Temporal activation of LRH-1 and RAR-γ in human pluripotent stem cells induces a functional naïve-like state.

Naïve pluripotency can be established in human pluripotent stem cells (hPSCs) by manipulation of transcription factors, signaling pathways, or a combination thereof. However, differences exist in the molecular and functional properties of naïve hPSCs generated by different protocols, which include varying similarities with pre-implantation human embryos, differentiation potential, and maintenance of genomic integrity. We show here that short treatment with two chemical agonists (2a) of nuclear receptors, liver receptor homologue-1 (LRH-1) and retinoic acid receptor gamma (RAR-γ), along with 2i/LIF (2a2iL) induces naïve-like pluripotency in human cells during reprogramming of fibroblasts, conversion of pre-established hPSCs, and generation of new cell lines from blastocysts. 2a2iL-hPSCs match several defined criteria of naïve-like pluripotency and contribute to human-mouse interspecies chimeras. Activation of TGF-ß signaling is instrumental for acquisition of naïve-like pluripotency by the 2a2iL induction procedure, and transient activation of TGF-ß signaling substitutes for 2a to generate naïve-like hPSCs. We reason that 2a2iL-hPSCs are an easily attainable system to evaluate properties of naïve-like hPSCs and for various applications.

Human Proteome Project and Human Pluripotent Stem Cells: Odd Bedfellows or a Perfect Match?

The Chromosome-Centric Human Proteome Project (C-HPP) aims at the identification of missing proteins (MPs) and the functional characterization of functionally unannotated PE1 (uPE1) proteins. A major challenge in addressing this goal is that many human proteins and MPs are silent in adult cells. A promising approach to overcome such challenge is to exploit the advantage of novel tools such as pluripotent stem cells (PSCs), which are capable of differentiation into three embryonic germ layers, namely, the endoderm, mesoderm, and ectoderm. Here we present several examples of how the Human Y Chromosome Proteome Project (Y-HPP) benefited from this approach to meet C-HPP goals. Furthermore, we discuss how integrating CRISPR engineering, human-induced pluripotent stem cell (hiPSC)-derived disease modeling systems, and organoid technologies provides a unique platform for Y-HPP and C-HPP for MP identification and the functional characterization of human proteins, especially uPE1s.

A novel autosomal recessive GJB2-associated disorder: Ichthyosis follicularis, bilateral severe sensorineural hearing loss, and punctate palmoplantar keratoderma.

Ichthyosis follicularis, a distinct cutaneous entity reported in combination with atrichia, and photophobia has been associated with mutations in MBTPS2. We sought the genetic cause of a novel syndrome of ichthyosis follicularis, bilateral severe sensorineural hearing loss and punctate palmoplantar keratoderma in two families. We performed whole exome sequencing on three patients from two families. The pathogenicity and consequences of mutations were studied in the Xenopus oocyte expression system and by molecular modeling analysis. Compound heterozygous mutations in the GJB2 gene were discovered: a pathogenic c.526A>G; p.Asn176Asp, and a common frameshift mutation, c.35delG; p.Gly12Valfs*2. The p.Asn176Asp missense mutation was demonstrated to significantly reduce the cell-cell gap junction channel activity and increase the nonjunctional hemichannel activity in the Xenopus oocyte expression system. Molecular modeling analyses of the mutant Cx26 protein revealed significant changes in the structural characteristics and electrostatic potential of the Cx26, either in hemichannel or gap junction conformation. Thus, association of a new syndrome of an autosomal recessive disorder of ichthyosis follicularis, bilateral severe sensorineural hearing loss and punctate palmoplantar keratoderma with mutations in GJB2, expands the phenotypic spectrum of the GJB2-associated disorders. The findings attest to the complexity of the clinical consequences of different mutations in GJB2.

Inherited Interleukin 2-Inducible T-Cell (ITK) Kinase Deficiency in Siblings With Epidermodysplasia Verruciformis and Hodgkin Lymphoma.

