EGFR marks a subpopulation of dermal mesenchymal cells highly expressing IGF1 which enhances hair follicle regeneration.

The skin harbours transcriptionally and functionally heterogeneous mesenchymal cells that participate in various physiological activities by secreting biochemical cues. In this study, we aimed to identify a new subpopulation of dermal mesenchymal cells that enhance hair follicle regeneration through a paracrine mechanism. Integrated single-cell RNA sequencing (scRNA-seq) data analysis revealed epidermal growth factor receptor (EGFR) as a marker of distinct fibroblast subpopulation in the neonatal murine dermis. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) were used to validate the existence of the cell population in Krt14-rtTA-H2BGFP mouse. The difference of gene expression between separated cell subpopulation was examined by real-time PCR. Potential effect of the designated factor on hair follicle regeneration was observed after the application on excisional wounds in Krt14-rtTA-H2BGFP mouse. Immunofluorescence staining demonstrated the existence of dermal EGFR+ cells in neonatal and adult mouse dermis. The EGFR+ mesenchymal population, sorted by FACS, displayed a higher expression level of Igf1 (insulin-like growth factor 1). Co-localisation of IGF1 with EGFR in the mouse dermis and upregulated numbers of hair follicles in healed wounds following the application of exogenous IGF1 illustrated the contribution of EGFR+ cells in promoting wound-induced hair follicle neogenesis. Our results indicate that EGFR identifies a subpopulation of dermal fibroblasts that contribute to IGF1 promotion of hair follicle neogenesis. It broadens the understanding of heterogeneity and the mesenchymal cell function in skin and may facilitate the potential translational application of these cells.

The RNA polymerase of cytoplasmically replicating Zika virus binds with chromatin DNA in nuclei and regulates host gene transcription.

Zika virus (ZIKV) targets the neural progenitor cells (NPCs) in brain during intrauterine infections and consequently causes severe neurological disorders, such as microcephaly in neonates. Although replicating in the cytoplasm, ZIKV dysregulates the expression of thousands of host genes, yet the detailed mechanism remains elusive. Herein, we report that ZIKV encodes a unique DNA-binding protein to regulate host gene transcription in the nucleus. We found that ZIKV NS5, the viral RNA polymerase, associates tightly with host chromatin DNA through its methyltransferase domain and this interaction could be specifically blocked by GTP. Further study showed that expression of ZIKV NS5 in human NPCs markedly suppressed the transcription of its target genes, especially the genes involved in neurogenesis. Mechanistically, ZIKV NS5 binds onto the gene body of its target genes and then blocks their transcriptional elongation. The utero electroporation in pregnant mice showed that NS5 expression significantly disrupts the neurogenesis by reducing the number of Sox2- and Tbr2-positive cells in the fetal cortex. Together, our findings demonstrate a molecular clue linking to the abnormal neurodevelopment caused by ZIKV infection and also provide intriguing insights into the interaction between the host cell and the pathogenic RNA virus, where the cytoplasmic RNA virus encodes a DNA-binding protein to control the transcription of host cell in the nuclei.

Combination RSL3 Treatment Sensitizes Ferroptosis- and EGFR-Inhibition-Resistant HNSCCs to Cetuximab.

Head and neck squamous cell carcinomas (HNSCCs) are a type of cancer originating in the mucosal epithelium of the mouth, pharynx, and larynx, the sixth most common cancer in the world. However, there is no effective treatment for HNSCCs. More than 90% of HNSCCs overexpress epidermal growth factor receptors (EGFRs). Although small molecule inhibitors and monoclonal antibodies have been developed to target EGFRs, few EGFR-targeted therapeutics are approved for clinical use. Ferroptosis is a new kind of programmed death induced by the iron catalyzed excessive peroxidation of polyunsaturated fatty acids. A growing body of evidence suggests that ferroptosis plays a pivotal role in inhibiting the tumor process. However, whether and how ferroptosis-inducers (FINs) play roles in hindering HNSCCs are unclear. In this study, we analyzed the sensitivity of different HNSCCs to ferroptosis-inducers. We found that only tongue squamous cell carcinoma cells and laryngeal squamous cell carcinoma cells, but not nasopharyngeal carcinoma cells, actively respond to ferroptosis-inducers. The different sensitivities of HNSCC cells to ferroptosis induction may be attributed to the expression of KRAS and ferritin heavy chain (FTH1) since a high level of FTH1 is associated with the poor prognostic survival of HNSCCs, but knocked down FTH1 can promote HNSCC cell death. Excitingly, the ferroptosis-inducer RSL3 plays a synthetic role with EGFR monoclonal antibody Cetuximab to inhibit the survival of nasopharyngeal carcinoma cells (CNE-2), which are insensitive to both ferroptosis induction and EGFR inhibition due to a high level of FTH1 and a low level of EGFR, respectively. Our findings prove that FTH1 plays a vital role in ferroptosis resistance in HNSCCs and also provide clues to target HNSCCs resistant to ferroptosis induction and/or EGFR inhibition.

