Development Diseases Research Articles

CRISPR/Cas9 Gene Disruption Studies in F0 Xenopus Tadpoles: Understanding Development and Disease in the Frog.

CRISPR/Cas9 has become the favorite method for gene knockouts in a range of vertebrate model organisms due to its ease of use and versatility. Gene-specific guide RNAs can be designed to a unique genomic sequence and used to target the Cas9 endonuclease, which causes a double-stranded break at the desired locus. Repair of the breaks through non-homologous end joining often results in the deletion or insertion of several nucleotides, which frequently result in nonsense mutations. Xenopus frogs have long been an excellent model organism in which to study gene function, and they have proven to be useful in gene-editing experiments, especially the diploid species, X. tropicalis. In this chapter, we present our protocols for gene disruption in Xenopus, which we regularly use to investigate developmental processes and model human genetic disease.

Pediatric retinal vascular disorders: From translational sciences to clinical practice.

Pediatric retinal vascular diseases are a spectrum with overlapping phenotypes and related genes. Retinal vascular development is biphasic. Vasculogenesis is responsible for the formation of primordial vessels leading to the four major arcades in the posterior retina. Angiogenesis, which is vascular endothelial growth factor dependent, is responsible for the formation of new vessels through budding from existing vessels, forming the peripheral vessels, increasing the capillary density of the central retina, and forming the superficial and deep capillary plexus. This process is controlled by WNT signaling, which is important for cell proliferation, division, and migration. Disorders of WNT signaling, such as familial exudative vitreoretinopathy (FEVR), have overlapping clinical findings. Conversely, pathogenic variants in some of the FEVR-related genes are reported in conditions such as retinopathy of prematurity (ROP), persistent fetal vasculature, and Coats disease. The various overlapping features and underlying genetic basis in the pathogenesis of pediatric retinal vascular developmental diseases suggest that genetic variants may provide a framework or a background for these conditions, upon which further insults can affect the development at any phase (such as prematurity and oxygenation in ROP), influencing and determining the final phenotype.

Protocol for the Generation of Human EPS-Blastoids Using a Three-Dimensional Two-Step Induction System.

Stem cell-derived embryos in vitro allow the exploration of the very early stages of human embryogenesis in vitro and are thus promising for widespread applications in developmental biology, related developmental disease modeling, and drug discovery. Several cell resources have been utilized, with different efficiencies and methods for generating human blastoids, a structure similar to natural blastocysts. Human EPS cells were reported to contribute to the embryonic and extraembryonic lineages and therefore can be a practical and efficient cell resource for constructing human blastoids. Here, we developed a three-dimensional, two-step induction system for generating human blastoids using human EPS cells. According to morphological and transcriptomic analysis, EPS-blastoids recapitulate the key developmental processes and cell lineages of human blastocysts. Moreover, in vitro extended culture for 8 and 10days of EPS-blastoids can result in postimplantation embryonic structures. In this chapter, we describe a protocol that covers the generation, maintenance, and developmental phenocopying of human EPS blastoids.

Fixation methods and immunolabeling for cilia proteins in ciliary and extraciliary locations.

Primary cilia are complex organelles, usually singularly located on cell surfaces that are now known to be important for signaling and whose defect is implicated in a category of developmental diseases known as ciliopathies. They are composed of a microtubule axoneme and contain a cilia membrane that is unique and distinct from the plasma membrane. Primary cilia also have their own transport system termed the intraflagellar transport (IFT) system that allows for proteins to be trafficked along the microtubule axoneme in either an anterograde or retrograde manner. Proteins that localize to the primary cilium are referred to as ciliary proteins and have been implicated directly or indirectly in ciliogenesis or ciliary function. It is now recognized that cilia proteins can localize to different compartments of cilia, but can also localize to multiple sites outside of cilia (extraciliary sites). This complexity results in a need for a better understanding of ciliary protein fixation and immunolabeling protocols, as different methods are required to visualize different cilia proteins and reveal novel or unique localizations. Here, we detail a variety of fixation methods and their effects on ciliary protein immunolabeling.

Defects and asymmetries in the visual pathway of non-human primates with natural strabismus and amblyopia.

