FOSL1 is a novel mediator of endotoxin/lipopolysaccharide-induced pulmonary angiogenic signaling.

Systemic sepsis is a known risk factor for bronchopulmonary dysplasia (BPD) in premature infants, a disease characterized by dysregulated angiogenesis and impaired vascular and alveolar development. We have previoulsy reported that systemic endotoxin dysregulates pulmonary angiogenesis resulting in alveolar simplification mimicking BPD in neonatal mice, but the underlying mechanisms remain unclear. We undertook an unbiased discovery approach to identify novel signaling pathways programming sepsis-induced deviant lung angiogenesis. Pulmonary endothelial cells (EC) were isolated for RNA-Seq from newborn C57BL/6 mice treated with intraperitoneal lipopolysaccharide (LPS) to mimic systemic sepsis. LPS significantly differentially-regulated 269 genes after 6 h, and 1,934 genes after 24 h. Using bioinformatics, we linked 6 h genes previously unknown to be modulated by LPS to 24 h genes known to regulate angiogenesis/vasculogenesis to identify pathways programming deviant angiogenesis. An immortalized primary human lung EC (HPMEC-im) line was generated by SV40 transduction to facilitate mechanistic studies. RT-PCR and transcription factor binding analysis identified FOSL1 (FOS like 1) as a transcriptional regulator of LPS-induced downstream angiogenic or vasculogenic genes. Over-expression and silencing studies of FOSL1 in immortalized and primary HPMEC demonstrated that baseline and LPS-induced expression of ADAM8, CXCR2, HPX, LRG1, PROK2, and RNF213 was regulated by FOSL1. FOSL1 silencing impaired LPS-induced in vitro HPMEC angiogenesis. In conclusion, we identified FOSL1 as a novel regulator of sepsis-induced deviant angiogenic signaling in mouse lung EC and human fetal HPMEC.

Histone deacetylase 6 regulates endothelial MyD88-dependent canonical TLR signaling, lung inflammation, and alveolar remodeling in the developing lung.

Lung endothelial cell (EC) immune activation during bacterial sepsis contributes to acute lung injury and bronchopulmonary dysplasia in premature infants. The epigenetic regulators of sepsis-induced endothelial immune activation, lung inflammation, and alveolar remodeling remain unclear. Herein, we examined the role of the cytoplasmic histone deacetylase, HDAC6, in regulating EC Toll-like receptor 4 (TLR4) signaling and modulating sepsis-induced lung injury in a neonatal model of sterile sepsis. In human primary microvascular endothelial cells (HPMEC), lipopolysaccharide (LPS)-induced MAPK, IKK-ß, and p65 phosphorylation as well as inflammatory cytokine expression were exaggerated with the HDAC6 inhibitor tubastatin A, and by dominant-negative HDAC6 with a mutated catalytic domain 2. Expression of HDAC6 wild-type protein suppressed LPS-induced myeloid differentiation primary response 88 (MyD88) acetylation, p65 (Lys310) acetylation, MyD88/TNF receptor-associated factor 6 (TRAF6) coimmunoprecipitation, and proinflammatory TLR4 signaling in HPMEC. In a neonatal mouse model of sepsis, the HDAC6 inhibitor tubastatin A amplified lung EC TLR4 signaling and vascular permeability. HDAC6 inhibition augmented LPS-induced MyD88 acetylation, MyD88/TRAF6 binding, p65 acetylation, canonical TLR4 signaling, and inflammation in the developing lung. Sepsis-induced decreases in the fibroblast growth factors FGF2 and FGF7 and increase in matrix metalloproteinase-9 were worsened with HDAC6 inhibition, while elastin expression was equally suppressed. Exaggerated sepsis-induced acute lung inflammation observed with HDAC6 inhibition worsened alveolar simplification evidenced by increases in mean linear intercepts and decreased radial alveolar counts. Our studies reveal that HDAC6 is a constitutive negative regulator of cytoplasmic TLR4 signaling in EC and the developing lung. The therapeutic efficacy of augmenting HDAC6 activity in neonatal sepsis to prevent lung injury needs to be evaluated.

Functional characterization of SLC26A3 c.392C>G (p.P131R) mutation in intestinal barrier function using CRISPR/CAS9-created cell models.

