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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.

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 β- 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+ β- 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 β- and α-cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.

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 β-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.

Supplemental Figure 1. Comparison of transduction efficiency of JAK2V617F retrovirus between WT and Alox5-/- donor bone marrow cells. Bone marrow cells from WT or Alox5-/- donor mice were transduced with JAK2V617F retrovirus, and transduced with the same virus again 24 hours later. Prior to injection into recipient mice, the percentages of GFP+ cells were analyzed by FACS. Some cells were cultured for two more days (72 hours after initial viral transduction), and then the percentages of GFP+ cells were analyzed.

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.

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. Here, 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 pre-diabetic period. Single-cell RNA-Seq and differential expression analyses of islets from pre-diabetic rats reveal subsets of β and a 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+ β and a 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 β and a cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.

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.

BACKGROUND Influenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different. METHODS With 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. Results Nasal 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. Conclusions Our 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. Funding Massachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) “COVID-19 Expansion for AIRe Program.”

Type 1 diabetes is a chronic autoimmune disease, characterized by the immune-mediated destruction of insulin-producing β 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.

This article selects the listed companies in China&rsquo, 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&rsquo, s environmental performance. The higher the media attention, the greater the company&rsquo, 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&rsquo, 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.

Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.

There is pressing urgency to better understand the pathogenesis of the severe acute respiratory syndrome (SARS) coronavirus (CoV) clade SARS-CoV-2. SARS-CoV-2, like SARS-CoV, utilizes ACE2 to bind host cells. While initial SARS-CoV-2 cell entry and infection depend on ACE2 in concert with the protease TMPRSS2 for spike (S) protein activation, the specific cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human and non-human primate (NHP) single-cell RNA-sequencing (scRNA-seq) datasets to uncover the cell subsets that may serve as cellular targets of SARS-CoV-2. We identify ACE2/TMPRSS2 co-expressing cells within type II pneumocytes, absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discover that ACE2 is an interferon-stimulated gene (ISG) in human barrier tissue epithelial cells. Thus, SARS-CoV-2 may exploit IFN-driven upregulation of ACE2, a key tissue-protective mediator during lung injury, to enhance infection.

The Capripoxvirus (CaPV) has a large double‑stranded DNA genome and a restricted host‑range. 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 phosphoribosyl‑transferase as a selection maker. The rGTPV vector was transfected into sheep fetal fibroblast/lamb testis cells pre‑infected with GTPV G14‑STV44‑55 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, anti‑Brucella‑specific 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.

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.

SummaryThe 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.

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.

Summary 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., Graphical Abstract, Highlights • Meta-analysis of human, non-human primate, and mouse single-cell RNA-seq datasets for putative SARS-CoV-2 targets • Type II pneumocytes, nasal secretory cells, and absorptive enterocytes are ACE2 + TMPRSS2 + • Interferon and influenza increase ACE2 in human nasal epithelia and lung tissue • Mouse Ace2 is not upregulated by interferon, raising implications for disease modeling, Analysis of single-cell RNA-seq datasets from human, non-human primate, and mouse barrier tissues identifies putative cellular targets of SARS-CoV-2 on the basis of ACE2 and TMPRSS2 expression. ACE2 represents a previously unappreciated interferon-stimulated gene in human, but not mouse, epithelial tissues, identifying anti-viral induction of a host tissue-protective mechanism, but also a potential means for viral exploitation of the host response.

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 months 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 β-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.

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 β-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.

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., Author Summary 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. We therefore 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. The accumulation of visceral adipose tissue, the relative decrease in alternatively activated macrophages, and the relative increase in circulating pro-inflammatory cytokines during early infancy provide the environment for the development of severe dengue in immune-susceptible infants.

