softening.

Homogeneous and heterogeneous crystallization of CaCO3 simultaneously occur in seed-induced crystallization during water softening, while suppressing homogeneous crystallization is necessary due to the production of fine particulates that poorly precipitate. However, homogeneous crystallization is difficult to distinguish from heterogeneous crystallization. Consequently, a central focus in improving water softening is understanding their competing activities. In this study, a novel method for distinguishing homogeneous and heterogeneous calcium carbonate crystallization is described that utilizes magnetite as seed particles. Results showed that saturation index (SI) was the primary driver of both homogeneous and heterogeneous crystallizations. Heterogeneous crystallization was preferentially promoted at low SI, while homogeneous crystallization was promoted at high SI. The highest suppression effect to homogeneous crystallization occurred at SI of about 1.01. Seed dosage and mean particle size were the primary parameters related to the competition of the crystallization types. Higher seed dosage and smaller seed particle sizes promoted heterogeneous crystallization and suppressed homogeneous crystallization. Due to the good adaptability of heterogeneous crystallization at low SI, the absorption of CO2 from the air into the solutions also improved the efficiency of hardness removal. The introduction of seed particles did not change crystalline product phases, with calcite being the only observed phase and possessing rhombohedral forms with highly regular and smooth edges. Water softening pilot test results showed that SI of 1.5 was more favorite for CaCO3 layer formation on seed surface and hardness removal in comparison with SI of 1.0 and 2.0. Overall, the results from this study demonstrate that the introduction of seed particles is a promising approach to suppress the homogeneous crystallization of CaCO3. Moreover, these results can serve as a framework for improved seed-induced crystallization during water softening.

Psychological experience and coping strategies of pregnant women with acute pancreatitis: a qualitative descriptive study.

BACKGROUND: To clarify the psychological experience and coping strategies in patients with acute pancreatitis in pregnancy (APIP) and propose interventional measures to improve pregnancy outcomes in these women. With an increasing trend of pregnant women in advanced ages and multiparous women, the incidence of APIP has significantly increased. Pregnancy accompanied by concurrent pancreatitis may subject these women to notable psychological stress, which is a factor that has been infrequently reported in previous studies. METHODS: APIP patients were interviewed from December 2020 to June 2021. Data were collected through semi-structured interviews based on an outline, including six questions. The interviews were recorded and analyzed using qualitative content analysis until data saturation was reached. RESULTS: Ten APIP patients were interviewed and four themes were identified, including excessive psychological burden, uncomfortable experience, urgent requirement for adequate medical resources, and importance of social support. CONCLUSION: Patients with APIP suffer from significant psychological stress due to their medical conditions and management. They desired adequate medical resources and social support. The local health department, hospital administrators, and medical staff should understand the psychological requirements and provide adequate healthcare and education that are easily accessible to these APIP patients. In addition, family support should also be encouraged to promote APIP patients' recovery.

Tumor marker-based RecistTM is superior to RECIST as criteria to predict the long-term benefits of targeted therapy in advanced non-small-cell lung cancer with driver gene mutations.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are standard first-line treatments for advanced non-small-cell lung cancer (NSCLC) with driver gene mutations. The Response Evaluation Criteria in Solid Tumors (RECIST) are limited in predicting long-term patient benefits. A tumour marker-based evaluation criteria, RecistTM, was used to investigate the potential for assessing targeted-therapy efficacy in lung cancer treatment. METHODS: We retrospectively analysed patients with stage IIIA-IV NSCLC and driver gene mutations, whose baseline tumour marker levels exceeded the pre-treatment cut-off value three-fold and who received TKI-targeted therapy as a first-line treatment. We compared efficacy, progression-free survival (PFS), and overall survival (OS) between RecistTM and RECIST. FINDINGS: The median PFS and OS differed significantly among treatment-response subgroups based on RecistTM but not RECIST. The predicted 1-, 2-, and 3-year disease-progression risk, according to area under the receiver operating characteristic curve, as well as the 1-, 3-, and 5-year mortality risk, differed significantly between RecistTM and RECIST. The median PFS and OS of tmCR according to RecistTM, was significantly longer than (CR+PR) according to RECIST. Imaging analysis revealed that the ΔPFS was 11.27 and 6.17 months in the intervention and non-intervention groups, respectively, suggesting that earlier intervention could extend patients' PFS. INTERPRETATION: RecistTM can assess targeted-therapy efficacy in patients with advanced NSCLC and driver gene mutations, along with tumour marker abnormalities. RecistTM surpasses RECIST in predicting short- and long-term patient benefits, and allows the early identification of patients resistant to targeted drugs, enabling prompt intervention and extending the imaging-demonstrated time to progression.

Analysis of the Relation Between Balance Control Subsystems: A Structural Equation Modeling Approach.

Balance plays a crucial role in human life and social activities. Maintaining balance is a relatively complex process that requires the participation of various balance control subsystems (BCSes). However, previous studies have primarily focused on evaluating an individual's overall balance ability or the ability of each BCS in isolation, without considering how they influence (or interact with) each other. The first study used clinical scales to evaluate the functions of the four BCSes, namely Reactive Postural Control (RPC), Anticipatory Postural Adjustment (APA), Dynamic Gait (DG), and Sensory Orientation (SO), and psychological factors such as fear of falling (FOF). A hierarchical structural equation modeling (SEM) was used to investigate the relationship between the BCSes and their association with FOF. The second study involved using posturography to measure and extract parameters from the center of pressure (COP) signal. SEM with sparsity constraint was used to analyze the relationship between vision, proprioception, and vestibular sense on balance based on the extracted COP parameters. The first study revealed that the RPC, APA, DG and SO indirectly influenced each other through their overall balance ability, and their association with FOF was not the same. APA has the strongest association with FOF, while RPC has the least association with FOF. The second study revealed that sensory inputs, such as vision, proprioception, and vestibular sensing, directly affected each other, but their associations were not identical. Among them, proprioception plays the most important role in the three sensory subsystems. This study provides the first numerical evidence that the BCSes are not independent of each other and exist in direct or indirect interplay. This approach has important implications for the diagnosis and management of balance-related disorders in clinical settings and improving our understanding of the underlying mechanisms of balance control.

Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Kisspeptin-10 modulates the proliferation and differentiation of the rhesus monkey derived stem cell line: R366.4.

The rhesus monkey embryonic stem cell line R366.4 has been identified to differentiate into a number of cell types. However, it has not been well characterized for its response to drugs affecting reproductive endocrinology. Kisspeptins (KPs) are ligands for the GPR-54, which is known to modulate reproductive function. The current study was designed to determine the effect of the KP-10 peptide on R366.4 cells and to investigate the role of KP-GPR54 in the cell proliferation process. Four different doses (0.1, 1, 10, and 100 nM) of KP-10 and control were selected to evaluate cell growth parameters and cellular morphological changes over a 72 hr period. The cells were treated with kisspeptin-10 during the early rosette stage. Proliferation rates, analyzed by flow cytometry and cell count methods, were found to be decreased after treatment. Moreover, the number of rosettes was found to decrease following KP-10 treatments. Morphological changes consisting of neuronal projections were also witnessed. This suggested that KP-10 had an antiproliferative effect on R366.4 cells leading to a differentiation state and morphological changes consistent with neuronal stem cell development. The R366.4 stem cell line differentiates based on kisspeptin signaling and may be used to investigate reproductive cell endocrinology in vitro.