Dissecting cell identity via network inference and in silico gene perturbation

Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory network

Gene regulatory network reconfiguration in direct lineage reprogramming

In direct lineage conversion, transcription factor (TF) overexpression reconfigures gene regulatory networks (GRNs) to reprogram cell identity. We previously developed CellOracle, a computational method to infer GRNs from single-cell transcriptome and epigenome data. Using inferred GRNs, CellOracle simulates gene expression changes in response to TF perturbation, enabling in silico interrogation of network reconfiguration. Here, we combine CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct reprogramming paradigm. By linking early network state to reprogramming outcome, we reveal distinct network configurations underlying successful and failed fate conversion. Via in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting their identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations, providing new mechanistic insights into lineage reprogramming

Bergman kernel and complex singularity exponent

We give a precise estimate of the Bergman kernel for the model domain defined by $\Omega_F=\{(z,w)\in \mathbb{C}^{n+1}:{\rm Im}w-|F(z)|^2>0\},$ where $F=(f_1,...,f_m)$ is a holomorphic map from $\mathbb{C}^n$ to $\mathbb{C}^m$, in terms of the complex singularity exponent of $F$.Comment: to appear in Science in China, a special issue dedicated to Professor Zhong Tongde's 80th birthda

Single-cell analysis reveals regional reprogramming during adaptation to massive small bowel resection in mice

BACKGROUND & AIMS: The small intestine (SI) displays regionality in nutrient and immunological function. Following SI tissue loss (as occurs in short gut syndrome, or SGS), remaining SI must compensate, or adapt ; the capacity of SI epithelium to reprogram its regional identity has not been described. Here, we apply single-cell resolution analyses to characterize molecular changes underpinning adaptation to SGS. METHODS: Single-cell RNA sequencing was performed on epithelial cells isolated from distal SI of mice following 50% proximal small bowel resection (SBR) vs sham surgery. Single-cell profiles were clustered based on transcriptional similarity, reconstructing differentiation events from intestinal stem cells (ISCs) through to mature enterocytes. An unsupervised computational approach to score cell identity was used to quantify changes in regional (proximal vs distal) SI identity, validated using immunofluorescence, immunohistochemistry, qPCR, western blotting, and RNA-FISH. RESULTS: Uniform Manifold Approximation and Projection-based clustering and visualization revealed differentiation trajectories from ISCs to mature enterocytes in sham and SBR. Cell identity scoring demonstrated segregation of enterocytes by regional SI identity: SBR enterocytes assumed more mature proximal identities. This was associated with significant upregulation of lipid metabolism and oxidative stress gene expression, which was validated via orthogonal analyses. Observed upstream transcriptional changes suggest retinoid metabolism and proximal transcription factor Creb3l3 drive proximalization of cell identity in response to SBR. CONCLUSIONS: Adaptation to proximal SBR involves regional reprogramming of ileal enterocytes toward a proximal identity. Interventions bolstering the endogenous reprogramming capacity of SI enterocytes-conceivably by engaging the retinoid metabolism pathway-merit further investigation, as they may increase enteral feeding tolerance, and obviate intestinal failure, in SGS

Morbid Obesity as a Risk Factor for Hospitalization and Death Due to 2009 Pandemic Influenza A(H1N1) Disease

BACKGROUND: Severe illness due to 2009 pandemic A(H1N1) infection has been reported among persons who are obese or morbidly obese. We assessed whether obesity is a risk factor for hospitalization and death due to 2009 pandemic influenza A(H1N1), independent of chronic medical conditions considered by the Advisory Committee on Immunization Practices (ACIP) to increase the risk of influenza-related complications. METHODOLOGY/PRINCIPAL FINDINGS: We used a case-cohort design to compare cases of hospitalizations and deaths from 2009 pandemic A(H1N1) influenza occurring between April-July, 2009, with a cohort of the U.S. population estimated from the 2003-2006 National Health and Nutrition Examination Survey (NHANES); pregnant women and children <2 years old were excluded. For hospitalizations, we defined categories of relative weight by body mass index (BMI, kg/m(2)); for deaths, obesity or morbid obesity was recorded on medical charts, and death certificates. Odds ratio (OR) of being in each BMI category was determined; normal weight was the reference category. Overall, 361 hospitalizations and 233 deaths included information to determine BMI category and presence of ACIP-recognized medical conditions. Among >or=20 year olds, hospitalization was associated with being morbidly obese (BMI>or=40) for individuals with ACIP-recognized chronic conditions (OR = 4.9, 95% CI 2.4-9.9) and without ACIP-recognized chronic conditions (OR = 4.7, 95%CI 1.3-17.2). Among 2-19 year olds, hospitalization was associated with being underweight (BMI<or=5(th) percentile) among those with (OR = 12.5, 95%CI 3.4-45.5) and without (OR = 5.5, 95%CI 1.3-22.5) ACIP-recognized chronic conditions. Death was not associated with BMI category among individuals 2-19 years old. Among individuals aged >or=20 years without ACIP-recognized chronic medical conditions death was associated with obesity (OR = 3.1, 95%CI: 1.5-6.6) and morbid obesity (OR = 7.6, 95%CI 2.1-27.9). CONCLUSIONS/SIGNIFICANCE: Our findings support observations that morbid obesity may be associated with hospitalization and possibly death due to 2009 pandemic H1N1 infection. These complications could be prevented by early antiviral therapy and vaccination

Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease

Characteristics of Patients with Oseltamivir-Resistant Pandemic (H1N1) 2009, United States

During April 2009–June 2010, thirty-seven (0.5%) of 6,740 pandemic (H1N1) 2009 viruses submitted to a US surveillance system were oseltamivir resistant. Most patients with oseltamivir-resistant infections were severely immunocompromised (76%) and had received oseltamivir before specimen collection (89%). No evidence was found for community circulation of resistant viruses; only 4 (unlinked) patients had no oseltamivir exposure

Hepatocyte Growth Factor Modulates Interleukin-6 Production in Bone Marrow Derived Macrophages: Implications for Inflammatory Mediated Diseases

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response

Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis

Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration