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Background. To observe different roles of direct bilirubin (Dbil) on portopulmonary hypertension (POPH) and idiopathic pulmonary arterial hypertension (IPAH). Methods. Thirty incident patients with POPH and 180 with IPAH (matched by the WHO functional classification in a 1 : 6 ratio) between March 2010 and December 2020 were included. The receiver operating curve and Kaplan–Meier method were applied to estimate the ability to distinguish between the two and survival, respectively. Univariate and forward multiple stepwise regression analyses were performed to access the relationship between pulmonary vascular resistance (PVR) and clinical indices. Results. Compared to IPAH, the POPH group had better hemodynamics including PVR (7.08 ± 3.95 vs. 14.89 ± 7.11, P < 0.001 ) and higher total bilirubin (Tbil) and Dbil. Tbil and Dbil had a negative correlation with PVR in the POPH group (r = −0.394, P = 0.031 ; r = −0.364, P = 0.048 , respectively) but positive correlation in the IPAH group (r = 0.218, P = 0.003 ; r = 0.178, P = 0.018 , respectively). Increased neutrophil counts (r = 0.394, P = 0.031 ) and elevated NT-proBNP (r = 0.433, P < 0.001 ) would help predict the elevation of PVR in POPH and IPAH groups independent of Dbil, respectively. Dbil could distinguish POPH from IPAH (AUC = 0.799, P = 0.009 ), and the ability was elevated when taking aspartate aminotransferase together (AUC = 0.835, P < 0.001 ). The overall survival was better in POPH than in IPAH (7 dead cases of POPH and 96 of IPAH, P = 0.002 ). Survival was better in POPH than in IPAH in the group of Dbil ≥7 μmol/L ( P = 0.001 ) but showed no significant difference between POPH and IPAH in the group of Dbil P = 0.192 ). Conclusions. The POPH group had a better hemodynamic profile than IPAH. Dbil was associated oppositely with the elevation of PVR in POPH and IPAH. Patients with POPH had better survival than those with IPAH in the total cohort and in the group of Dbil ≥7 μmol/L, but limited dead cases of POPH should be noted.

Prolactin (PRL) is a polypeptide hormone that is mainly synthesized and secreted by the lactotroph cells of the pituitary. There are two main isoforms of PRL: 23-kDa PRL (named full-length PRL) and vasoinhibins (including 5.6-18 kDa fragments). Both act as circulating hormones and cytokines to stimulate or inhibit vascular formation at different stages and neovascularization, including endothelial cell proliferation and migration, protease production, and apoptosis. However, their effects on vascular function and cardiovascular diseases are different or even contrary. In addition to the structure, secretion regulation, and signal transduction of PRL/vasoinhibins, this review focuses on the pathological mechanism and clinical significance of PRL/vasoinhibins in cardiovascular diseases.

Investigating fitness interactions in natural populations remains a considerable challenge. We take advantage of the unique population structure of Vibrio parahaemolyticus, a bacterial pathogen of humans and shrimp, to perform a genome-wide screen for coadapted genetic elements. We identified 90 interaction groups involving 1,560 coding genes. 82 of these interaction groups are between accessory genes, many of which have functions related to carbohydrate transport and metabolism. Only 8 interaction groups involve both core and accessory genomes. The largest includes 1,540 SNPs in 82 genes and 338 accessory genome elements, many involved in lateral flagella and cell wall biogenesis. The interactions have a complex hierarchical structure encoding at least four distinct ecological strategies. Preliminary experiments imply that the strategies influence biofilm formation and bacterial growth rate in vitro. One strategy involves a divergent profile in multiple genome regions, implying that strains have irreversibly specialized, while the others involve fewer genes and are more plastic. Our results imply that most genetic alliances are ephemeral but that increasingly complex strategies can evolve and eventually cause speciation.

Investigating fitness interactions in natural populations remains a considerable challenge. We take advantage of the unique population structure of Vibrio parahaemolyticus, a bacterial pathogen of humans and shrimp, to perform a genome-wide screen for coadapted genetic elements. We identified 90 interaction groups (IGs) involving 1,560 coding genes. 82 IGs are between accessory genes, many of which have functions related to carbohydrate transport and metabolism. Only 8 involve both core and accessory genomes. The largest includes 1,540 SNPs in 82 genes and 338 accessory genome elements, many involved in lateral flagella and cell wall biogenesis. The interactions have a complex hierarchical structure encoding at least four distinct ecological strategies. One strategy involves a divergent profile in multiple genome regions, while the others involve fewer genes and are more plastic. Our results imply that most genetic alliances are ephemeral but that increasingly complex strategies can evolve and eventually cause speciation.

