The scale and capabilities of single-cell RNA-sequencing methods have expanded rapidly in recent years, enabling major discoveries and large-scale cell mapping efforts. However, these methods have ...not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single-cell and/or single-nucleus profiling-selecting representative methods based on their usage and our expertise and resources to prepare libraries-including two low-throughput and five high-throughput methods. We tested the methods on three types of samples: cell lines, peripheral blood mononuclear cells and brain tissue, generating 36 libraries in six separate experiments in a single center. To directly compare the methods and avoid processing differences introduced by the existing pipelines, we developed scumi, a flexible computational pipeline that can be used with any single-cell RNA-sequencing method. We evaluated the methods for both basic performance, such as the structure and alignment of reads, sensitivity and extent of multiplets, and for their ability to recover known biological information in the samples.
Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules ...governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.
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•A scRNA-seq study reveals shared and distinct features of human MMRd and MMRp CRC•Co-variation of single-cell transcriptional programs across specimens predicts immune hubs•A myeloid-rich inflammatory hub is identified below the colonic lumen in human CRC•CXCR3-ligand+ cells form foci with activated T cells in human MMRd CRC
Single-cell transcriptomics-based co-variation analysis of human colorectal cancer identifies a spatially resolved myeloid-rich inflammatory hub that is shared by mismatch repair-deficient (MMRd) and mismatch repair-proficient (MMRp) tumors and CXCR3-ligand+ multicellular foci distinct for MMRd tumors.
Telomerase and telomeres are important for indefinite replication of stem cells. Recently, telomeres of somatic cells were found to be reprogrammed to elongate in induced pluripotent stem cells ...（iPSCs）. The role of telomeres in developmental pluripotency in vivo of embryonic stem cells （ESCs） or iPSCs, however, has not been directly addressed. We show that ESCs with long telomeres exhibit authentic developmental pluripotency, as evidenced by generation of complete ESC pups as well as germline-competent chimeras, the most stringent tests available in rodents. ESCs with short telomeres show reduced teratoma formation and chimera production, and fail to generate complete ESC pups. Telomere lengths are highly correlated （r 〉 0.8） with the developmental pluripotency of ESCs. Short telomeres decrease the proliferative rate or capacity of ESCs, alter the expression of genes related to telomere epigenetics, down-regulate genes important for embryogenesis and disrupt germ cell differentiation. Moreover, iPSCs with longer telomeres generate chimeras with higher efficiency than those with short telomeres. Our data show that functional telomeres are essential for the developmental pluripotency of ESCs/iPSCs and suggest that telomere length may provide a valuable marker to evaluate stem cell pluripotency, particularly when the stringent tests are not feasible.
Herpes simplex virus 1 (HSV-1), a leading cause of genital herpes, infects oral or genital mucosal epithelial cells before infecting the peripheral sensory nervous system. The spread of HSV-1 beyond ...the sensory nervous system and the resulting broader spectrum of disease are not well understood. Using a mouse model of genital herpes, we found that HSV-1-infection-associated lethality correlated with severe fecal and urinary retention. No inflammation or infection of the brain was evident. Instead, HSV-1 spread via the dorsal root ganglia to the autonomic ganglia of the enteric nervous system (ENS) in the colon. ENS infection led to robust viral gene transcription, pathological inflammatory responses, and neutrophil-mediated destruction of enteric neurons, ultimately resulting in permanent loss of peristalsis and the development of toxic megacolon. Laxative treatment rescued mice from lethality following genital HSV-1 infection. These results reveal an unexpected pathogenesis of HSV associated with ENS infection.
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•HSV-1-associated lethality in mice correlates with severe fecal and urinary retention•HSV-1 spreads from nociceptors in the vagina to the enteric nervous system (ENS)•Viral replication in the ENS leads to inflammation and neutrophil recruitment•Neutrophil-mediated destruction of the ENS leads to lethality from genital herpes
Genital herpesvirus infections cause urinary and gastrointestinal retention, which is poorly understood. Using a mouse model of genital infection, Khoury-Hanold et al. demonstrate that HSV spreads via nociceptors to the enteric nervous system (ENS). The resulting neutrophil-mediated ENS destruction and loss of peristalsis lead to toxic megacolon and death.