Strabismus and amblyopia are common ophthalmologic developmental diseases caused by abnormal visual experiences. However, the underlying pathogenesis and visual defects are still not fully understood. Most studies have used experimental interference to establish disease-associated animal models, while ignoring the natural pathophysiological mechanisms. This study was designed to investigate whether natural strabismus and amblyopia are associated with abnormal neurological defects. We screened one natural strabismic monkey ( Macaca fascicularis) and one natural amblyopic monkey from hundreds of monkeys, and retrospectively analyzed one human strabismus case. Neuroimaging, behavioral, neurophysiological, neurostructural, and genovariation features were systematically evaluated using magnetic resonance imaging (MRI), behavioral tasks, flash visual evoked potentials (FVEP), electroretinogram (ERG), optical coherence tomography (OCT), and whole-genome sequencing (WGS), respectively. Results showed that the strabismic patient and natural strabismic and amblyopic monkeys exhibited similar abnormal asymmetries in brain structure, i.e., ipsilateral impaired right hemisphere. Visual behavior, visual function, retinal structure, and fundus of the monkeys were impaired. Aberrant asymmetry in binocular visual function and structure between the strabismic and amblyopic monkeys was closely related, with greater impairment of the left visual pathway. Several similar known mutant genes for strabismus and amblyopia were also identified. In conclusion, natural strabismus and amblyopia are accompanied by abnormal asymmetries of the visual system, especially visual neurophysiological and neurostructural defects. Our results suggest that future therapeutic and mechanistic studies should consider defects and asymmetries throughout the entire visual system.

The role of EZH1 and EZH2 in development and cancer

Polycomb Repressive Complex 2 (PRC2) exhibits key roles in mammalian development through its temporospatial repression of gene expression. EZH1 or EZH2 is the catalytic subunit of PRC2 that mediates the mono-, di- and tri-methylation of histone H3 lysine 27 (H3K27me1/2/3), H3K27me2/me3 being a hallmark of facultative heterochromatin. PRC2 is a chromatinmodifying enzyme that is recruited to a limited number of "nucleation sites", spreads H3K27 methylation and fosters chromatin compaction. EZH1 and EZH2 exhibit differences in their expression patterns, levels of histone methyltransferase activity (HMT) in the context of PRC2, and DNA/nucleosome binding activity. This suggests that their roles in heterochromatin formation are disparate. Dysregulation of PRC2 activity leads to aberrant gene expression and is implicated in cancer and developmental diseases. In this review, we discuss the distinct function of PRC2/EZH1 and PRC2/EZH2 in the early and late developmental stages. We then discuss the cancers associated with PRC2/EZH1 and PRC2/EZH2. [BMB Reports 2022; 55(12): 595-601].

Piezo protein determines stem cell fate by transmitting mechanical signals.

Piezo ion channel is a mechanosensitive protein on the cell membrane, which contains Piezo1 and Piezo2. Piezo channels are activated by mechanical forces, including stretch, matrix stiffness, static pressure, and shear stress. Piezo channels transmit mechanical signals that cause different downstream responses in the differentiation process, including integrin signaling pathway, ERK1/2 MAPK signaling pathway, Notch signaling, and WNT signaling pathway. In the fate of stem cell differentiation, scientists found differences in Piezo channel expression and found that Piezo channel expression is related to developmental diseases. Here, we briefly review the structure and function of Piezo channels and the relationship between Piezo and mechanical signals, discussing the current understanding of the role of Piezo channels in stem cell fate and associated molecules and developmental diseases. Ultimately, we believe this review will help identify the association between Piezo channels and stem cell fate.

Dynamic regulation and key roles of ribonucleic acid methylation.

Ribonucleic acid (RNA) methylation is the most abundant modification in biological systems, accounting for 60% of all RNA modifications, and affects multiple aspects of RNA (including mRNAs, tRNAs, rRNAs, microRNAs, and long non-coding RNAs). Dysregulation of RNA methylation causes many developmental diseases through various mechanisms mediated by N 6-methyladenosine (m6A), 5-methylcytosine (m5C), N 1-methyladenosine (m1A), 5-hydroxymethylcytosine (hm5C), and pseudouridine (Ψ). The emerging tools of RNA methylation can be used as diagnostic, preventive, and therapeutic markers. Here, we review the accumulated discoveries to date regarding the biological function and dynamic regulation of RNA methylation/modification, as well as the most popularly used techniques applied for profiling RNA epitranscriptome, to provide new ideas for growth and development.