BACKGROUND: Congenital chloride diarrhea (CCD) in a newborn is a rare autosomal recessive disorder with life-threatening complications, requiring early diagnostics and treatment to prevent severe dehydration and infant mortality. SLC26A3 rs386833481 (c.392C>G; p.P131R) gene polymorphism is an important genetic determinant of CCD. Here, we report the influence of the non-synonymous SLC26A3 variant rs386833481 gene polymorphism on the function of the epithelial barrier and the potential mechanisms of these effects. RESULTS: We found that P131R-SLC26A3 increased dysfunction of the epithelial barrier compared with wild type SLC26A3 in human colonic Caco-2 and mouse colonic CMT-93 cells. When P131R-SLC26A3 was subsequently reverted to wild type, the epithelial barrier function was restored similar to wild type cells. Further study demonstrated that variant P131R-SLC26A3 disrupts function of epithelial barrier through two distinct molecular mechanisms: (a) decreasing SLC26A3 expression through a ubiquitination pathway and (b) disrupting a key interaction with its partner ZO-1/CFTR, thereby increasing the epithelial permeability. CONCLUSION: Our study provides an important insight of SLC26A3 SNPs in the regulation of the epithelial permeability and indicates that SLC26A3 rs386833481 is likely a causative mutation in the dysfunction of epithelial barrier of CCD, and correction of this SNP or increasing SLC26A3 function could be therapeutically beneficial for chronic diarrhea diseases.

Epigenetic regulation of NfatC1 transcription and osteoclastogenesis by nicotinamide phosphoribosyl transferase in the pathogenesis of arthritis.

Nicotinamide phosphoribosyltransferase (NAMPT) functions in NAD synthesis, apoptosis, and inflammation. Dysregulation of NAMPT has been associated with several inflammatory diseases, including rheumatoid arthritis (RA). The purpose of this study was to investigate NAMPT's role in arthritis using mouse and cellular models. Collagen-induced arthritis (CIA) in DBA/1J Nampt +/- mice was evaluated by ELISA, micro-CT, and RNA-sequencing (RNA-seq). In vitro Nampt loss-of-function and gain-of-function studies on osteoclastogenesis were examined by TRAP staining, nascent RNA capture, luciferase reporter assays, and ChIP-PCR. Nampt-deficient mice presented with suppressed inflammatory bone destruction and disease progression in a CIA mouse model. Nampt expression was required for the epigenetic regulation of the Nfatc1 promoter and osteoclastogenesis. Finally, RNA-seq identified 690 differentially expressed genes in whole ankle joints which associated (P < 0.05) with Nampt expression and CIA. Selected target was validated by RT-PCR or functional characterization. We have provided evidence that NAMPT functions as a genetic risk factor and a potential therapeutic target to RA.

Identification of Novel Regulatory Genes in APAP Induced Hepatocyte Toxicity by a Genome-Wide CRISPR-Cas9 Screen.

Acetaminophen (APAP) is a commonly used analgesic responsible for more than half of acute liver failure cases. Identification of previously unknown genetic risk factors would provide mechanistic insights and novel therapeutic targets for APAP-induced liver injury. This study used a genome-wide CRISPR-Cas9 screen to evaluate genes that are protective against, or cause susceptibility to, APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR-Cas9 gene knockouts were treated with 15 mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap of expression data from APAP overdose studies, and 3) predicted affected biological pathways. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. This study illustrated the power of a genome-wide CRISPR-Cas9 screen to systematically identify novel genes involved in APAP-induced hepatotoxicity and to provide potential targets to develop novel therapeutic modalities.

δ-Tocotrienol feeding modulates gene expression of EIF2, mTOR, protein ubiquitination through multiple-signaling pathways in chronic hepatitis C patients.

