Trans-omic profiling uncovers molecular controls of early human cerebral organoid formation.

Defining the molecular networks orchestrating human brain formation is crucial for understanding neurodevelopment and neurological disorders. Challenges in acquiring early brain tissue have incentivized the use of three-dimensional human pluripotent stem cell (hPSC)-derived neural organoids to recapitulate neurodevelopment. To elucidate the molecular programs that drive this highly dynamic process, here, we generate a comprehensive trans-omic map of the phosphoproteome, proteome, and transcriptome of the exit of pluripotency and neural differentiation toward human cerebral organoids (hCOs). These data reveal key phospho-signaling events and their convergence on transcriptional factors to regulate hCO formation. Comparative analysis with developing human and mouse embryos demonstrates the fidelity of our hCOs in modeling embryonic brain development. Finally, we demonstrate that biochemical modulation of AKT signaling can control hCO differentiation. Together, our data provide a comprehensive resource to study molecular controls in human embryonic brain development and provide a guide for the future development of hCO differentiation protocols.

Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

Risk factors, management, and clinical outcomes of invasive Mycoplasma and Ureaplasma infections after lung transplantation.

Mollicute infections, caused by Mycoplasma and Ureaplasma species, are serious complications after lung transplantation; however, understanding of the epidemiology and outcomes of these infections remains limited. We conducted a single-center retrospective study of 1156 consecutive lung transplants performed from 2010-2019. We used log-binomial regression to identify risk factors for infection and analyzed clinical management and outcomes. In total, 27 (2.3%) recipients developed mollicute infection. Donor characteristics independently associated with recipient infection were age ≤40 years (prevalence rate ratio [PRR] 2.6, 95% CI 1.0-6.9), White race (PRR 3.1, 95% CI 1.1-8.8), and purulent secretions on donor bronchoscopy (PRR 2.3, 95% CI 1.1-5.0). Median time to diagnosis was 16 days posttransplant (IQR: 11-26 days). Mollicute-infected recipients were significantly more likely to require prolonged ventilatory support (66.7% vs 21.4%), undergo dialysis (44.4% vs 6.3%), and remain hospitalized ≥30 days (70.4% vs 27.4%) after transplant. One-year posttransplant mortality in mollicute-infected recipients was 12/27 (44%), compared to 148/1129 (13%) in those without infection (P <.0001). Hyperammonemia syndrome occurred in 5/27 (19%) mollicute-infected recipients, of whom 3 (60%) died within 10 weeks posttransplant. This study highlights the morbidity and mortality associated with mollicute infection after lung transplantation and the need for better screening and management protocols.

The diagnostic accuracy of Gram stain on formalin-fixed paraffin-embedded sections of skin tissue in the diagnosis of bacterial skin infection.

BACKGROUND AND OBJECTIVE: To evaluate the sensitivity, specificity, and likelihood ratios of Gram stain on formalin-fixed, paraffin-embedded (GS-FFPE) sections of skin in diagnosing bacterial skin infection. METHODS: We reviewed a retrospective series of skin specimens reported at our institution wherein histopathological assessment included Gram stain and fresh tissue was concurrently submitted for microscopy and culture. The clinicopathological correlation was the reference standard, whereby the presence of infection was deduced from the final diagnosis in each patient's case notes. RESULTS: Our sample included 168 cases (105 positive for infection). GS-FFPE showed a sensitivity of 0.43 (95% confidence interval 0.29, 0.57), a specificity of 0.98 (0.95, 1.01), a positive likelihood ratio of 21.50 (19.76, 23.24), and a negative likelihood ratio of 0.58 (0.41, 0.75). CONCLUSIONS: GS-FFPE has poor sensitivity, and a negative result should not be used as evidence to exclude infection. In contrast, it has excellent specificity and, unless the pretest probability of infection is very low, a positive result would make infection much more likely. The value of the GS-FFPE lies in cases where sterile tissue was not submitted for microbiological studies, or sterile tissue culture was negative, and there is at least a low-to-moderate pretest probability of infection.

Phenotypic Analysis of Early Neurogenesis in a Mouse Chimeric Embryo and Stem Cell-Based Neuruloid Model.

Analyzing the impact of genetic mutations on early neurogenesis of mammalian embryos in conventional mouse mutant models is laborious and time-consuming. To overcome these constraints and to fast-track the phenotypic analysis, we developed a protocol that harnesses the amenability of engineering genetic modifications in embryonic stem cells from which mid-gestation mouse chimeras and in vitro neuruloids are generated. These stem cell-based chimera and neuruloid experimental models allow phenotyping at early developmental time points of neurogenesis.

Cryptococcal endophthalmitis complicated by immune reconstitution inflammatory syndrome in a renal transplant recipient: A case report and review of the literature.

A 59 year old male renal transplant recipient developed endogenous cryptococcal endophthalmitis which was complicated by immune reconstitution inflammatory syndrome (IRIS). Herein we report a novel diagnostic test using lateral flow assay, the management of cryptococcal endophthalmitis and the novel complication of intraocular IRIS in a solid organ transplant recipient.

Rat epiblast-derived stem cells recapitulate the attributes of pre-gastrulation epiblast.

Iwatsuki and colleagues have generated self-renewing pluripotent stem cells from the pre-gastrulation epiblast of the rat embryo and from other cellular sources: rat embryonic stem cells (rESCs) and epiblast-like cells derived from the rESCs. These rat epiblast-derived stem cells (rEpiSCs) display germ-line competence that is characteristic of mouse formative stem cells and early signature of specification of germ layer lineages typical of primed state mouse epiblast stem cells.

Time space and single-cell resolved tissue lineage trajectories and laterality of body plan at gastrulation.

Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.

Technical challenges of studying early human development.

Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.

Necrotizing Soft Tissue Infections in South Australia: A 15-Year Review.

Necrotizing soft tissue infections (NSTIs) are associated with high morbidity and mortality. We retrospectively examined the impact of empiric antimicrobials coupled with early surgery on mortality in NSTI. Early surgery independently reduced 30-day mortality (odds ratio, .16; 95% confidence interval, .05-.51; P < .001) that was not further augmented by empiric antimicrobial choice.

Programming of Multicellular Patterning with Mechano-Chemically Microstructured Cell Niches.

Multicellular patterning of stem-cell-derived tissue models is commonly achieved via self-organizing activities triggered by exogenous morphogenetic stimuli. However, such tissue models are prone to stochastic behavior, limiting the reproducibility of cellular composition and forming non-physiological architectures. To enhance multicellular patterning in stem cell-derived tissues, a method for creating complex tissue microenvironments endowed with programmable multimodal mechano-chemical cues, including conjugated peptides, proteins, morphogens, and Young's moduli defined over a range of stiffnesses is developed. The ability of these cues to spatially guide tissue patterning processes, including mechanosensing and the biochemically driven differentiation of selected cell types, is demonstrated. By rationally designing niches, the authors engineered a bone-fat assembly from stromal mesenchyme cells and regionalized germ layer tissues from pluripotent stem cells. Through defined niche-material interactions, mechano-chemically microstructured niches enable the spatial programming of tissue patterning processes. Mechano-chemically microstructured cell niches thereby offer an entry point for enhancing the organization and composition of engineered tissues, potentiating structures that better recapitulate their native counterparts.

Eye Abnormalities in Children with Fetal Alcohol Spectrum Disorders: A Systematic Review.

PURPOSE: Although eye abnormalities are reported in fetal alcohol spectrum disorders (FASD), no systematic review based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines has been undertaken. Our aim was to document the range and prevalence of eye abnormalities reported in children with prenatal alcohol exposure (PAE) and/or FASD. METHODS: Searches of electronic databases and manual searches. Eligible articles were observational studies in children with PAE and/or FASD; peer reviewed journal articles in the English language; and studies reporting quantitative or frequency data on functional/structural eye abnormalities. Pooled prevalence, odds ratio, and mean differences were calculated. RESULTS: Of the 1,068 retrieved articles 36 were eligible, including articles on children with diagnosed fetal alcohol syndrome/FASD (N = 31); PAE (N = 3); and FASD or PAE without FASD (N = 2). Structural and functional eye abnormalities were identified, the most prevalent being short palpebral fissure length (66.1%), visual impairment (55.5%), epicanthus (53.5%), subnormal stereoacuity (53.0%), abnormal retinal tortuosity (50.5%), impaired fixation ability (33.3%), telecanthus (31.7%), optic nerve hypoplasia (30.2%), and small optic discs (27.0%). Compared to non-exposed controls, strabismus, subnormal vision, ptosis, short palpebral fissure length, microphthalmos, smaller optic disc area, and retinal vessel tortuosity were more prevalent in children with FASD. CONCLUSIONS: Examination of eyes and vision should be considered in children with PAE and suspected or diagnosed FASD to enable early identification and optimal management. This first comprehensive, systematic literature review demonstrates the variety and frequency of eye abnormalities reported in PAE/FASD.

Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies.

Recent advances of single-cell transcriptomics technologies and allied computational methodologies have revolutionized molecular cell biology. Meanwhile, pioneering explorations in spatial transcriptomics have opened up avenues to address fundamental biological questions in health and diseases. Here, we review the technical attributes of single-cell RNA sequencing and spatial transcriptomics, and the core concepts of computational data analysis. We further highlight the challenges in the application of data integration methodologies and the interpretation of the biological context of the findings.

Mammalian gastrulation: signalling activity and transcriptional regulation of cell lineage differentiation and germ layer formation.

The interplay of signalling input and downstream transcriptional activity is the key molecular attribute driving the differentiation of germ layer tissue and the specification of cell lineages within each germ layer during gastrulation. This review delves into the current understanding of signalling and transcriptional control of lineage development in the germ layers of mouse embryo and non-human primate embryos during gastrulation and highlights the inter-species conservation and divergence of the cellular and molecular mechanisms of germ layer development in the human embryo.

Meta-Analysis Identifies BDNF and Novel Common Genes Differently Altered in Cross-Species Models of Rett Syndrome.

Rett syndrome (RTT) is a rare disorder and one of the most abundant causes of intellectual disabilities in females. Single mutations in the gene coding for methyl-CpG-binding protein 2 (MeCP2) are responsible for the disorder. MeCP2 regulates gene expression as a transcriptional regulator as well as through epigenetic imprinting and chromatin condensation. Consequently, numerous biological pathways on multiple levels are influenced. However, the exact molecular pathways from genotype to phenotype are currently not fully elucidated. Treatment of RTT is purely symptomatic as no curative options for RTT have yet to reach the clinic. The paucity of this is mainly due to an incomplete understanding of the underlying pathophysiology of the disorder with no clinically useful common disease drivers, biomarkers, or therapeutic targets being identified. With the premise of identifying universal and robust disease drivers and therapeutic targets, here, we interrogated a range of RTT transcriptomic studies spanning different species, models, and MECP2 mutations. A meta-analysis using RNA sequencing data from brains of RTT mouse models, human post-mortem brain tissue, and patient-derived induced pluripotent stem cell (iPSC) neurons was performed using weighted gene correlation network analysis (WGCNA). This study identified a module of genes common to all datasets with the following ten hub genes driving the expression: ATRX, ADCY7, ADCY9, SOD1, CACNA1A, PLCG1, CCT5, RPS9, BDNF, and MECP2. Here, we discuss the potential benefits of these genes as therapeutic targets.

Spatial molecular anatomy of germ layers in the gastrulating cynomolgus monkey embryo.

During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors, and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation. We reconstructed three-dimensional digital models from serial sections of cynomolgus monkey (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21. Spatial transcriptomics identifies gene expression profiles unique to the germ layers. Cross-species comparison reveals a developmental coordinate of germ layer segregation between mouse and primates, and species-specific transcription programs during gastrulation. These findings offer insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

Mouse organogenesis atlas at single-cell resolution.

The generation of spatial transcriptomes of whole embryo has been limited in scale and resolution due to various technological restrictions. In this issue of Cell, Chen et al. introduce a DNA nanoball-based sample-capture technology for spatial transcriptome analysis to generate a molecular atlas of mouse organogenesis at single-cell resolution.

Identification and Visualization of Protein Expression in Whole Mouse Embryos by Immunofluorescence.

Mouse embryo studies are pivotal for the understanding of early development. Analysis of the spatial and temporal changes of protein expression during development of a mouse embryo allows us to identify the genetic basis of errors of development in animal disease models. Immunofluorescence is a powerful technique to study the localization and variation in expression pattern of specific proteins in cells, tissues, and organs. Detecting the antigens with their specific antibodies labeled with fluorescent probes allows visualization of proteins at the cellular level. Here, we provide the optimized protocol of immunostaining whole mouse embryos at embryonic stages E7.5 to E11.5.