Targeting the lung endothelial niche to promote angiogenesis and regeneration: A review of applications.

Lung endothelial cells comprise the pulmonary vascular bed and account for the majority of cells in the lungs. Beyond their role in gas exchange, lung ECs form a specialized microenvironment, or niche, with important roles in health and disease. In early development, progenitor ECs direct alveolar development through angiogenesis. Following birth, lung ECs are thought to maintain their regenerative capacity despite the aging process. As such, harnessing the power of the EC niche, specifically to promote angiogenesis and alveolar regeneration has potential clinical applications. Here, we focus on translational research with applications related to developmental lung diseases including pulmonary hypoplasia and bronchopulmonary dysplasia. An overview of studies examining the role of ECs in lung regeneration following acute lung injury is also provided. These diseases are all characterized by significant morbidity and mortality with limited existing therapeutics, affecting both young children and adults.

Ossifying Fibroma: A Case Report of an Unusual Presentation of Angiodysplastic Disease with Review of Literature.

Ossifying fibroma is a nonneoplastic developmental disease of osseous tissue seen rarely in association with Sturge-Weber syndrome. It is a lesion of unknown aetiology, uncertain pathogenesis, and diverse histopathology. The aim of this study is to report an unusual case of in a 11-year-old male of SWS. The rarity of the case and the fact that ossifying fibroma may be associated with Sturge-Weber syndrome propelled us to report it. Physical examination showed facial asymmetry (due to hemifacial swelling) without any tenderness, fluctuation, ocular pain, or ophthalmoplegia. Imaging studies revealed a solid mass involving the left maxilla and orbital floor. A conservative therapeutic approach to these lesions may be sufficient to relieve signs and symptoms effectively. Periodic follow-up is indicated to detect recurrences or malignant changes in the early stages.

A Review on Biomaterials for Organoid Modeling and Tumor Spheroids

Organoids are miniature forms of organs to demonstrate spatio-temporal cellular structure and tissue function. The organoids creation revolutionized developmental biology and provided the opportunity to study and modify human development and disease in laboratory setting. Recently, new biomaterial-guided culture systems have represented the versatility for designing and producing of organoids in a constant and reproducible manner. Since 2D cell culture models often lack in vivo tissue architecture, recent detailed research has allowed many 3D culture models development demonstrating the characteristics of in vivo organ structure and function. Organoid models are able to create 3D structures complex that maintain multiple cell types and also hide the relevant organ functions in vivo, and therefore, the development of organoids in particular has revolutionized developmental biology, disease modeling, and drug discovery. The new biomaterials production has been important for development of in vitro 3D models. Further work with biomaterials has been on the creation of hybrid polymers that combine the advantages of both synthetic and natural polymers to take place of communal materials such as Matrigel and polydimethylsiloxane (PDMS). The creation of 3D culture systems has also revolutionized in vitro drug testing. Furthermore, recreating the three-dimensional environment of tumors and the functional arrangement of cancer cells has been a major motivation for developing new tumor models. Under defined culture conditions, cancer cells can form three-dimensional structures known as spheroids and advances in development of embryonic to self-organize into three-dimensional cultures known as organoids. These newly designed biomaterials using for tumor modeling will make an important contribution to understand the main mechanisms of cancer.

Ginsenoside Rg1 regulates thiram-induced chondrocytes' apoptosis and angiogenesis in broiler chickens.