Autophagy receptor CCDC50 tunes the STING-mediated interferon response in viral infections and autoimmune diseases.

DNA sensing and timely activation of interferon (IFN)-mediated innate immunity are crucial for the defense against DNA virus infections and the clearance of abnormal cells. However, overactivation of immune responses may lead to tissue damage and autoimmune diseases; therefore, these processes must be intricately regulated. STING is the key adaptor protein, which is activated by cyclic GMP-AMP, the second messenger derived from cGAS-mediated DNA sensing. Here, we report that CCDC50, a newly identified autophagy receptor, tunes STING-directed type I IFN signaling activity by delivering K63-polyubiquitinated STING to autolysosomes for degradation. Knockout of CCDC50 significantly increases herpes simplex virus 1 (HSV-1)- or DNA ligand-induced production of type I IFN and proinflammatory cytokines. Ccdc50-deficient mice show increased production of IFN, decreased viral replication, reduced cell infiltration, and improved survival rates compared with their wild-type littermates when challenged with HSV-1. Remarkably, the expression of CCDC50 is downregulated in systemic lupus erythematosus (SLE), a chronic autoimmune disease. CCDC50 levels are negatively correlated with IFN signaling pathway activation and disease severity in human SLE patients. CCDC50 deficiency potentiates the cGAS-STING-mediated immune response triggered by SLE serum. Thus, our findings reveal the critical role of CCDC50 in the immune regulation of viral infections and autoimmune diseases and provide insights into the therapeutic implications of CCDC50 manipulation.

Post-Translational Modification of MRE11: Its Implication in DDR and Diseases.

Maintaining genomic stability is vital for cells as well as individual organisms. The meiotic recombination-related gene MRE11 (meiotic recombination 11) is essential for preserving genomic stability through its important roles in the resection of broken DNA ends, DNA damage response (DDR), DNA double-strand breaks (DSBs) repair, and telomere maintenance. The post-translational modifications (PTMs), such as phosphorylation, ubiquitination, and methylation, regulate directly the function of MRE11 and endow MRE11 with capabilities to respond to cellular processes in promptly, precisely, and with more diversified manners. Here in this paper, we focus primarily on the PTMs of MRE11 and their roles in DNA response and repair, maintenance of genomic stability, as well as their association with diseases such as cancer.

A Shortage of FTH Induces ROS and Sensitizes RAS-Proficient Neuroblastoma N2A Cells to Ferroptosis.

Ferroptosis, an iron-dependent form of programmed cell death, has excellent potential as an anti-cancer therapeutic strategy in different types of tumors, especially in RAS-mutated ones. However, the function of ferroptosis for inhibiting neuroblastoma, a common child malignant tumor with minimal treatment, is unclear. This study investigated the anti-cancer function of ferroptosis inducer Erastin or RSL3 in neuroblastoma N2A cells. Our results show that Erastin or RSL3 induces ROS level and cell death and, therefore, reduces the viability of RAS-proficient N2A cells. Importantly, inhibitors to ferroptosis, but not apoptosis, ameliorate the high ROS level and viability defect in Erastin- or RSL3-treated cells. In addition, our data also show that N2A cells are much more sensitive to ferroptosis inducers than primary mouse cortical neural stem cells (NSCs) or neurons. Moreover, a higher level of ROS and PARylation is evidenced in N2A, but not NSCs. Mechanically, ferritin heavy chain 1 (Fth), the ferroxidase function to oxidate redox-active Fe2+ to redox-inactive Fe3+, is likely responsible for the hypersensitivity of N2A to ferroptosis induction since its expression is lower in N2A compared to NSCs; ectopic expression of Fth reduces ROS levels and cell death, and induces expression of GPX4 and cell viability in N2A cells. Most importantly, neuroblastoma cell lines express a significantly low level of Fth than almost all other types of cancer cell lines. All these data suggest that Erastin or RSL3 induce ferroptosis cell death in neuroblastoma N2A cells, but not normal neural cells, regardless of RAS mutations, due to inadequate FTH. This study, therefore, provides new evidence that ferroptosis could be a promising therapeutic target for neuroblastoma.