Human adenovirus type 4 (HAdV-4) is an epidemic virus that contributes to serious acute respiratory disease (ARD) in both pediatric and adult patients. However, no licensed drug or vaccine is currently available to the civilian population. The identification of neutralizing epitopes of HAdV-4 should allow the development of a novel antiviral vaccine and a novel gene transfer vector, and an effective neutralizing monoclonal antibody (MAb) will be useful in developing appropriate antiviral drugs. In this study, we report that MAb MN4b shows strong neutralizing activity against HAdV-4. MN4b recognizes a conformational epitope (418AGSEK422) within hypervariable region 7 (HVR7). Mutations within this site permitted HAdV-4 mutants to escape neutralization by MN4b and to resist neutralization by animal and human anti-HAdV-4 sera. A recombinant virus, rAd3-A4R7-1, containing the identified neutralizing epitope in the HVR7 region of HAdV-3 hexon, successfully induced antiserum that inhibited HAdV-4 infection. These results indicate that a small surface loop of HAdV-4 hexon is a critical neutralization epitope for this virus. The generation of MN4b and the identification of this neutralizing epitope may be useful in developing therapeutic treatment, a subunit vaccine, and a novel vector that can escape preexisting neutralization for HAdV-4. IMPORTANCE Neutralizing antibodies are considered good tools for the prevention of human adenovirus type 4 (HAdV-4) infections. The identification of the epitopes recognized by such neutralizing antibodies is important for the generation of recombinant antiviral vaccines. However, until now, no neutralizing epitope has been reported for HAdV-4. Here, we developed a serotype-specific neutralizing MAb directed against HAdV-4, MN4b. We provide evidence that MN4b recognizes a conformational epitope within HVR7 of HAdV-4 hexon. Antisera generated to this conformational epitope displayed on HAdV-3 hexon inhibited infection of AD293 cells by HAdV-4. Our findings are very important for the development of therapeutic treatment, a subunit vaccine, and a novel vector for HAdV-4.

Human adenovirus types 3 and 7 (HAdV-3 and HAdV-7) occur epidemically and contribute greatly to respiratory diseases, but there is no currently available licensed recombinant HAdV-3/HAdV-7 bivalent vaccine. Identification of serotype-specific neutralizing antibody (NAb) epitopes for HAdV-3 and HAdV-7 will be beneficial for development of recombinant HAdV-3/HAdV-7 bivalent vaccines. In this study, four NAb epitopes within hexon hypervariable regions (HVRs) were predicted for HAdV-3 and HAdV-7, respectively, by using bioinformatics. Eight hexon chimeric adenovirus vectors with the alternation of only one predicted neutralizing epitope were constructed. Further in vitro and in vivo neutralization assays indicated that E2 (residing in HVR2) and E3 (residing in HVR5) are NAb epitopes for HAdV-7, and E3 plays a more important role in generating NAb responses. Cross-neutralization assays indicated that all four predicted epitopes, R1 to R4, are NAb epitopes for HAdV-3, and R1 (residing in HVR1) plays the most important role in generating NAb responses. Humoral immune responses elicited by the recombinant rAdH7R1 (containing the R1 epitope) were significantly and durably suppressed by HAdV-3-specific NAbs. Surprisingly, the rAdΔE3GFP-specific neutralizing epitope responses induced by rAdMHE3 (R3 replaced by E3) and rAdMHE4 (R4 replaced by E4) were weaker than those of rAdMHE1 (R1 replaced by E1) or rAdMHE2 (R2 relaced by E2) in vitro and in vivo . Furthermore, rAdMHE4 replicated more slowly in HEp-2 cells, and the final yield was about 10-fold lower than that of rAdΔE3GFP. The current findings contribute not only to the development of new adenovirus vaccine candidates, but also to the construction of new gene delivery vectors.

Expression of the human T-cell leukemia virus type 1 (HTLV-1) oncoprotein Tax is correlated with cellular transformation, contributing to the development of adult T-cell leukemia. In this study, we investigated the role of Tax in the regulation of the ZNF268 gene, which plays a role in the differentiation of blood cells and the pathogenesis of leukemia. We demonstrated that ZNF268 mRNA was repressed in HTLV-1-infected cells. We also showed that stable and transient expression of HTLV-1 Tax led to repression of ZNF268. In addition, by using reporter constructs that bear the human ZNF268 promoter and its mutants, we showed that Tax repressed ZNF268 promoter in a process dependent on a functional cAMP-responsive element. By using Tax, cAMP-responsive element-binding protein (CREB)-1, CREB-2, and their mutants, we further showed that Tax repressed ZNF268 through the CREB/activating transcription factor pathway. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated the formation of the complex of Tax·CREB-1 directly at the cAMP-responsive element both in vitro and in vivo. These findings suggest a role for ZNF268 in aberrant T-cell proliferation observed in HTLV-1-associated diseases.