Tibial dyschondroplasia (TD) is a developmental cartilaginous disease due to thiram toxicity. The abnormity of chondrocytes and insufficient angiogenesis within the growth plate are the major factors leading to the occurrence of TD in most cases. In the current study, we evaluated the beneficial effects of ginsenoside (Rg1) against thiram-induced TD for knowing the possible underlying mechanisms in broiler chickens through in vivo and in vitro assessment. Arbor acres broilers (1-day-old, n = 120) were randomly divided for the in vivo evaluation. The control broilers were fed under normal conditions during the whole experiment cycle (18days). The TD broilers were fed with 50mg/kg thiram, while the treatment group was given 40mg/kg of Rg1. According to our findings, thiram caused a decrease in production performance and tibia parameters (p < 0.05), which were significantly reversed by Rg1 administration. In addition, the results from the histological evaluation showed that the proliferative zone had a smaller number of blood vessels, surrounded by inviable chondrocytes, proving apoptosis during the occurrence of TD, while Rg1 treatment significantly increased blood vessels and decreased apoptotic cells. Furthermore, it was found that Rg1 effectively ameliorated the angiogenesis by regulation of HIF-1α/VEGFA/VEGFR2 signaling pathway and the chondrocytes' apoptosis via the mitochondrial pathway. Hence, these findings suggest that Rg1 might be a perfect choice in the prevention and treatment of TD via regulating chondrocytes apoptosis and angiogenesis. Also, it might be a potential therapeutic drug for humans to overcome different bone disorders, involving chondrocytes.

Constitutive and Inducible Expression of Genes Related to Salicylic Acid and Ethylene Pathways in a Moderately Resistant Tomato Cultivar Leads to Delayed Development of Meloidogyne javanica

Knowledge of the molecular changes in resistant and susceptible cultivars during nematode attack is essential for developing plant resistance. Increased expression of genes related to the synthesis and signaling of salicylic acid, jasmonic acid, and ethylene is known to induce expression of genes related to defense against plant parasitic nematodes. Here, we inoculated approximately 3000 s-stage juveniles (J2s) of Meloidogyne javanica to moderately resistant and highly susceptible tomato cultivars (ALYSTE F-1 and Dutch Mobil, respectively) to compare the developmental disease stages. The roots of each cultivar were collected daily until 30 days after inoculation (DAI). The roots were stained with acid-fuchsin and dissected under a microscope. The results showed that a few parasitic J2s were converted to J3s in the moderately resistant cultivar at 14 DAI, at which time, the highly susceptible cultivar had the highest number of J3s. Comparison of hormonal pathways in the two cultivars revealed that the expression of genes related to the ethylene pathway in ALYSTE F-1 was more strongly upregulated than in Dutch Mobil at 14 DAI. Moreover, the jasmonic acid pathway in the roots of both cultivars decreased at 14 DAI. The expression of genes related to salicylic acid synthesis and signaling was not significantly different between the two cultivars with regard to their non-inoculated controls, respectively, but ALYSTE F-1 in general showed constitutively higher levels of these genes compared to Dutch Mobil at 14 DAI. These results suggest that constitutive and induced expression of genes related to the salicylic acid pathway and ethylene pathway, respectively, delay the development of M. javanica J2s in ALYSTE F-1.

GENOMIC VIEW ON EPILEPSY AND AUTISM CANDIDATE GENES

The most frequently linked behavioural and neurological disorders to epilepsy are common complex disorders. Numerous disease-associated genes were discovered as a result of extensive parallel sequencing of individual or cohort genomes and exomes. Here, we evaluate the potential genes for epilepsy with an emphasis on exome and gene panel data. The findings demonstrate that, along with the analysis of genes expressed in the brain and the post synaptic proteome. 1. Candidate genes for non-metabolic epilepsies and autism are frequently AT-rich, and 2. Genes implicated in synaptic processes and developmental brain diseases typically have large transcript sizes and local AT-richness. These findings suggest that essential candidate genes for epilepsy and autism are preferentially located in late-replicating, GC-poor chromosomal regions (isochores). These findings suggest that the genetic changes causing various brain illnesses are restricted to responsive chromatin regions containing important genes for the brain. These findings suggest that essential candidate genes for epilepsy and autism are preferentially located in late-replicating, GC-poor chromosomal regions (isochores). These findings suggest that the genetic changes causing various brain illnesses are restricted to responsive chromatin regions containing genes essential for the proper functioning of the brain. Keywords: Remote sensing, Climate change, Artifcial Intelligence, Carbon emissions, Ocean acidification

Single-cell mass cytometry analysis reveals stem cell heterogeneity.