Biallelic mutations in the ITK gene cause a T-cell primary immunodeficiency with Epstein-Barr virus (EBV)-lymphoproliferative disorders. We describe a novel association of a homozygous ITK mutation with ß-human papillomavirus (HPV)-positive epidermodysplasia verruciformis. Thus, loss of function in ITK can result in broad dysregulation of T-cell responses to oncogenic viruses, including ß-HPV and EBV.

Identification of a missense variant in CLDN2 in obstructive azoospermia.

Obstructive azoospermia (OA), defined as an obstruction in any region of the male genital tract, accounts for 40% of all azoospermia cases. Of all OA cases, ~30% are thought to have a genetic origin, however, hitherto, the underlying genetic etiology of the majority of these cases remain unknown. To address this, we took a family-based whole-exome sequencing approach to identify causal variants of OA in a multiplex family with epidydimal obstruction. A novel gain-of-function missense variant in CLDN2 (c.481G>C; p.Gly161Arg) was found to co-segregate with the phenotype, consistent with the X-linked inheritance pattern observed in the pedigree. To assess the pathogenicity of this variant, the wild and mutant protein structures were modeled and their potential for strand formation in multimeric form was assessed and compared. The results showed that dimeric and tetrameric arrangements of Claudin-2 were not only reduced, but were also significantly altered by this single residue change. We, therefore, envisage that this amino acid change likely forms a polymeric discontinuous strand, which may lead to the disruption of tight junctions among epithelial cells. This missense variant is thus likely to be responsible for the disruption of the blood-epididymis barrier, causing dislodged epithelial cells to clog the genital tract, hence causing OA. This study not only sheds light on the underlying pathobiology of OA, but also provides a basis for more efficient diagnosis in the clinical setting.

Maple syrup urine disease mutation spectrum in a cohort of 40 consanguineous patients and insilico analysis of novel mutations.

Maple syrup urine disease is the primary aminoacidopathy affecting branched-chain amino acid (BCAA) metabolism. The disease is mainly caused by the deficiency of an enzyme named branched-chained α-keto acid dehydrogenase (BCKD), which consist of four subunits (E1α, E1ß, E2, and E3), and encoded by BCKDHA, BCKDHB, DBT, and DLD gene respectively. BCKD is the main enzyme in the catabolism pathway of BCAAs. Hight rate of autosomal recessive disorders is expected from consanguineous populations like Iran. In this study, we selected two sets of STR markers linked to the four genes, that mutation in which can result in MSUD disease. The patients who had a homozygous haplotype for selected markers of the genes were sequenced. In current survey, we summarized our recent molecular genetic findings to illustrate the mutation spectrum of MSUD in our country. Ten novel mutations including c.484 A > G, c.834_836dup CAC, c.357del T, and c. (343 + 1_344-1) _ (742 + 1_743-1)del in BCKDHB, c.355-356 ins 7 nt ACAAGGA, and c.703del T in BCKDHA, and c.363delCT/c.1238 T > C, c. (433 + 1_434-1) _ (939 + 1_940-1)del, c.1174 A > C, and c.85_86ins AACG have been found in DBT gene. Additionally, structural models of MSUD mutations have been performed to predict the pathogenicity of the newly identified variants.

Red/ox states of human protein disulfide isomerase regulate binding affinity of 17 beta-estradiol.

Human protein disulfide isomerase (hPDI) is a key redox-regulated thiol-containing protein operating as both oxidoreductase and molecular chaperone in the endoplasmic reticulum of cells. hPDI thiol-disulfide interchange reactions lead to the adoption of two distinct red/ox conformations with different substrate preferences. hPDI also displays high binding capacity for some endogenous steroid hormones including 17 beta-estradiol (E2) and thus contributes to the regulation of their intracellular concentration, storage and actions. The primary focus of this study was to investigate the impact of E2 binding on functional activity of recombinant hPDI. Then, we examined the effect of E2 binding on structural alteration of hPDI red/ox conformations and its influence on affinity and position of interaction using experimental and computational analysis. Our results revealed that interaction of one E2 per each hPDI molecule led to the inhibition of hPDI reductase activity and conformational changes in both oxidation states. Mutually, E2-binding position were also redox-regulated with higher affinity in oxidized hPDI compare to the reduced form. The importance of histidine-256 protonation states in distinct binding preferences of E2 were also demonstrated in hPDI red/ox conformations. These findings might pave the way for better understanding of the mechanisms behind the redox-dependent hormone-binding activity of hPDI.