Isolation and characterization of endothelial progenitor cells from canine bone marrow.

Endothelial progenitor cells (EPC) are located predominantly in the bone marrow. These cells are useful for treating human vascular diseases; they also are a possible target for restricting blood vessel growth for tumors. Little is known about canine EPC. We investigated a bone marrow EPC isolation method that combines the whole bone marrow culture method and the differential adherent speed method using stillborn canines. MTT proliferation, flow cytometry detection, Dil-ac-LDL uptake, FITC-UEA-1 binding and matrigel assays were used to identify and characterize EPC. We isolated two types of EPC: early EPC and late EPC. We found that isolated cells produced typical colony and cobblestone morphology, and were positive for CD31, CD34, CD133 and VEGFR-2. Significant differences were observed in the intensity of expression between early and late EPC, which suggests their different roles during angiogenesis and vasculogenesis. Both early and late EPC were positive for Dil-ac-LDL and FITC-UEA-1, and displayed tube formation when re-suspended in matrigel, both of which are important functional criteria for identifying EPC. Our method is a novel, effective and efficient way to produce enriched EPC.

NBS1 interacts with Notch signaling in neuronal homeostasis.

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

Corrigendum: Expanded CURB-65: a new score system predicts severity of community-acquired pneumonia with superior efficiency.

This corrects the article DOI: 10.1038/srep22911.

Improvement of virus removal using ultrafiltration membranes modified with grafted zwitterionic polymer hydrogels.

Potable water reuse has been adopted by cities suffering water scarcity in recent years. The microbial safety in water reuse, especially with respect to pathogenic viruses, is still a concern for water consumers. Membrane filtration can achieve sufficient removal of pathogenic viruses without disinfection byproducts, but the required energy is intensive. In this study, we graft-polymerized zwitterionic SPP ([3-(methacryloylamino) propyl] dimethyl (3-sulfopropyl) ammonium hydroxide) on a 150 kDa ultrafiltration polyethersulfone membrane to achieve a significantly higher virus removal. The redox-initiated graft-polymerization was performed in an aqueous solution during filtration of the monomer and initiators, allowing for functionalizing the membrane pores with hydrophilic polySPP. Bacteriophage MS2 and human adenovirus type 2 (HAdV-2) were used as surrogates for pathogenic human norovirus and human adenovirus. The grafting resulted in ∼18% loss of the membrane permeability but an increase of 4 log10 in HAdV-2 removal and 3 log10 in MS2 removal. The pristine and the grafted membranes were both conditioned with soluble microbial products (SMP) extracted from a full-scale membrane bioreactor (MBR) in order to test the virus removal after fouling the membranes. After fouling, the HAdV-2 removal by the grafted membrane was 1 log10 higher than that of the pristine membrane. For MS2, the grafted membrane after fouling with SMP achieved an additional 5 log10 removal compared to the unmodified membrane. The simple graft-polymerization functionalization of commercialized membrane achieving enhanced virus removal efficiency highlights the promise of membrane filtration for pathogen control in potable water reuse.

Influence of solution chemistry on the inactivation of particle-associated viruses by UV irradiation.

MS2 inactivation by UV irradiance was investigated with the focus on how the disinfection efficacy is influenced by bacteriophage MS2 aggregation and adsorption to particles in solutions with different compositions. Kaolinite and Microcystis aeruginosa were used as model inorganic and organic particles, respectively. In the absence of model particles, MS2 aggregates formed in either 1mM NaCl at pH=3 or 50-200mM ionic strength CaCl2 solutions at pH=7 led to a decrease in the MS2 inactivation efficacy because the virions located inside the aggregate were protected from the UV irradiation. In the presence of kaolinite and Microcystis aeruginosa, MS2 adsorbed onto the particles in either 1mM NaCl at pH=3 or 50-200mM CaCl2 solutions at pH=7. In contrast to MS2 aggregates formed without the presence of particles, more MS2 virions adsorbed on these particles were exposed to UV irradiation to allow an increase in MS2 inactivation. In either 1mM NaCl at pH from 4 to 8 or 2-200mM NaCl solutions at pH=7, the absence of MS2 aggregation and adsorption onto the model particles explained why MS2 inactivation was not influenced by pH, ionic strength, and the presence of model particles in these conditions. The influence of virus adsorption and aggregation on the UV disinfection efficiency found in this research suggests the necessity of accounting for particles and cation composition in virus inactivation for drinking water.