Photoreceptors are specialized neurons that rely on Ca2+ to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca2+ homeostasis is ...disrupted. Ca2+ homeostasis is maintained partly by mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU), which can influence cytosolic Ca2+ signals, stimulate energy production, and trigger apoptosis. Here we discovered that zebrafish cone photoreceptors express unusually low levels of MCU. We expected that this would be important to prevent mitochondrial Ca2+ overload and consequent cone degeneration. To test this hypothesis, we generated a cone-specific model of MCU overexpression. Surprisingly, we found that cones tolerate MCU overexpression, surviving elevated mitochondrial Ca2+ and disruptions to mitochondrial ultrastructure until late adulthood. We exploited the survival of MCU overexpressing cones to additionally demonstrate that mitochondrial Ca2+ uptake alters the distributions of citric acid cycle intermediates and accelerates recovery kinetics of the cone response to light. Cones adapt to mitochondrial Ca2+ stress by decreasing MICU3, an enhancer of MCU-mediated Ca2+ uptake, and selectively transporting damaged mitochondria away from the ellipsoid toward the synapse. Our findings demonstrate how mitochondrial Ca2+ can influence physiological and metabolic processes in cones and highlight the remarkable ability of cone photoreceptors to adapt to mitochondrial stress.
Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely ...thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response.
To provide clinicians with a review of key considerations relating to the physical and behavioral well-being of children raised by their grandparents.
As the number of children being raised by their ...grandparents in the United States steadily increases, the needs of these families require greater attention. These children and their custodial grandparents face unique health, social, legal, and financial challenges. Children being raised by their grandparents are at higher risk for developmental and behavioral problems because of prior or current adverse family environments. Moreover, there is evidence that custodial grandparents may experience negative health, social, and financial outcomes that may constrain their ability to provide the best care for their grandchildren.
Pediatricians should not only be aware of the medical and developmental status of children who are being reared by their grandparents, but also assess the needs, abilities, and potential limitations of these custodial grandparents. In addition to providing useful parenting advice and direct support to custodial grandparents, pediatricians should refer these families as needed to local grandparenting groups, social service agencies, experienced legal counsel, and relevant national organizations for support and guidance.
Background: According to the Census Bureau, more than 7 million grandchildren (GC) in the United States were being raised solely by their 2.7 million grandparents (GPs) in 2012. Parenting a GC can be ...challenging for GPs, especially since behavioral challenges and neurodevelopmental disorders are more prevalent in this population. Although GPs are traditionally allowed to "spoil" their grandchildren, effective limit-setting and discipline techniques are essential for GPs assuming a primary parenting role. Given that corporal punishment (CP) was tacitly accepted and widely practiced among older generations, it is unclear to what extent grandparents raising grandchildren (GRGs) struggle with discipline in general and endorse the use of CP. The objective of the study is to assess, in a large national cohort, to what extent GRGs report difficulties disciplining their GC, and to what extent they believe corporal punishment is an appropriate form of discipline. Methods: An anonymous online parenting questionnaire focused on GRGs was developed. GRGs were recruited by contacting state and local GP support groups across the country as well as state and local social service agencies that support the elderly. Results: 747 GPs that self-identified as the primary caregiver of one or more of their grandchildren completed a detailed online questionnaire focused on demographics, parenting experiences and views regarding discipline. After excluding children with autism &/or intellectual disability and limiting the sample to children <17 years of age, the final sample was 516 (Table 1). Overall, 18% of GPs believe that CP is an appropriate method of discipline (Table 2); no differences were noted with GP or GC age or gender. Approximately half of GRGs indicated disciplining their grandchild was more difficult than expected; older children were perceived as more difficult than expected to discipline (p<.03); no other gender or age differences were noted. Overall, more than half of GRGs stated they were less strict with their GC before their GC began living together. Conclusion(s): In a large national sample of GPs who are the primary caregiver for their GC, the majority indicated that discipline is more difficult than expected, and some indicated that CP was acceptable. Given these findings, pediatricians need to ask GRG about discipline challenges as part of their neurobehavioral surveillance and counsel them accordingly. Many GRGs would likely benefit from parent training in behavior management strategies and/or other resources.
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