Cellular heterogeneity is fundamental to both developmental differentiation and disease establishment. Recent advances in high-throughput single-cell technology have been rapidly revolutionizing the resolution of our understanding of development and disease. However, while the study of single-cell transcriptomes is easily accessible, the analysis of single-cell proteomes is still in its infancy. In this study, we describe simultaneous profiling of multiple regulatory proteins at a single-cell level using mass cytometry or cytometry by time of flight. We develop mass cytometry reagents to study key transcription factors, signaling proteins and chromatin modifiers that regulate mouse embryonic stem cells. Our data reveal that the protein level of stem cell regulators significantly varies and that cell signaling pathways are extensively cross-activated across defined culture conditions of embryonic stem cells. In addition, the mass cytometry data enabled us to identify distinct multiple cell states of embryonic stem cells and determine their variation across culture conditions. We discuss the mass cytometry method, our results of the multi-protein analysis in embryonic stem cells and potential future perspectives for single-cell protein analysis.

QUANTIFICATION OF LEAD CONTAMINATION IN DIFFERENT SAUDI CHOCOLATE BRANDS USING ATOMIC ABSORPTION TECHNIQUE

Introduction: Although chocolate and candies are widely preferred snacks for children and adults, they can carry a risk of lead (Pb) contamination. The lead is considered as a toxic heavy metal that can cause many diseases to adults, including renal, reproductive, nervous, immune, and cardiovascular systems diseases. In children, lead can cause behavioral, developmental diseases and learning deficits. Aim and objectives:Our aim in this study is to measure the lead concentrations in different Saudi chocolate brands from different Saudi cities in order to compare it to the American FDA recommended levels of lead.Secondary objective: to assess if chocolate lead contamination contributes to the high blood lead levels that was found in our pediatric population. Methods: Ten different chocolate brands products were brought from different Saudi cities markets these samples were digested using (MARS 6) Microwave Digestion system. The clear digested samples were diluted to 50 ml and subjected for the trace elements determination by Flame atomic absorption spectrometry using AAS-7000. Results:All chocolate products that we investigated have shown lead levels between (0.00006 to 0.00654 ppm) which are below the recommended levels. Conclusion:Depending on our findings, the different chocolate brands that we have studied are within the acceptable ranges of lead contamination, as recommended by the American FDA. Further studies should focus on other causes of lead exposure as we have a high blood lead levels in our pediatric population.

Interventions to promote development in the next 1000 days: A mapping review.

The next 1000 days - the period from 2 to 5 years of age - has been highlighted as a key developmental stage in the life-course. A mapping review was conducted to categorize existing literature on interventions in the next 1000 days that promote key developmental outcomes, including publications between 1990 and July 2020. A total of 805 intervention studies were included for data extraction in the review. The number of intervention studies has increased substantially from 2010. Most interventions were from high-income countries, with few (5%) from low- and lower-middle-income countries. Interventions including typically developing children (n = 593, 74%) were mostly (80%) implemented in early childhood care and education (ECCE) settings, with 15% taking place in the home or with families and 5% in community or healthcare settings. Children's literacy and language outcomes were the target of 27% of these interventions, while 25% of interventions targeted early childhood development more holistically or targeted multiple developmental domains. Social-emotional development and social skills were the target of 15% of interventions, motor development 13%, numeracy 8% and cognitive development 8%. For children with any developmental delay, disability, disease or exposure (n = 212), interventions frequently targeted Autism Spectrum Disorder (24%), language or literacy delays (21%), developmental delays or disability more generally (20%); 16% targeted behavioral (or conduct) problems; and 5% targeted attention deficit hyperactivity disorder. Almost half (49%) took place in ECCE settings; 24% occurred in the home or with families, or in community (13%) and/or healthcare (14%) settings. This review highlights the need for more intervention research in low- and middle-income countries and for interventions supporting development in the next 1000 days. While the evidence base for interventions to promote development in this age group continues to expand, the most vulnerable children are not benefiting from this evidence.