BACKGROUND: δ-Tocotrienol is a naturally occurring proteasome inhibitor, which has the capacity to inhibit proliferation and induce apoptosis in several cancer cells obtained from several organs of humans, and other cancer cell lines. Moreover, results of plasma total mRNAs after δ-tocotrienol feeding to hepatitis C patients revealed significant inhibition in the expression of pro-inflammatory cytokines (TNF-α, VCAM1, proteasome subunits) and induction in the expression of ICAM1 and IFN-γ after post-treatment. This down-regulation of proteasome subunits leads to autophagy, apoptosis of immune cells and several genes. The present study describes RNA-sequence analysis of plasma total mRNAs obtained from δ-tocotrienol treatment of hepatitis C patients on gene expression regulated by proteasome. METHODS: Pooled specimens of plasma total mRNAs of pre-dose versus post-dose of δ-tocotrienol treatment of hepatitis C patients were submitted to RNA-sequence analyses. The data based on > 1 and 8-fold expression changes of 2136 genes were uploaded into "Ingenuity Pathway Analyses (IPA)" for core analysis, which describes possible canonical pathways, upstream regulators, diseases and functional metabolic networks. RESULTS: The IPA of "molecules" indicated fold change in gene expression of 953 molecules, which covered several categories of biological biomarkers. Out of these, gene expression of 220 related to present study, 12 were up-regulated, and 208 down-regulated after δ-tocotrienol treatment. The gene expression of transcription regulators (ceramide synthase 3 and Mohawk homeobox) were up-regulated, and gene expression of 208 molecules were down-regulated, involved in several biological functions (HSP90AB1, PSMC3, CYB5R4, NDUFB1, CYP2R1, TNFRF1B, VEGFA, GPR65, PIAS1, SFPQ, GPS2, EIF3F, GTPBP8, EIF4A1, HSPA14, TLR8, TUSSC2). IPA of "causal network" indicated gene regulators (676), in which 76 down-regulated (26 s proteasomes, interleukin cytokines, and PPAR-ligand-PPA-Retinoic acid-RXRα, PPARγ-ligand-PPARγ-Retinoic acid-RARα, IL-21, IL-23) with significant P-values. The IPA of "diseases and functions" regulators (85) were involved with cAMP, STAT2, 26S proteasome, CSF1, IFNγ, LDL, TGFA, and microRNA-155-5p, miR-223, miR-21-5p. The IPA of "upstream analysis" (934) showed 57 up-regulated (mainly 38 microRNAs) and 64 gene regulators were down-regulated (IL-2, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-24, IL-27, IL-32), interferon ß-1a, interferon γ, TNF-α, STAT2, NOX1, prostaglandin J2, NF-κB, 1κB, TCF3, and also miRNA-15, miRNA-124, miRNA-218-5P with significant activation of Z-Score (P < 0.05). CONCLUSIONS: This is first report describing RNA-sequence analysis of δ-tocotrienol treated plasma total mRNAs obtained from chronic hepatitis C patients, that acts via multiple-signaling pathways without any side-effects. These studies may lead to development of novel classes of drugs for treatment of chronic hepatitis C patients.

MEG3-4 is a miRNA decoy that regulates IL-1ß abundance to initiate and then limit inflammation to prevent sepsis during lung infection.

Long noncoding RNAs (lncRNAs) regulate gene expression. We investigated the role of lncRNAs in the inflammatory response to bacterial infection in the lungs. We identified the lncRNA MEG3 as a tissue-specific modulator of inflammatory responses during bacterial infection. Among the 10 transcript isoforms of MEG3, transcript 4 (referred to as MEG3-4) encodes the isoform with the lowest abundance in mouse lungs. Nonetheless, we found that MEG3-4 bound to the microRNA miR-138 in a competitive manner with mRNA encoding the proinflammatory cytokine interleukin-1ß (IL-1ß), thereby increasing IL-1ß abundance and intensifying inflammatory responses to bacterial infection in alveolar macrophages and lung epithelial cells in culture and in lung tissue in mice. MEG3-4-mediated sponging of miR-138 in the cytoplasm increased the autocrine activity of IL-1ß that subsequently induced a negative feedback mechanism mediated by nuclear factor κB that decreased MEG3-4 abundance and inflammatory cytokine production. This timely reduction in MEG3-4 abundance tempered proinflammatory responses in mice with pulmonary bacterial infection, preventing the progression to sepsis. Together, these findings reveal that MEG3-4 dynamically modulates pulmonary inflammatory responses through transcriptional regulation of immune response genes, extending the decoy and sponge mechanism associated with lncRNAs to antibacterial immunity, which affects both response and disease progression.

Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown.