In silico analysis of novel mutations in maple syrup urine disease patients from Iran.

Maple Syrup Urine Disease (MSUD) is a rare autosomal recessive disorder of branched-chain amino acid (BCAA) metabolism. The disease is mainly caused by mutations either in the BCKDHA, BCKDHB, DBT or DLD genes encoding components of the E1α, E1ß, E2 and E3 subunits of branched-chain α-keto acid dehydrogenase complex (BCKDC), respectively. BCKDC is a mitochondrial enzyme which is responsible for the normal breakdown of BCAA. The rate of consanguineous marriage in Iran is 38.6 %, so the prevalence of autosomal recessive disorders is higher in comparison to other countries. Consanguinity increases the chance of the presence of pathogenic mutations in a homoallelic state. This phenomenon has made homozygosity mapping a powerful tool for finding the probable causative gene in heterogeneous disorders like IEM (Inborn Errors of Metabolism). In this study, two sets of multiplex polymorphic STR (Short Tandem Repeat) markers linked to the above-mentioned genes were selected to identify the probable pathogenic gene in the studied families. The families who showed a homozygous haplotype for the STR markers of the BCKDHB gene were subsequently sequenced. Four novel mutations including c.633 + 1G > A, c.988G > A, c.833_834insCAC, and a homozygous deletion of whole exon 3 c. (274 + 1_275-1) _(343 + 1_344-1), as well as one recently reported (c. 508G > T) mutation have been identified. Interestingly, three families shared a common haplotype structure along with the c. 508G > T mutation. Also, four other families revealed another similar haplotype with c.988G > A mutation. Founder effect can be a suggestive mechanism for the disease. Additionally, structural models of MSUD mutations have been performed to predict the pathogenesis of the newly identified variants.

A fresh look at the male-specific region of the human Y chromosome.

The Chromosome-centric Human Proteome Project (C-HPP) aims to systematically map the entire human proteome with the intent to enhance our understanding of human biology at the cellular level. This project attempts simultaneously to establish a sound basis for the development of diagnostic, prognostic, therapeutic, and preventive medical applications. In Iran, current efforts focus on mapping the proteome of the human Y chromosome. The male-specific region of the Y chromosome (MSY) is unique in many aspects and comprises 95% of the chromosome's length. The MSY continually retains its haploid state and is full of repeated sequences. It is responsible for important biological roles such as sex determination and male fertility. Here, we present the most recent update of MSY protein-encoding genes and their association with various traits and diseases including sex determination and reversal, spermatogenesis and male infertility, cancers such as prostate cancers, sex-specific effects on the brain and behavior, and graft-versus-host disease. We also present information available from RNA sequencing, protein-protein interaction, post-translational modification of MSY protein-coding genes and their implications in biological systems. An overview of Human Y chromosome Proteome Project is presented and a systematic approach is suggested to ensure that at least one of each predicted protein-coding gene's major representative proteins will be characterized in the context of its major anatomical sites of expression, its abundance, and its functional relevance in a biological and/or medical context. There are many technical and biological issues that will need to be overcome in order to accomplish the full scale mapping.

The Dynamic Proteome of Oligodendrocyte Lineage Differentiation Features Planar Cell Polarity and Macroautophagy Pathways.

