SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

Identification and functional analysis of the GTPV bidirectional promoter region.

The goat pox chick embryo-attenuated virus (GTPV) has been developed as an effective vaccine that can elicit protective immune responses. It possesses a large genome and a robust ability to express exogenous genes. Thus, this virus is an ideal vector for recombinant live vaccines for infectious diseases in ruminant animals. In this study, we identified a novel bidirectional promoter region of GTPV through screening named PbVV(±). PbVV(±) is located between ETF-l and VITF-3, which are transcribed in opposite directions. A new recombinant goat pox virus (rGTPV) was constructed, in which duplicate PbVV(+) was used as a promoter element to enhance Brucella OMP31 expression, and duplicate PbVV(-) was used as a promoter element to regulate enhanced green fluorescent protein (EGFP) at the same time as the selection marker. PbVV(-) promoter activity was compared to that of the P7.5 promoter of vaccinia virus, as measured by EGFP expression; the fluorescence intensity of EGFP expressed in cells was confirmed by fluorescence microscopy and flow cytometry. PbVV(+) promoter activity was measured by Brucella OMP31 expression. Interaction with the anti-Brucella-OMP31 monoclonal antibody was confirmed by western blotting, and OMP31 mRNA expression was assessed by qRT-PCR. The results of this study will be useful for the further study of effective multivalent vaccines based on rGTPV. This study also provides a theoretical basis for overcoming the problem of low expression of exogenous genes.

ADAM9 promotes type I interferon-mediated innate immunity during encephalomyocarditis virus infection.

Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.

Outward foreign direct investment and energy intensity: evidence from the listed companies in China.

Since 2005, China's outward foreign direct investment (OFDI) has increased year by year, which corresponds to the continuous decline of energy intensity. But there is limited literature concerning their relationship nowadays. To answer whether or not OFDI can reduce energy intensity, this paper selects data from 29 provinces in China from 2006 to 2015 and establishes a fixed-effects model to analyze the relationship. Further, this paper divides OFDI into technology-intensive ones and non-technology-intensive ones in order to distinguish the impact of outward foreign direct investment from different types of enterprises on the energy intensity. Combined with the micro-data of A-share listed companies in Shanghai and Shenzhen stock exchanges, we find that OFDI plays a significant role in reducing the energy intensity in China, and OFDI of high-technology-intensive enterprises has a greater effect on the decrease of energy intensity than that of low-technology-intensive enterprises. This paper classifies OFDI from the perspective of enterprise technology intensity, which enriches the existing research results in the field of international cooperation and energy intensity. It also overcomes the limitations of previous literature data and provides new evidence for encouraging high-tech enterprises to strengthen international cooperation from a micro level.

An Early Islet Transcriptional Signature Is Associated With Local Inflammation in Autoimmune Diabetes.

Identifying the early islet cellular processes of autoimmune type 1 diabetes (T1D) in humans is challenging given the absence of symptoms during this period and the inaccessibility of the pancreas for sampling. In this article, we study temporal events in pancreatic islets in LEW.1WR1 rats, in which autoimmune diabetes can be induced with virus infection, by performing transcriptional analysis of islets harvested during the prediabetic period. Single-cell RNA-sequencing and differential expression analyses of islets from prediabetic rats reveal subsets of ß- and α-cells under stress as evidenced by heightened expression, over time, of a transcriptional signature characterized by interferon-stimulated genes, chemokines including Cxcl10, major histocompatibility class I, and genes for the ubiquitin-proteasome system. Mononuclear phagocytes show increased expression of inflammatory markers. RNA-in situ hybridization of rat pancreatic tissue defines the spatial distribution of Cxcl10+ ß- and α-cells and their association with CD8+ T cell infiltration, a hallmark of insulitis and islet destruction. Our studies define early islet transcriptional events during immune cell recruitment to islets and reveal spatial associations between stressed ß- and α-cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.