The molecular mechanisms controlling human hair growth and scalp hair loss are poorly understood. By screening about 350,000 individuals in two populations from the Volga-Ural region of Russia, we identified a gene mutation in families who show an inherited form of hair loss and a hair growth defect. Affected individuals were homozygous for a deletion in the LIPH gene on chromosome 3q27, caused by short interspersed nuclear element–retrotransposon–mediated recombination. The LIPH gene is expressed in hair follicles and encodes a phospholipase called lipase H (alternatively known as membrane-associated phosphatidic acid–selective phospholipase A1α), an enzyme that regulates the production of bioactive lipids. These results suggest that lipase H participates in hair growth and development.

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.

A 7.3 kb BamH I genomic fragment from fowlpox virus (FPV) strain 282E4 has been sequenced. After comparison with the FPV sequences collected in Genbank, and the entire genomic sequence of vaccinia virus (VV) strain Compenhagen, a ll.2 kb BamH I fragment from FPV strain Munich HP-438 has been found homologous to the fragment. Every predicted open reading frame (ORF) within the fragment has been compared with the ORFs of VV strain Copenhagen at the amino acid level. The signal peptide and transmembrane region of each peptide encoded by each corresponding ORF predicted in the fragment have been studied as well.

The myeloproliferative neoplasms (MPNs) are hematologic malignancies with a chronic clinical course and a risk of developing thrombosis and acute leukemia. JAK2 is a member of the Janus family of non-receptor tyrosine kinases, and the somatic activating point mutation of JAK2 (JAK2V617F) has been found in Philadelphia chromosome-negative MPNs in approximately 95% of polycythemia (PV), 60% of essential thrombocytosis (ET), and 50% of JAK2 V617F-positive MPD (PMF) patients. Although several JAK2 inhibitors are in the early stage of clinical trials and have been shown to help to improve symptoms and quality of life in patients, their long-term effectiveness in patients remains to be determined. New therapeutic strategies need to be developed for treating PV more effectively. Because hematopoietic stem cells (HSCs) harbor JAK2V617F in PV patients, PV should be compared to Philadelphia chromosome-positive chronic myeloid leukemia (CML) that is also derived from HSCs and has a myeloproliferative phenotype similar to PV in mice. Therefore, we reasoned that a gene essential for CML development might also play a critical role in PV development. We have shown that the survival and self-renewal of CML-initiating cells require the arachidonate 5-lipoxygenase gene (Alox5) and that Alox5 is essential for CML development. Therefore, in this study we investigated the role of Alox5 in pathogenesis of PV in mice. We showed that JAK2V617F upregulates Alox5 expression both in vitro (Ba/F3 cells) and in vivo (the spleen cells in PV mice). To examine whether Alox5 is required for induction of PV by JAK2V617F, we transduced bone marrow (BM) cells from wild type (WT) or Alox5 homozygous knockout (Alox5-/-) mice with JAK2V617F-GFP, followed by transplantation into lethally-irradiated recipient mice. We found that the average percentage of JAK2V617F-expressing white blood cell counts (GFP+ WBCs) in recipients of JAK2V617F-transduced Alox5-/- donor BM cells was significantly lower than that in recipients of JAK2V617F-transduced WT donor BM cells (P< 0.001). Total numbers of WBCs, red blood cells (RBCs), hemoglobin (HGB) and hematocrit (HCT) were also significantly lower in the absence of Alox5. With time (at 9 months after induction of PV), the effect of Alox5 deficiency on PV development became more significant. Because JAK2V617F-transformed HSCs is shown to be a PV-initiating cell population, we further tested whether loss of Alox5 affects this cell population in PV mice. We found that the percentage and total number of HSCs (Lin-Sca-1+c-Kit+) in mice receiving JAK2V617F-transduced Alox5-/- BM cells was significantly lower than that in mice receiving JAK2V617F-transduced WT BM cells. These findings suggest that Alox5 could be an effective target gene for PV. We tested this idea by treating PV mice with an Alox5 inhibitor zileuton (300 mg/kg, once a day) or a placebo. Western blot analysis of protein lysates from the spleens of PV mice showed that 5-lipoxygines (5-LO, a protein product of the Alox5 gene) is upregulated by JAK2V617F, and inhibition of Alox5 function by zileuton restored the 5-LO level back to the control level. In addition, the number of WBCs in placebo-treated mice rose significantly with time, whereas the number of WBCs in zileuton-treated mice did not. The inhibition of PV development by zileuton correlated with much smaller infiltrated spleen in zileuton-treated PV mice than in placebo-treated PV mice. Furthermore, we compared the levels of JAK2V617F-expressing cells (GFP+) in PV mice. FACS analysis showed that the percentage of Gr-1+ or Mac+ PV cells in peripheral blood was significantly lower in zileuton-treated PV mice than in placebo-treated PV mice. Consistent with the inhibition of PV cells by zileuton in mice, survival of zileuton-treated PV mice was significantly improved compared to placebo-treated PV mice (Fig. 3I). At 5 months after induction of PV, about 40% placebo-treated PV mice died, but all zileuton-treated mice survived. To understand the underlying mechanisms, we treated JAK2V617F-expressing BaF/3 cells with zileuton, and found that zileuton inhibited activation of AKT and β-catenin by JAK2V617F, and did not affect activation of PI3K, MAPK and JAK2. Together, these results demonstrate that Alox5 represents a critical pathway in JAK2V617F-induced PV and that targeting of Alox5 provides a new strategy for treating PV. Disclosures: No relevant conflicts of interest to declare.