Although a deficiency of surfactant protein B (SFTPB) has been associated with lung injury, SFTPB expression has not yet been linked with nicotinamide phosphoribosyltransferase (NAMPT), a potential biomarker of acute lung injury (ALI). The effects of Nampt in the pulmonary epithelial cell on both SFTPB expression and lung inflammation were investigated in a LPS-induced ALI mouse model. Pulmonary epithelial cell-specific knockdown of Nampt gene expression, achieved by the crossing of Nampt gene exon 2 floxed mice with mice expressing epithelial-specific transgene Cre or by the use of epithelial-specific expression of anti-Nampt antibody cDNA, significantly attenuated LPS-induced ALI. Knockdown of Nampt expression was accompanied by lower levels of bronchoalveolar lavage (BAL) neutrophil infiltrates, total protein and TNF-α levels, as well as lower lung injury scores. Notably, Nampt knockdown was also associated with significantly increased BAL SFTPB levels relative to the wild-type control mice. Down-regulation of NAMPT increased the expression of SFTPB and rescued TNF-α-induced inhibition of SFTPB, whereas overexpression of NAMPT inhibited SFTPB expression in both H441 and A549 cells. Inhibition of NAMPT up-regulated SFTPB expression by enhancing histone acetylation to increase its transcription. Additional data indicated that these effects were mainly mediated by NAMPT nonenzymatic function via the JNK pathway. This study shows that pulmonary epithelial cell-specific knockdown of NAMPT expression attenuated ALI, in part, via up-regulation of SFTPB expression. Thus, epithelial cell-specific knockdown of Nampt may be a potential new and viable therapeutic modality to ALI.-Bi, G., Wu, L., Huang, P., Islam, S., Heruth, D. P., Zhang, L. Q., Li, D.-Y., Sampath, V., Huang, W., Simon, B. A., Easley, R. B., Ye, S. Q. Up-regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell-specific NAMPT knockdown.

Ontogeny-related pharmacogene changes in the pediatric liver transcriptome.

OBJECTIVES: The majority of drug dosing studies are based on adult populations, with modification of the dosing for children based on size and weight. This rudimentary approach for drug dosing children is limited, as biologically a child can differ from an adult in far more aspects than just size and weight. Specifically, understanding the ontogeny of childhood liver development is critical in dosing drugs that are metabolized through the liver, as the rate of metabolism determines the duration and intensity of a drug's pharmacologic action. Therefore, we set out to determine pharmacogenes that change over childhood development, followed by a secondary agnostic analysis, assessing changes transcriptome wide. MATERIALS AND METHODS: A total of 47 human liver tissue samples, with between 10 and 13 samples in four age groups spanning childhood development, underwent pair-end sequencing. Kruskal-Wallis and Spearman's rank correlation tests were used to determine the association of gene expression levels with age. Gene set analysis based on the pathways in KEGG utilized the gamma method. Correction for multiple testing was completed using q-values. RESULTS: We found evidence for increased expression of 'very important pharmacogenes', for example, coagulation factor V (F5) (P=6.7×10(-7)), angiotensin I converting enzyme (ACE) (P=6.4×10(-3)), and solute carrier family 22 member 1 (SLC22A1) (P=7.0×10(-5)) over childhood development. In contrast, we observed a significant decrease in expression of two alternative CYP3A7 transcripts (P=1.5×10(-5) and 3.0×10(-5)) over development. The analysis of genome-wide changes detected transcripts in the following genes with significant changes in mRNA expression (P<1×10(-9) with false discovery rate<5×0(-5)): ADCY1, PTPRD, CNDP1, DCAF12L1 and HIP1. Gene set analysis determined ontogeny-related transcriptomic changes in the renin-angiotensin pathway (P<0.002), with lower expression of the pathway, in general, observed in liver samples from younger participants. CONCLUSION: Considering that the renin-angiotensin pathway plays a central role in blood pressure and plasma sodium concentration, and our observation that ACE and PTPRD expression increased over the spectrum of childhood development, this finding could potentially impact the dosing of an entire class of drugs known as ACE-inhibitors in pediatric patients.

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.