BaseFormer: Transformer based Base-Caller for Fast and Accurate Next Generation Sequencing.

Gene sequencing technology is a tool which greatly impacts modern biology and medicine. The next-generation sequencing (NGS) lies at the heart of gene sequencing for its massively increasing throughput, but it is difficult to analyze the large quantities of fluorescent images with high accuracy because the fluorescent signals are weak with varying noise signals, and current designs are limited on accuracy and speed. In this paper, we proposed a novel deep learning based gene sequencing pipeline with semi-automatic labelling method. The obtained results are promising, especially on the high-density data, as the BaseFormer surpasses the traditional methods in terms of cluster quality (Q30: 88 %), throughput (16.5% better), and with similar and low error rate (down to 0.137% on average, best at 0.068 % on high-density data).

Anti-SARS-CoV-2 Activity of Adamantanes In Vitro and in Animal Models of Infection.

Coronavirus disease 2019 (COVID-19) has had devastating effects worldwide, with particularly high morbidity and mortality in outbreaks on residential care facilities. Amantadine, originally licensed as an antiviral agent for therapy and prophylaxis against influenza A virus, has beneficial effects on patients with Parkinson's disease and is used for treatment of Parkinson's disease, multiple sclerosis, acquired brain injury, and various other neurological disorders. Recent observational data suggest an inverse relationship between the use of amantadine and COVID-19. Adamantanes, including amantadine and rimantadine, are reported to have in vitro activity against severe acute respiratory syndrome coronavirus (SARS-CoV) and, more recently, SARS-CoV-2. We hypothesized that adamantanes have antiviral activity against SARS-CoV-2, including variant strains. To assess the activity of adamantanes against SARS-CoV-2, we used in vitro and in vivo models of infection. We established that amantadine, rimantadine, and tromantadine inhibit the growth of SARS-CoV-2 in vitro in cultured human epithelial cells. While neither rimantadine nor amantadine reduces lung viral titers in mice infected with mouse-adapted SARS-CoV-2, rimantadine significantly reduces viral titers in the lungs in golden Syrian hamsters infected with SARS-CoV-2. In summary, rimantadine has antiviral activity against SARS-CoV-2 in human alveolar epithelial cells and in the hamster model of SARS-CoV-2 lung infection. The evaluation of amantadine or rimantadine in human randomized controlled trials can definitively address applications for the treatment or prevention of COVID-19.

Human nasal wash RNA-Seq reveals distinct cell-specific innate immune responses in influenza versus SARS-CoV-2.

BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) "COVID-19 Expansion for AIRe Program."

Type I IFN-Driven Immune Cell Dysregulation in Rat Autoimmune Diabetes.

Type 1 diabetes is a chronic autoimmune disease, characterized by the immune-mediated destruction of insulin-producing ß cells of pancreatic islets. Essential components of the innate immune antiviral response, including type I IFN and IFN receptor (IFNAR)-mediated signaling pathways, likely contribute to human type 1 diabetes susceptibility. We previously showed that LEW.1WR1 Ifnar1 -/- rats have a significant reduction in diabetes frequency following Kilham rat virus (KRV) infection. To delineate the impact of IFNAR loss on immune cell populations in KRV-induced diabetes, we performed flow cytometric analysis in spleens from LEW.1WR1 wild-type (WT) and Ifnar1 -/- rats after viral infection but before the onset of insulitis and diabetes. We found a relative decrease in CD8+ T cells and NK cells in KRV-infected LEW.1WR1 Ifnar1 -/- rats compared with KRV-infected WT rats; splenic regulatory T cells were diminished in WT but not Ifnar1 -/- rats. In contrast, splenic neutrophils were increased in KRV-infected Ifnar1 -/- rats compared with KRV-infected WT rats. Transcriptional analysis of splenic cells from KRV-infected rats confirmed a reduction in IFN-stimulated genes in Ifnar1 -/- compared with WT rats and revealed an increase in transcripts related to neutrophil chemotaxis and MHC class II. Single-cell RNA sequencing confirmed that MHC class II transcripts are increased in monocytes and macrophages and that numerous types of splenic cells harbor KRV. Collectively, these findings identify dynamic shifts in innate and adaptive immune cells following IFNAR disruption in a rat model of autoimmune diabetes, providing insights toward the role of type I IFNs in autoimmunity.