Cross-regulome profiling of RNA polymerases highlights the regulatory role of polymerase III on mRNA transcription by maintaining local chromatin architecture

BackgroundMammalian cells have three types of RNA polymerases (Pols), Pol I, II, and III. However, the extent to which these polymerases are cross-regulated and the underlying mechanisms remain unclear.ResultsWe employ genome-wide profiling after acute depletion of Pol I, Pol II, or Pol III to assess cross-regulatory effects between these Pols. We find that these enzymes mainly affect the transcription of their own target genes, while certain genes are transcribed by the other polymerases. Importantly, the most active type of crosstalk is exemplified by the fact that Pol III depletion affects Pol II transcription. Pol II genes with transcription changes upon Pol III depletion are enriched in diverse cellular functions, and Pol III binding sites are found near their promoters. However, these Pol III binding sites do not correspond to transfer RNAs. Moreover, we demonstrate that Pol III regulates Pol II transcription and chromatin binding of the facilitates chromatin transcription (FACT) complex to alter local chromatin structures, which in turn affects the Pol II transcription rate.ConclusionsOur results support a model suggesting that RNA polymerases show cross-regulatory effects: Pol III affects local chromatin structures and the FACT-Pol II axis to regulate the Pol II transcription rate at certain gene loci. This study provides a new perspective for understanding the dysregulation of Pol III in various tissues affected by developmental diseases.

Role of Climate Change During the Covid-19 Pandemic

Climate change has become a topic of concern around the world. Many aspects of human well-being are affected by climate change. The social, economic, political, ecological and cultural development was influenced directly or indirectly. This paper has examined the role of climate change during the current period of the COVID-19 pandemic. The documentary analysis method was chosen. Various books, research reports, WHO databases and internet sources have been used extensively. Various literature shows that climate change may have played a significant role in the widespread effects of the COVID-19 pandemic. COVID-19 affects people with compromised immunity. The weakened immunity results from some diseases and treatments that weaken the body’s immunity. These include chronic lung diseases such as chronic asthma, some cancers, high blood pressure (hypertension), HIV / AIDS, and some developmental diseases. As a result, people become vulnerable to COVID-19. Poor diet also causes people to have weak immunity and become less resistant to the coronavirus. Poor nutrition results from changes in the climate pattern; Unavailability of sufficient and high-quality food, low production of sufficient food, and inaccessibility of high-quality food due to poverty. The world must take serious steps to stop excessive greenhouse gas emissions. If not, the human species will die as new diseases and viruses will emerge to take our lives as COVID-19 did.

Whole exome screening of neurodevelopmental regression disorders in a cohort of Egyptian patients

Developmental regression describes a child who begins to lose his previously acquired milestones skills after he has reached a certain developmental stage and though affects his childhood development. It is associated with neurodegenerative diseases including leukodystrophy and neuronal ceroid lipofuscinosis diseases (NCLs), one of the most frequent childhood-onset neurodegenerative disorders. The current study focused on screening causative genes of developmental regression diseases comprising neurodegenerative disorders in Egyptian patients using next-generation sequencing (NGS)-based analyses as well as developing checklist to support clinicians who are not familiar with these diseases. A total of 763 Egyptian children (1 to 11 years), mainly diagnosed with developmental regression, seizures, or visual impairment, were studied using whole exome sequencing (WES). Among 763 Egyptian children, 726 cases were early clinically and molecularly diagnosed, including 482 cases that had pediatric stroke, congenital infection, and hepatic encephalopathy; meanwhile, 192 had clearly dysmorphic features, 31 showed central nervous system (CNS) malformation, 17 were diagnosed by leukodystrophy, 2 had ataxia telangiectasia, and 2 were diagnosed with tuberous sclerosis. The remained 37 out of 763 candidates were suspected with NCLs symptoms; however, 28 were confirmed to be NCLs patients, 1 was Kaya-Barakat-Masson syndrome, 1 was diagnosed as infantile neuroaxonal dystrophy, and 7 cases required further molecular diagnosis. This study provided an NGS-based approach of the genetic causes of developmental regression and neurodegenerative diseases as it comprised different variants and de novo mutations with complex phenotypes of these diseases which in turn help in early diagnoses and counseling for affected families.