Expanded CURB-65: a new score system predicts severity of community-acquired pneumonia with superior efficiency.

Aim of this study was to develop a new simpler and more effective severity score for community-acquired pneumonia (CAP) patients. A total of 1640 consecutive hospitalized CAP patients in Second Affiliated Hospital of Zhejiang University were included. The effectiveness of different pneumonia severity scores to predict mortality was compared, and the performance of the new score was validated on an external cohort of 1164 patients with pneumonia admitted to a teaching hospital in Italy. Using age ≥ 65 years, LDH > 230 u/L, albumin < 3.5 g/dL, platelet count < 100 × 10(9)/L, confusion, urea > 7 mmol/L, respiratory rate ≥ 30/min, low blood pressure, we assembled a new severity score named as expanded-CURB-65. The 30-day mortality and length of stay were increased along with increased risk score. The AUCs in the prediction of 30-day mortality in the main cohort were 0.826 (95% CI, 0.807-0.844), 0.801 (95% CI, 0.781-0.820), 0.756 (95% CI, 0.735-0.777), 0.793 (95% CI, 0.773-0.813) and 0.759 (95% CI, 0.737-0.779) for the expanded-CURB-65, PSI, CURB-65, SMART-COP and A-DROP, respectively. The performance of this bedside score was confirmed in CAP patients of the validation cohort although calibration was not successful in patients with health care-associated pneumonia (HCAP). The expanded CURB-65 is objective, simpler and more accurate scoring system for evaluation of CAP severity, and the predictive efficiency was better than other score systems.

Random sequential adsorption of human adenovirus 2 onto polyvinylidene fluoride surface influenced by extracellular polymeric substances.

Virus removal by membrane bioreactors depends on virus-membrane and virus-foulant interactions. The adsorption of human adenovirus 2 (HAdV-2) on polyvinylidene fluoride (PVDF) membrane and a major membrane foulant, extracellular polymeric substances (EPS), were measured in a quartz crystal microbalance. In 3-100mM CaCl2 solutions, irreversible adsorption of HAdV-2 was observed on both pristine and EPS-fouled PVDF surfaces. The HAdV-2 adsorption kinetics was successfully fitted with the random sequential adsorption (RSA) model. The applicability of the RSA model for HAdV-2 adsorption is confirmed by comparing the two fitting parameters, adsorption rate constant k(a) and area occupied by each adsorbed HAdV-2 particle a, with experimentally measured parameters. A linear correlation between the fitting parameter k(a) and the measured attachment efficiency was found, suggesting that the RSA model correctly describes the interaction forces dominating the HAdV-2 adsorption. By comparing the fitting parameter d(ads) with the hydrodynamic diameter of HAdV-2, we conclude that virus-virus and virus-surface interactions determine the area occupied by each adsorbed HAdV-2 particle, and thus influence the adsorption capacity. These results provide insights into virus retention and will benefit improving virus removal in membrane filtration.

Mechanisms of MS2 bacteriophage removal by fouled ultrafiltration membrane subjected to different cleaning methods.

An ultrafiltration unit with a polyvinylidene fluoride (PVDF) membrane of 40 nm nominal pore size was used to study bacteriophage MS2 removal under different membrane conditions: pristine membrane, membrane fouled by soluble microbial product (SMP) extracted from membrane bioreactor (MBR) feedwater, backwashed membrane, and chemically cleaned membrane. The order of MS2 removal by these membranes was as follows: fouled membrane > backwashed membrane > chemically cleaned membrane ≈ pristine membrane. A linear correlation between membrane relative permeability and MS2 removal was found. Mass balance analysis showed a high percentage of MS2 in the concentrate for the fouled membrane as compared with the pristine membrane. Quartz crystal microbalance (QCM) results showed faster kinetics of MS2 adhesion to the pristine membrane than to the SMP-fouled membrane. In agreement with QCM results, an attractive force between MS2 and the pristine membrane was detected using an atomic force microscope (AFM), whereas a repulsive force was detected for the interaction between MS2 and the fouled membrane. The presence of SMP on the membrane surface led to higher rejection of MS2 due to both pore blocking and repulsion between MS2 and the SMP layer. Chemical cleaning removed most of the SMP foulant and as a result led to a lower MS2 removal.