BACKGROUND: Generation of oligodendrocytes is a sophisticated multistep process, the mechanistic underpinnings of which are not fully understood and demand further investigation. To systematically profile proteome dynamics during human embryonic stem cell differentiation into oligodendrocytes, we applied in-depth quantitative proteomics at different developmental stages and monitored changes in protein abundance using a multiplexed tandem mass tag-based proteomics approach. FINDINGS: Our proteome data provided a comprehensive protein expression profile that highlighted specific expression clusters based on the protein abundances over the course of human oligodendrocyte lineage differentiation. We identified the eminence of the planar cell polarity signalling and autophagy (particularly macroautophagy) in the progression of oligodendrocyte lineage differentiation-the cooperation of which is assisted by 106 and 77 proteins, respectively, that showed significant expression changes in this differentiation process. Furthermore, differentially expressed protein analysis of the proteome profile of oligodendrocyte lineage cells revealed 378 proteins that were specifically upregulated only in 1 differentiation stage. In addition, comparative pairwise analysis of differentiation stages demonstrated that abundances of 352 proteins differentially changed between consecutive differentiation time points. CONCLUSIONS: Our study provides a comprehensive systematic proteomics profile of oligodendrocyte lineage cells that can serve as a resource for identifying novel biomarkers from these cells and for indicating numerous proteins that may contribute to regulating the development of myelinating oligodendrocytes and other cells of oligodendrocyte lineage. We showed the importance of planar cell polarity signalling in oligodendrocyte lineage differentiation and revealed the autophagy-related proteins that participate in oligodendrocyte lineage differentiation.

Human closed and open apex premolar teeth express different toll-like receptor.

BACKGROUND: The innate immune activation which promotes inflammation responses in the dental pulp tissue leads to the progression of dentin caries. Accordingly, toll-like receptors (TLRs) are key molecules of the innate immune system that identify pathogen-associated molecular patterns (PAMPs) on microorganisms and may have a critical role in a dental injury. Therefore, this study aimed to investigate the expression of TLR2, TLR3, and TLR4 in the human dental pulp of opened and closed apex teeth. METHODS: Human dental pulps were derived from the healthy opened and closed apex premolar, in which extraction was indicated for orthodontic reasons. The extraction of RNA was performed and the gene expression determined by real-time polymerase chain reaction (RT-PCR). The result from real-time PCR was confirmed using western blot analysis. RESULTS: Real-time PCR data analysis showed that the expression TLR2 and TLR4 were significantly increased in closed apex premolar teeth compared to open apex teeth, whereas TLR3 expression was not significantly different in these two groups (p < .05). CONCLUSION: The results of the present study suggested increased expression of TLR2 and TLR4 by the maturation of the apex, which may be due to the presence of microorganisms in the normal or destructed dental pulp tissue. Thus, identifying the expression of TLRs molecules in dental pulp tissue helps to develop a deeper knowledge of the immune responses in the oral cavity.

Research Performance in Stem Cell Science and Regenerative Medicine in Iran: A National Comprehensive Observation.

BACKGROUND: Accomplishments in stem cell science and tissue engineering have resulted in a remarkable revolution in the context of future medicine. However, a general insight over the progress of stem cell research in Iran is still lacking. A better understanding of current needs in this field is required to design a better national roadmap. METHODS: In this study, we defined the geographical regions based on the significance of their contribution to stem cell research; then, using the Scopus database, we retrieved reports from Iran and other countries concerning stem cell science and regenerative medicine published from 1994 until the end of 2016. RESULTS: A significant number of citations belong to North America (6554029 citations equal to 49% of the total citations) and Europe (4425465 citations equal to 33% of the total citations) and the rest of citations were related to Asia (2423352 citations equal to 18% of the total citations). East Asian (2168472 citations equal to 76% of the total citations related to Asia) documents were cited more than those from the Middle East (ME) (494141 citations equal to 17% of the total citations related to Asia) and North and Central Asia (196382 citations equal to 7% of the total citations related to Asia). Iran as a country in the ME attracted 17% of the total citations related to the Asian countries winning the second position in this regard. The overview of total number of citations showed a sharp increase and upward trend in citation numbers for all the Iranian institutes from 2007 that resulted in the expansion of stem cell science in all major cities of Iran such as Shiraz (8%), Mashhad (5%), Isfahan (5%) and Ahvaz (5%). H-index of Tehran University of Medical Science, which has the highest total citations and document numbers, is the highest among all Iranian research institutes. Citation per paper of Royan Institute (RI) is the highest among the top 10 Iranian institutes, by 13 citations per paper. CONCLUSION: Stem cell research in Iran is rapidly developing. Since 2007, the number of published documents in major research institutes increased; thus, there is necessity for analysis of the status of publications in this field and choosing a better direction based on needs. Furthermore, it is necessary to expand and organize international collaborations to enrich our research and benefit from different team experiences.

Mutations in PLOD3, encoding lysyl hydroxylase 3, cause a complex connective tissue disorder including recessive dystrophic epidermolysis bullosa-like blistering phenotype with abnormal anchoring fibrils and type VII collagen deficiency.

Epidermolysis bullosa (EB), the paradigm of heritable skin fragility disorders, is associated with mutations in as many as 20 distinct genes. One of the clinical variants, recessive dystrophic EB (RDEB), demonstrates sub-lamina densa blistering accompanied by alterations in anchoring fibrils due to mutations in COL7A1. In this study, we characterized a patient with widespread connective tissue abnormalities, including skin blistering similar to that in RDEB. Whole exome sequencing, combined with genome-wide homozygosity mapping, identified a homozygous missense mutation in PLOD3 encoding lysyl hydroxylase 3 (LH3). No mutations in COL7A1, the gene previously associated with RDEB, were detected. The level of LH3 was dramatically reduced in the skin and fibroblast cultures from the patient. The blistering in the skin occurred below the lamina densa and was associated with variable density and morphology of anchoring fibrils. The level of type VII collagen expression in the skin was markedly reduced. Analysis of hydroxylysine and its glycosylated derivatives (galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine) revealed marked reduction in glycosylated hydroxylysine. Collectively, these findings indicate that PLOD3 mutations can result in a dystrophic EB-like phenotype in the spectrum of connective tissue disorders and add it to the list of candidate genes associated with skin fragility.

Type 1 Diabetes Mellitus: Cellular and Molecular Pathophysiology at A Glance.

Type 1 diabetes mellitus (T1DM) is a disease where destruction of the insulin producing pancreatic beta-cells leads to increased blood sugar levels. Both genetic and environmental factors play a part in the development of T1DM. Currently, numerous loci are specified to be the responsible genetic factors for T1DM; however, the mechanisms of only a few of these genes are known. Although several environmental factors are presumed responsible for progression of T1DM, to date, most of their mechanisms remain undiscovered. After several years of hyperglycemia, late onsets of macrovascular (e.g., cardiovascular) and microvascular (e.g., neurological, ophthalmological, and renal) complications may occur. This review and accompanying figures provides an overview of the etiological factors for T1DM, its pathogenesis at the cellular level, and attributed complications.

Comparative In Vitro Evaluation of Human Dental Pulp and Follicle Stem Cell Commitment.

OBJECTIVE: Pulp and periodontal tissues are well-known sources of mesenchymal stem cells (MSCs) that provide a promising place in tissue engineering and regenerative medicine. The molecular mechanisms underlying commitment and differentiation of dental stem cells that originate from different dental tissues are not fully understood. In this study, we have compared the expression levels of pluripotency factors along with immunological and developmentally-related markers in the culture of human dental pulp stem cells (hDPSCs), human dental follicle stem cells (hDFSCs), and human embryonic stem cells (hESCs). MATERIALS AND METHODS: In this experimental study, isolated human dental stem cells were investigated using quantitative polymerase chain reaction (qPCR), immunostaining, and fluorescence-activated cell sorting (FACS). Additionally, we conducted gene ontology (GO) analysis of differentially expressed genes and compared them between dental stem cells and pluripotent stem cells. RESULTS: The results demonstrated that pluripotency (OCT4 and SOX2) and immunological (IL-6 and TLR4) factors had higher expressions in hDFSCs, with the exception of the JAGGED-1/NOTCH1 ratio, c-MYC and NESTIN which expressed more in hDPSCs. Immunostaining of OCT4, SOX2 and c-MYC showed cytoplasmic and nucleus localization in both groups at similar passages. GO analysis showed that the majority of hDFSCs and hDPSCs populations were in the synthesis (S) and mitosis (M) phases of the cell cycle, respectively. CONCLUSION: This study showed different status of heterogeneous hDPSCs and hDFSCs in terms of stemness, differentiation fate, and cell cycle phases. Therefore, the different behaviors of dental stem cells should be considered based on clinical treatment variations.

