Single-cell RNA sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes, but current methods are incompatible with bacteria. Here, we introduce microSPLiT ...(microbial split-pool ligation transcriptomics), a high-throughput scRNA-seq method for Gram-negative and Gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000
cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction and also identified unexpected gene expression states, including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities that are otherwise not amenable to single-cell analysis, such as natural microbiota.
The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. ...Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.
Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic ...analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals 47%). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.
Background
Cadherin‐associated protein p120 catenin regulates cell adhesion and migration in cell cultures and is required for axial elongation in embryos. Its roles in adhesion and cell migration ...are regulated by phosphorylation. We determined the effects of phosphorylation of six serine and three threonine residues in p120 catenin during zebrafish (Danio rerio) embryogenesis.
Results
We knocked down endogenous p120 catenin‐δ1 with an antisense RNA‐splice‐site morpholino (Sp‐MO) causing defects in axis elongation. These defects were rescued by co‐injections of mRNAs for wildtype mouse p120 catenin‐δ1‐3A or various mutated forms. Several mRNAs containing serine or threonine codons singly or doubly mutated to phosphomimetic glutamic acid rescued, and some nonphosphorylatable mutants did not.
Conclusions
We discovered that phosphorylation of serine residue S252 or S879 is required for convergent extension of zebrafish embryos, since rescue occurred only when these residues were mutated to glutamic acid. In addition, the phosphorylation of either S268 or S269 is required, not both, consistent with the presence of only a single one of these residues in two isoforms of zebrafish and Xenopus laevis. In summary, phosphorylation of multiple serine and threonine residues of p120 catenin activates migration of presomitic mesoderm of zebrafish embryos facilitating elongation of the dorsal axis.
Key Findings
Knockdown of zebrafish p120 catenin‐δ1 with a morpholino blocks migration of presomitic mesoderm and extension of the dorsal axis.
We compared rescue of morphology by injecting mouse p120 catenin‐δ1‐3A (CTNND1‐3A) mRNAs mutated to phosphomimetic (E) and nonphosphorylatable (F) versions of serine and threonine residues.
Convergent extension of zebrafish embryos was partially rescued by phosphorylation of T310, S905, or T916. Importantly, it required phosphorylation of serine residues S252, S879, and either of S268 or S269.
We see normal morphogenesis as a dynamic interplay of motile filopodia and lamellipodia forming at the leading edge of the presomitic mesoderm cell as well as turnover of new cadherin‐mediated anchoring adherens junctions that facilitate a treadmilling action used by presomitic cells to migrate toward the dorsal aspect of the embryo.
Abstract
We performed a comprehensive analysis of the transcriptional changes occurring during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using single cell ...RNA-seq, we sequenced > 20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. Differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (
MYH7
and
MYH6
) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (
A2M
,
H19
,
IGF2
) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.
Affordably tracking the transmission of respiratory infectious diseases in urban transport infrastructures can inform individuals about potential exposure to diseases and guide public policymakers to ...prepare timely responses based on geographical transmission in different areas in the city. Towards that end, we designed and tested a method to detect SARS-CoV-2 RNA in the air filters of public buses, revealing that air filters could be used as passive fabric sensors for the detection of viral presence. We placed and retrieved filters in the existing HVAC systems of public buses to test for the presence of trapped SARS-CoV-2 RNA using phenol-chloroform extraction and RT-qPCR. SARS-CoV-2 RNA was detected in 14% (5/37) of public bus filters tested in Seattle, Washington, from August 2020 to March 2021. These results indicate that this sensing system is feasible and that, if scaled, this method could provide a unique lens into the geographically relevant transmission of SARS-CoV-2 through public transit rider vectors, pooling samples of riders over time in a passive manner without installing any additional systems on transit vehicles.
Display omitted
•Passive, non-destructive sensing of viral presence on existing urban transit infrastructure is possible.•Analysis of varying methods to overcome environmental factors•Existing metro transit infrastructure can be used for viral surveillance.•Amenable to pooled testing as a supplement to wastewater monitoring
Thomas Leyden called Shiseido a "leader" in the use of solar power and said the firm is "adding to the movement of this industry." The system Shiseido installed is similar to ones at Tiffany & Co.'s ...distribution centers in Whippany and Parsippany, N.J. Also, SunPower is working with Macy's Inc. to install solar power systems at 28 of the retailer's California stores.
PDF