Corporate Environmental Performance in China: The Moderating Effects of the Media versus the Approach of Local Governments.

This article selects the listed companies in China's A-share heavy pollution industry from 2014 to 2018 as samples, uses a random effect model to empirically test the relationship between media attention and corporate environmental performance and examines the impacts of local government environmental protection and property nature on that relationship. Results are as follow: (1) Media attention can significantly affect a company's environmental performance. The higher the media attention, the greater the company's supervision and the better its environmental performance. (2) In areas where the government pays less attention to environmental protection, the impact of media on corporate environmental performance is more obvious, but in other areas, the impact of media on environmental performance cannot be reflected; (3) The media attention is very significant for the environmental performance improvement of state-owned enterprises, and it is not obvious in non-state-owned enterprises. (4) A further breakdown of the study found that the role of media attention in corporate environmental performance is only significant in the sample of local governments that have low environmental protection and are state-owned enterprises. This research incorporates the local government's emphasis on environmental protection into the research field of vision, expands the research scope of media and corporate environmental performance, and also provides new clues and evidence for promoting the active fulfillment of environmental protection responsibilities by companies and local governments.

Expression and functional analysis of Brucella outer membrane protein 25 in recombinant goat pox virus.

The Capripoxvirus (CaPV) has a large double­stranded DNA genome and a restricted host­range. At present, it is being investigated as an ideal vaccine vector. In the present study, a novel recombinant goat pox virus (rGTPV) was constructed to express Brucella outer membrane protein (OMP)25, and was validated by in vitro and in vivo immunization assays. A novel rGTPV vector was created, in which the thymidine kinase gene was used as a flanking sequence, I1L was inserted as a promoter element to enhance Brucella OMP25 expression, and p7.5 as another promoter element was used to regulate guanine phosphoribosyl­transferase as a selection maker. The rGTPV vector was transfected into sheep fetal fibroblast/lamb testis cells pre­infected with GTPV G14­STV44­55 to recombine. Brucella OMP25 protein was expressed in cells by rGTPV, and activated immune reactivity to Brucella OMP25 protein, as detected by western blotting. Furthermore, rGTPV elicited, anti­Brucella­specific immunoglobulin G responses, as determined by ELISA. Mice vaccinated with rGTPV did not exhibit pathology alterations in the kidney and liver. These results suggested that the novel rGTPV was able to efficiently drive Brucella OMP25 protein expression and activate immune reactivity, and may have applications in CaPV live vector vaccines and associated research.

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.

Alox5 Blockade Eradicates JAK2V617F-Induced Polycythemia Vera in Mice.

Myeloproliferative neoplasms such as polycythemia vera (PV), which are associated with the JAK mutation V617F, remain incurable despite progress in the use of JAK2 inhibitors for treatment of some of these diseases. In this study, we employed mice that undergo JAK2V617F-induced PV as a tool to explore new candidate targets for therapy. Our investigations focused on the lipid metabolic enzyme arachidonate 5-lipoxygenase (Alox5), which we found to be strongly upregulated by JAK2V617F in hematopoietic cells in vitro and in vivo Notably, genetic deletion of Alox5 or its inhibition in mice with a bioactive small-molecule inhibitor was sufficient to attenuate PV development. This therapeutic effect was associated with induction of a blockade in cell-cycle progression and also with apoptosis in PV cells. Genetic loss exerted an inhibitory effect on PV-initiating cells. Similarly, Alox5 inhibition was sufficient to suppress colony formation in human JAK2V617F-expressing CD34+ cells. Mechanistic investigations showed that Alox5 inhibition reduced AKT activation and decreased ß-catenin expression in JAK2V617F-expressing cells. Together, our results define Alox5 as a key genetic effector of JAK2V617F in driving PV, and they identify this enzyme as a candidate therapeutic target to treat this refractory myeloproliferative neoplasm. Cancer Res; 77(1); 164-74. ©2016 AACR.