Machine Learning and Network Analysis of Molecular Dynamics Trajectories Reveal Two Chains of Red/Ox-specific Residue Interactions in Human Protein Disulfide Isomerase.

The human protein disulfide isomerase (hPDI), is an essential four-domain multifunctional enzyme. As a result of disulfide shuffling in its terminal domains, hPDI exists in two oxidation states with different conformational preferences which are important for substrate binding and functional activities. Here, we address the redox-dependent conformational dynamics of hPDI through molecular dynamics (MD) simulations. Collective domain motions are identified by the principal component analysis of MD trajectories and redox-dependent opening-closing structure variations are highlighted on projected free energy landscapes. Then, important structural features that exhibit considerable differences in dynamics of redox states are extracted by statistical machine learning methods. Mapping the structural variations to time series of residue interaction networks also provides a holistic representation of the dynamical redox differences. With emphasizing on persistent long-lasting interactions, an approach is proposed that compiled these time series networks to a single dynamic residue interaction network (DRIN). Differential comparison of DRIN in oxidized and reduced states reveals chains of residue interactions that represent potential allosteric paths between catalytic and ligand binding sites of hPDI.

Gene-Targeted Next Generation Sequencing Identifies PNPLA1 Mutations in Patients with a Phenotypic Spectrum of Autosomal Recessive Congenital Ichthyosis: The Impact of Consanguinity.

Autosomal recessive congenital ichthyosis is a heterogeneous group of disorders associated with mutations in at least nine distinct genes. To ascertain the molecular basis of ichthyosis patients in Iran, a country of approximately 80 million people with a high prevalence of customary consanguineous marriages, we have developed a gene-targeted next generation sequencing array consisting of 38 genes reported in association with ichthyosis phenotypes. In a subset of nine extended consanguineous families, we found homozygous missense mutations in the PNPLA1 gene, six of them being distinct and, to our knowledge, previously unpublished. This gene encodes an enzyme with lipid hydrolase activity, important for development and maintenance of the barrier function of the epidermis. These six mutations, as well as four previously published mutations, reside exclusively within the patatin-like subdomain of PNPLA1 containing the catalytic site. The mutations clustered around the active center of the enzyme or resided at the surface of the protein possibly involved in the protein-protein interactions. Clinical features of the patients showed considerable intra- and interfamilial heterogeneity. Knowledge of the specific mutations allows identification of heterozygous carriers, assisting in genetic counseling, prenatal testing, and preimplantation genetic diagnosis in extended families at risk of recurrence of this disorder, the incidence of which is significantly increased in consanguineous marriages.

Quantitative proteomic analysis of human testis reveals system-wide molecular and cellular pathways associated with non-obstructive azoospermia.