Human Parvovirus Infection of Human Airway Epithelia Induces Pyroptotic Cell Death by Inhibiting Apoptosis.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes acute respiratory tract infections in young children. In this study, we confirmed that, when polarized/well-differentiated human airway epithelia are infected with HBoV1 in vitro, they develop damage characterized by barrier function disruption and cell hypotrophy. Cell death mechanism analyses indicated that the infection induced pyroptotic cell death characterized by caspase-1 activation. Unlike infections with other parvoviruses, HBoV1 infection did not activate the apoptotic or necroptotic cell death pathway. When the NLRP3-ASC-caspase-1 inflammasome-induced pathway was inhibited by short hairpin RNA (shRNA), HBoV1-induced cell death dropped significantly; thus, NLRP3 mediated by ASC appears to be the pattern recognition receptor driving HBoV1 infection-induced pyroptosis. HBoV1 infection induced steady increases in the expression of interleukin 1α (IL-1α) and IL-18. HBoV1 infection was also associated with the marked expression of the antiapoptotic genes BIRC5 and IFI6 When the expression of BIRC5 and/or IFI6 was inhibited by shRNA, the infected cells underwent apoptosis rather than pyroptosis, as indicated by increased cleaved caspase-3 levels and the absence of caspase-1. BIRC5 and/or IFI6 gene inhibition also significantly reduced HBoV1 replication. Thus, HBoV1 infection of human airway epithelial cells activates antiapoptotic proteins that suppress apoptosis and promote pyroptosis. This response may have evolved to confer a replicative advantage, thus allowing HBoV1 to establish a persistent airway epithelial infection. This is the first report of pyroptosis in airway epithelia infected by a respiratory virus.IMPORTANCE Microbial infection of immune cells often induces pyroptosis, which is mediated by a cytosolic protein complex called the inflammasome that senses microbial pathogens and then activates the proinflammatory cytokines IL-1 and IL-18. While virus-infected airway epithelia often activate NLRP3 inflammasomes, studies to date suggest that these viruses kill the airway epithelial cells via the apoptotic or necrotic pathway; involvement of the pyroptosis pathway has not been reported previously. Here, we show for the first time that virus infection of human airway epithelia can also induce pyroptosis. Human bocavirus 1 (HBoV1), a human parvovirus, causes lower respiratory tract infections in young children. This study indicates that HBoV1 kills airway epithelial cells by activating genes that suppress apoptosis and thereby promote pyroptosis. This strategy appears to promote HBoV1 replication and may have evolved to allow HBoV1 to establish persistent infection of human airway epithelia.

Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase.

BACKGROUND: Bone degenerative disorders like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Although there are studies suggesting that metabolic changes occur with stem cell differentiation, the molecular mechanisms governing energy metabolism and epigenetic modification are not understood fully. Here we reported the key role of nicotinamide phosphoribosyltransferase (Nampt), which is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide, in the osteogenic differentiation of bone marrow stromal cells. RESULTS: Differentiated bone marrow stromal cells isolated from Nampt+/- mice presented with diminished osteogenesis, as evaluated by alkaline phosphatase (ALP) staining, ALP activity and osteoblast-mediated mineralization, compared to cells from Nampt+/+ mice. Similar results were observed in differentiated Nampt-deficient C3H/10T1/2 and MC3T3-E1 cells. Further studies showed that Nampt promotes osteoblast differentiation through increased function and expression of Runx2 as tested by luciferase reporter assay, RT-PCR, and Western Blotting. Our data also demonstrated that Nampt regulates Runx2 transcription in part through epigenetic modification of H3-Lys9 acetylation. CONCLUSION: Our study demonstrated that Nampt plays a critical role in osteoblast differentiation through epigenetic augmentation of Runx2 transcription. NAMPT may be a potential therapeutic target of aging-related osteoporosis.

Novel Genetic Variants Associated with Child Refractory Esophageal Stricture with Food Allergy by Exome Sequencing.

BACKGROUND: Refractory esophageal stricture (RES) may be attributed to food allergy. Its etiology and pathogenesis are not fully understood. Identification of novel genetic variants associated with this disease by exome sequencing (exome-seq) may provide new mechanistic insights and new therapeutic targets. METHODS: To identify new and novel disease-associating variants, whole-exome sequencing was performed on an Illumina NGS platform in three children with RES as well as food allergy. RESULTS: A total of 91,024 variants were identified. By filtering out 'normal variants' against those of the 1000 Genomes Project, we identified 12,741 remaining variants which are potentially associated with RES plus food allergy. Among these variants, there are 11,539 single nucleotide polymorphisms (SNPs), 627 deletions, 551 insertions and 24 mixture variants. These variants are located in 1370 genes. They are enriched in biological processes or pathways such as cell adhesion, digestion, receptor metabolic process, bile acid transport and the neurological system. By the PubMatrix analysis, 50 out of the top 100 genes, which contain most variants, have not been previously associated with any of the 17 allergy-associated diseases. These 50 genes represent newly identified allergy-associated genes. Those variants of 627 deletions and 551 insertions have also not been reported before in RES with food allergy. CONCLUSIONS: Exome-seq is potentially a powerful tool to identify potential new biomarkers for RES with food allergy. This study has identified a number of novel genetic variants, opening new avenues of research in RES plus food allergy. Additional validation in larger and different patient populations and further exploration of the underlying molecular mechanisms are warranted.