The IFITMs Inhibit Zika Virus Replication.

Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses.

Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics.

The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.

The Pattern of Adipose Tissue Accumulation during Early Infancy Provides an Environment for the Development of Dengue Hemorrhagic Fever.

BACKGROUND: Dengue is the most prevalent arthropod-borne viral illness in humans with half of the world's population at risk. During early infancy, severe dengue can develop after a primary dengue virus infection. There has been a clinical observation that severe dengue during the first year of life is seen only in chubby infants. METHODOLOGY/PRINCIPAL FINDINGS: We examined the associations between the development of severe dengue and adipose tissue accumulation patterns during the first year of life in a prospective observational clinical study of infants and dengue virus infections. We found that adipose tissue contains two potential targets for dengue virus infection and production- adipocytes and adipose tissue macrophages. During the first year of life, total body adiposity and visceral adipose tissue stores were at their highest levels in early infancy. Early infancy was also characterized by a relative decrease in alternatively activated (anti-inflammatory) macrophages, and a relative increase in circulating pro-inflammatory cytokines. CONCLUSIONS/SIGNIFICANCE: The data has been used to propose a model where the adipose tissue accumulation pattern and pro-inflammatory environment during early infancy provide the conditions for the potential development of severe dengue in immune-susceptible infants.

Roles of p53 and ASF1A in the Reprogramming of Sheep Kidney Cells to Pluripotent Cells.

Since the first report of induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka, numerous attempts have been made to derive iPSCs from other species via the ectopic expression of defined factors. Sheep iPSCs (siPSCs) have significant potential for biotechnology and agriculture. Although several groups have described siPSCs, the reprogramming efficiency was extremely low. The exogenous transgenes could be not silenced in the iPSCs, which hampered their development and application. Here, we report that p53 knockdown and antisilencing function 1A (ASF1A) overexpression promoted iPSC generation from sheep kidney cells (SKCs). Compared with transduction with eight human defined transcription factors (Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28, hTERT, and SV40LT), the additional introduction of p53 RNA interference (RNAi) and/or ASF1A in the presence of small-molecule compounds [vitamin C (Vc) and valproic acid (VPA)] greatly improved the efficiency of sheep iPSC generation. The siPSCs exhibited morphological features similar to mouse embryonic stem cells (ESCs) and were positive for alkaline phosphatase and, pluripotent marker genes (Oct4, Nanog, Sox2, Rex1, TRA-1-60, TRA-1-81, and E-cadherin). Furthermore, these cells exhibited a normal karyotype of 54 chromosomes and were able to differentiate into all three germ layers both in vitro and in vivo. Moreover, the exogenous genes were silenced in siPSCs when p53 small hairpin RNA (shRNA) and ASF1A were added. Our results may help to reveal the role of p53 and ASF1A in sheep somatic cell reprogramming and provide an efficient approach to reprogramming sheep somatic cells.

Arachidonate 15-lipoxygenase is required for chronic myeloid leukemia stem cell survival.

Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34+ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.

LSK derived LSK- cells have a high apoptotic rate related to survival regulation of hematopoietic and leukemic stem cells.

A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin(-)Sca-1(+)c-Kit(-) (LSK(-)) cell population derived from HSC-containing Lin(-)Sca-1(+)c-Kit(+) (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK(-) population. In contrast, the LSK(-) population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK(-) population. The transition of LSK to LSK(-) cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK(-) cells in the regulation of LSK cells and LSCs.