Male infertility accounts for half of the infertility problems experienced by couples. Azoospermia, having no measurable level of sperm in seminal fluid, is one of the known conditions resulting in male infertility. In order to elucidate the complex molecular mechanisms causing male azoospermia, label-free quantitative shotgun proteomics was carried out on testicular tissue specimens from patients with obstructive azoospermia and non-obstructive azoospermia, including maturation arrest (MA) and Sertoli cell only syndrome (SCOS). The abundance of 520 proteins was significantly changed across three groups of samples. We were able to identify several functional biological pathways enriched in azoospermia samples and confirm selected differentially abundant proteins, using multiple histological methods. The results revealed that cell cycle and proteolysis, and RNA splicing were the most significant biological processes impaired by the substantial suppression of proteins related to the aforementioned categories in SCOS tissues. In the MA patient testes, generation of precursor metabolites and energy as well as oxidation-reduction were the most significantly altered processes. Novel candidate proteins identified in this study include key transcription factors, many of which have not previously been shown to be associated with azoospermia. Our findings can provide substantial insights into the molecular regulation of spermatogenesis and human reproduction. SIGNIFICANCE: The obtained data showed a drastic suppression of proteins involved in spliceosome, cell cycle and proteasome proteins, as well as energy and metabolic production in Sertoli cell only syndrome testis tissue, and to a lesser extent in maturation arrest samples. Moreover, we identified new transcription factors that are highly down-regulated in SCOS and MA patients, thus helping to understand the molecular complexity of spermatogenesis in male infertility. Our findings provide novel candidate protein targets associated with SCOS or MA azoospermia.

Identification of six novel mutations in Iranian patients with maple syrup urine disease and their in silico analysis.

Maple syrup urine disease (MSUD) is a rare inborn error of branched-chain amino acid metabolism. The disease prevalence is higher in populations with elevated rate of consanguineous marriages such as Iran. Different types of disease causing mutations have been previously reported in BCKDHA, BCKDHB, DBT and DLD genes known to be responsible for MSUD phenotype. In this study, two sets of multiplex polymorphic STR (Short Tandem Repeat) markers linked to the above genes were used to aid in homozygosity mapping in order to find probable pathogenic change(s) in the studied families. The families who showed homozygote haplotype for the BCKDHA gene were subsequently sequenced. Our findings showed that exons 2, 4 and 6 contain most of the mutations which are novel. The changes include two single nucleotide deletion (i.e. c. 143delT and c.702delT), one gross deletion covering the whole exon four c.(375+1_376-1)_(8849+1_885-1), two splice site changes (c.1167+1G>T, c. 288+1G>A), and one point mutation (c.731G>A). Computational approaches were used to analyze these two novel mutations in terms of their impact on protein structure. Computational structural modeling indicated that these mutations might affect structural stability and multimeric assembly of branched-chain α-keto acid dehydrogenase complex (BCKDC).

Increased robustness of early embryogenesis through collective decision-making by key transcription factors.

BACKGROUND: Understanding the mechanisms by which hundreds of diverse cell types develop from a single mammalian zygote has been a central challenge of developmental biology. Conrad H. Waddington, in his metaphoric "epigenetic landscape" visualized the early embryogenesis as a hierarchy of lineage bifurcations. In each bifurcation, a single progenitor cell type produces two different cell lineages. The tristable dynamical systems are used to model the lineage bifurcations. It is also shown that a genetic circuit consisting of two auto-activating transcription factors (TFs) with cross inhibitions can form a tristable dynamical system. RESULTS: We used gene expression profiles of pre-implantation mouse embryos at the single cell resolution to visualize the Waddington landscape of the early embryogenesis. For each lineage bifurcation we identified two clusters of TFs - rather than two single TFs as previously proposed - that had opposite expression patterns between the pair of bifurcated cell types. The regulatory circuitry among each pair of TF clusters resembled a genetic circuit of a pair of single TFs; it consisted of positive feedbacks among the TFs of the same cluster, and negative interactions among the members of the opposite clusters. Our analyses indicated that the tristable dynamical system of the two-cluster regulatory circuitry is more robust than the genetic circuit of two single TFs. CONCLUSIONS: We propose that a modular hierarchy of regulatory circuits, each consisting of two mutually inhibiting and auto-activating TF clusters, can form hierarchical lineage bifurcations with improved safeguarding of critical early embryogenesis against biological perturbations. Furthermore, our computationally fast framework for modeling and visualizing the epigenetic landscape can be used to obtain insights from experimental data of development at the single cell resolution.