MC-PPEA as a new and more potent inhibitor of CLP-induced sepsis and pulmonary inflammation than FK866.

Our previous study indicated that overexpression of nicotinamide phosphoribosyltransferase (NAMPT) aggravated acute lung injury, while knockdown of NAMPT expression attenuated ventilator-induced lung injury. Recently, we found that meta-carborane-butyl-3-(3-pyridinyl)-2E-propenamide (MC-PPEA, MC4), in which the benzoylpiperidine moiety of FK866 has been replaced by a carborane, displayed a 100-fold increase in NAMPT inhibition over FK866. Here, we determined the effects of MC4 and FK866 on cecal ligation and puncture (CLP) surgery-induced sepsis in C57BL/6J mice. MC4 showed stronger inhibitory effects than FK866 on CLP-induced mortality, serum tumor necrosis factor α (TNFα) levels, pulmonary myeloperoxidase activity, alveolar injury, and interleukin 6 and interleukin1ß messenger RNA levels. In vitro cell permeability and electric cell-substrate impedance sensing assays demonstrated that MC4 inhibited TNFα- and thrombin-mediated pulmonary endothelial cell permeability better than FK866. MC4 also exerted more potent effects than FK866, at concentrations as low as 0.3 nM, to attenuate TNFα-mediated intracellular cytokine expression, nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH levels, and nuclear factor kappa B p65 phosphorylation and nuclear translocation in A549 cells. Our results strongly suggest that the newly developed MC4 is a more potent suppressor of CLP-induced pulmonary inflammation and sepsis than FK866, with potential clinical application as a new treatment agent for sepsis and inflammation.

Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

Human parvovirus B19 (B19V) infection of primary human erythroid progenitor cells (EPCs) arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR) that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2) within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related) activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase.

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein implicated in the pathogenesis of acute respiratory distress syndrome, aging, cancer, coronary heart diseases, diabetes, nonalcoholic fatty liver disease, obesity, rheumatoid arthritis, and sepsis. However, the underlying molecular mechanisms of NAMPT in these physiological and pathological processes are not fully understood. Here, we provide experimental evidence that a Nampt gene homozygous knockout (Nampt-/-) resulted in lethality at an early stage of mouse embryonic development and death within 5-10 days in adult mice accompanied by a 25.24±2.22% body weight loss, after the tamoxifen induction of NamptF/F × Cre mice. These results substantiate that Nampt is an essential gene for life. In Nampt-/- mice versus Nampt+/+ mice, biochemical assays indicated that liver and intestinal tissue NAD levels were decreased significantly; histological examination showed that mouse intestinal villi were atrophic and disrupted, and visceral fat was depleted; mass spectrometry detected unusual higher serum polyunsaturated fatty acid containing triglycerides. RNA-seq analyses of both mouse and human pediatric liver transcriptomes have convergently revealed that NAMPT is involved in key basic cellular functions such as transcription, translation, cell signaling, and fundamental metabolism. Notably, the expression of all eight enzymes in the tricarboxylic acid cycle were decreased significantly in the Nampt-/- mice. These findings prompt us to posit that adult Nampt-/- mouse lethality is a result of a short supply of ATP from compromised intestinal absorption of nutrients from digested food, which leads to the exhaustion of body fat stores.

Identification of lung-specific genes by meta-analysis of multiple tissue RNA-seq data.

Lung-specific genes play critically important roles in lung development, lung physiology, and pathogenesis of lung-associated diseases. We performed a meta-analysis of multiple tissue RNA-seq data to identify lung-specific genes in order to better investigate their lung-specific functions and pathological roles. We identified 83 lung-specific genes consisting of 62 protein-coding genes, five pseudogenes and 16 noncoding RNA genes. About 49.4% of lung-specific genes were implicated in the pathogenesis of lung diseases and 21.7% were involved with lung development. The identification of genes with enriched expression in the lung will facilitate the elucidation of lung-specific functions and their roles in disease pathogenesis.

The Role of Nicotinamide Phosphoribosyltransferase in Pregnancy: A Review.