Abstract 1666 NF-κB activation has been linked to the promotion of an assortment of malignancies. While in vitro studies have supported a tumor-promoting role for NF-κB in leukemias, in vivo evidence has not been collected. NF-κB has also been proposed as a therapeutic target for Philadelphia chromosome-positive leukemias including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). In this study, we investigate the role the Nfkb1 gene plays in CML and B-ALL using mouse models for these leukemias induced by the BCR-ABL oncogene. Contrary to conventional thinking, we find a tumor-suppressing role for Nfkb1 in BCR-ABL leukemias. To induced CML, we transduced bone marrow cells from 5-FU-primed wild type or Nfkb1−/− mice with BCR-ABL-GFP retrovirus, followed by transplantation of the transduced cells into lethally irradiated recipient mice. We show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells developed CML more rapidly than the wild type recipients, correlating to elevated percentages (82.3% vs 38.5%) and total numbers of Gr-1+GFP+ cells in peripheral blood of the mice. We next tested whether the loss of Nfkb1 also accelerated B-ALL development under the B-ALL-inducing conditions, and show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells succumbed to B-ALL more quickly than the wild type recipients. These results suggest that Nfkb1 does not stimulate leukemia growth, and instead, it plays a tumor-suppressing role in CML and B-ALL. Because these findings are opposite from what people believe in the field, we confirmed our findings by conducting a rescue experiment. The Nfkb1 gene encodes the p105 protein; roughly half of p105 product is selectively proteolyzed to yield the p50 transcription factor. In this rescue experiment, we transduced Nfkb1−/− donor cells with BCR-ABL alone or BCR-ABL and p50 of Nfkb1 to induce leukemia, and we show that acceleration of leukemia development caused by the Nfkb1 loss is partially reversed by p50. To study the underlying mechanism, we focused on CARD11, because it plays a critical role in mediating NF-κB signaling in lymphocytes. We find that CARD11 expression is induced in myeloid and increased in lymphoid cell lines expressing BCR-ABL. Furthermore, we induced B-ALL using Card1−/− mice. We find that similar to the loss of Nfkb1, the loss of Card11 expression also accelerates the development of B-ALL. To increase human relevance of our findings, we overexpressed p50 in human K562 cells by retroviral transduction with p50-IRES-GFP or empty vector, and find that FACS-sorted p50/GFP K562 cells grow significantly slower than empty vector-transduced cells. These findings support an idea that Nfkb1 signaling suppresses BCR-ABL-induce leukemia at least partially through a CARD11-dependent mechanism. In summary, our results demonstrate that Nfkb1 plays a tumor-suppressing role in BCR-ABL-induced leukemias, arguing that we should be careful when targeting of Nfkb1 in leukemia treatment. Disclosures: No relevant conflicts of interest to declare.

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.