Nicotinamide phosphoribosyltransferase (NAMPT) was first reported in 1994 and has been explored in various human disease processes. However, until recently, very little has been done to define the role of NAMPT in pregnancy. NAMPT is a 52 kDa protein that has diverse functions in the human body, acting as a growth factor, cytokine, an enzyme, and an insulinomimetic agent. Initial studies examined NAMPT expression in fetal membranes and its effects on the amnion. Later research in nonpregnant studies showed an insulinomimetic effect, and attention focused on its role in gestational diabetes. In addition, as studies revealed NAMPT's function as an inflammatory cytokine, studies examined NAMPT in preeclampsia and fetal growth restriction. Several studies have confirmed that NAMPT is a marker of systemic infectious processes such as pyelonephritis and intrauterine infection. In this review, we present the current understanding of NAMPT's role in various pregnancy-related conditions as well as possible directions for future research.

Fetoplacental regional variations in the expression of angiopoietin-1, angiopoietin-2, and Tie2 in normal-term and near-term pregnancies.

BACKGROUND: Angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), and the receptor tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) are known to be involved in fetoplacental angiogenesis adequacy, which is a primary determinant of fetal growth. Regional variations in Ang1, Ang2, and Tie2 remain unknown, although fetoplacental vascularity and gene expressions differ between the placental center and the periphery. OBJECTIVE: The aim of this study was to test the hypothesis that there are regional variations in the expression of these angiopoietins in human placentas from uncomplicated term and near term pregnancies. STUDY DESIGN: In this prospective study, central and peripheral samples were collected from fresh placentas from normal-term and near-term pregnancies delivered by Cesarean section (n = 7, 36-41 week gestation) prior to the onset of labor. Regional differences in Ang1, Ang2, and Tie2 protein expressions were measured by Western blot and densitometric analyses with b-actin normalization, and their fetoplacental regional localization assessed by immunohistochemistry. The Ang1 and Ang2 ratios at central and peripheral sites were determined. Statistical analysis was performed using Student's t-test. RESULTS: Ang1 protein expression was higher in the placental periphery than in the center (2.48 ± 0.42 versus 1.74 ± 0.27, p = 0.01). In contrast, Ang2 protein expression was greater in the placental center than in the periphery (10.10 ± 1.82 versus 7.15 ± 1.12, respectively, p = 0.03). The Ang1-Ang2 ratio reflected these differential expressions. Tie2 protein expression was higher in the placental periphery than in the center (0.21 ± 0.02 versus 0.16 ± 0.02, p = 0.003). The immunoreactivity of Ang1 and Tie2 was stronger in the periphery than in the center, and that of Ang2 was stronger in the center than in the periphery. CONCLUSIONS: Ang1, Ang2, and Tie2 are differentially expressed in placental center and periphery. Ang1/Ang2 ratio reflects this regional variation in the angiogenic balance that has implications for fetoplacental villous angiogenesis. The results also demonstrate the importance of considering the location of placental sampling sites for any future investigations of fetoplacental villous angiogenesis.

Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers.

Members of the human CYP3A family of metabolizing enzymes exhibit developmental changes in expression whereby CYP3A7 is expressed in fetal tissues, followed by a transition to expression of CYP3A4 in the first months of life. Despite knowledge about the general pattern of CYP3A activity in human development, the mechanisms that regulate developmental expression remain poorly understood. Epigenetic changes, including cytosine methylation, have been suggested to play a role in the regulation of CYP3A expression. The objective of this study was to investigate changes in cytosine methylation of the CYP3A4 and CYP3A7 genes in human pediatric and prenatal livers. The methylation status of cytosine-phospho-guanine dinucleotides was determined in 16 pediatric liver samples using methyl-seq and confirmed by bisulfite sequencing of 48 pediatric and 34 prenatal liver samples. Samples were separated by age into five groups (prenatal, < 1 year of age, 1.8-6 years, 7-11 years, and 12-17 years). Methyl-seq anaylsis revealed that cytosines in the proximal promoter of CYP3A7 are hypomethylated in neonates compared with adolescents (P < 0.001). In contrast, a cytosine 383 base pair upstream of CYP3A4 is hypermethylated in liver samples from neonates compared with adolescents (P = 0.00001). Developmental changes in methylation of cytosines in the proximal promoters of CYP3A4 and CYP3A7 in pediatric livers were confirmed by bisulfite sequencing. In addition, the methylation status of cytosine in the CYP3A4 and CYP3A7 proximal promoters correlated with changes in developmental expression of mRNA for the two enzymes.