Background Benzo[a]pyrene (B[a]P) is a common environmental and foodborne pollutant. Although the carcinogenicity of high-dose B[a]P has been extensively reported, the effects of long-term B[a]P exposure at lower environmental doses on cancer development are less understood. Objectives We investigated the impact of B[a]P on human hepatocellular carcinoma (HCC) progression at various levels of exposure and identified a potential intervention target. Methods We used a model based on human HCC cells exposed to various concentrations of B[a]P (i.e., 0.01, 1, or 100 nM) for 1 month to examine the effects of B[a]P on cell growth, migration, invasion, and angiogenicity. A bioluminescent murine model was established to assess tumor metastasis in vivo. Results Chronic B[a]P exposure did not alter HCC cell growth but promoted cell migration and invasion both in vitro and in vivo. There was an negative association between B[a]P exposure and the survival of tumor-bearing mice. In addition, B[a]P-treated HCC cells recruited vascular endothelial cells and promoted tumor angiogenesis, possibly through elevating vascular endothelial growth factor secretion. Furthermore, the NF-κB pathway may be an adverse outcome pathway associated with the cumulative effects of B[a]P on HCC metastasis. Conclusions These findings a) indicate that B[a]P has effects on HCC progression; b) identify a possible adverse outcome pathway; and c) contribute to a better understanding of the adverse effects of chronic exposure of B[a]P to human health. Citation Ba Q, Li J, Huang C, Qiu H, Li J, Chu R, Zhang W, Xie D, Wu Y, Wang H. 2015. Effects of benzo[a]pyrene exposure on human hepatocellular carcinoma cell angiogenesis, metastasis, and NF-κB signaling. Environ Health Perspect 123:246–254; http://dx.doi.org/10.1289/ehp.1408524

Zinc finger proteins (ZNF) play important roles in various physiological processes. Here we report that ZNF300, a novel zinc finger protein, identified specifically in humans, promotes tumour development by modulating the NF-κB pathway. Inflammatory factors were found to induce ZNF300 expression in HeLa cell line, and ZNF300 expression further enhanced NF-κB signalling by activating TRAF2 and physically interacting with IKKβ. Furthermore, ZNF300 overexpression increased ERK1/2 phosphorylation and the expression of c-myc, IL-6, and IL-8 but decreased the expression of p21waf-1 and p27Kip1; whose down-regulation led to the opposite effect. Most importantly, ZNF300 overexpression stimulated cancer cell proliferation in vitro and significantly enhanced tumour development and metastasis in mouse xenograft model, while knocking down ZNF300 led to the opposite effects. We have identified a novel function for ZNF300 in tumour development that may uniquely link inflammation and NF-κB to tumourigenesis in humans but not in mice.

ZNF300 was recently identified as a member of the human KRAB/C2H2 zinc finger protein family. Little is known about the role of ZNF300 in human gene regulation networks. In this study, the DNA-binding property of ZNF300 was further analyzed. We found that the recombinant ZNF300 could bind to the binding site 5′-GCGGGGGCG-3′ of Egr1, another member of the KRAB/C2H2 zinc finger protein family. Similarly, recombinant Egr1 also showed a similar binding affinity to the ZNF300 binding site 5′-CTGGGGGCG-3′. Bioinformatics analysis revealed that there is an overlapping ZNF300/Egr1 binding site in the human IL-2Rβ promoter region, which was previously known to be recognized by endogenous Egr1. Electrophoretic mobility shift assays showed that endogenous ZNF300 could also bind to this site. A transient transfection assay revealed that both ZNF300 and Egr1 could transactivate the IL-2Rβ promoter, and that the activation was abrogated by a mutation of residues in the overlapping ZNF300/Egr1 binding site. Co-expression of ZNF300 and Egr1 led to enhanced IL-2Rβ promoter activity. Thus, ZNF300 is likely to be another regulator of the human IL-2Rβ promoter.

The human ZNF300 gene is a member of the KRAB/C2H2 zinc finger gene family, the members of which are known to be involved in various developmental and pathological processes. Here, we show that the ZNF300 gene encodes a 68-kDa nuclear protein that binds DNA in a sequence-specific manner. The ZNF300 DNA binding site, C(t/a)GGGGG(c/g)G, was defined via a random oligonucleotide selection assay, and the DNA binding site was further confirmed by electrophoretic mobility shift assays. A potential ZNF300 binding site was found in the promoter region of the human IL-2Rβ gene. The results of electrophoretic mobility shift assays indicated that ZNF300 bound to the ZNF300 binding site in the IL-2Rβ promoter in vitro. Transient co-transfection assays showed that ZNF300 could activate the IL-2Rβ promoter, and that the activation was abrogated by the mutation of residues in the ZNF300 binding site. Identifying the DNA binding site and characterizing the transcriptional regulation property of ZNF300 would provide critical insights into its potential as a transcriptional regulator.

The ZNF268 gene was originally isolated from an early human embryo cDNA library. Several different transcripts have been isolated for the ZNF268 gene and developmental expression studies suggest that ZNF268 plays a role in the development of human fetal liver and the differentiation of blood cells. In our effort to study the functions of ZNF268 in different organs during development and in pathogenesis, we have now identified 3 novel splicing isoforms, ZNF268e, ZNF268f and ZNF268g, in human fetal tissues and human tumor derived cell lines. The 8 alternatively spliced mRNAs discovered so for are predicted to encode 3 protein isoforms. Expression analysis showed that different mRNA isoforms have different expression profiles. In particular, ZNF268c mRNA was detected only in tumor cells, and ZNF268f appeared to be tissue-specific. By Western blot analysis, all 3 ZNF268 protein isoforms, ZNF268a, ZNF268b1 and ZNF268b2, were expressed in tumor cell lines, while only two protein products, ZNF268b1 and ZNF268b2, were detected in human fetal tissues. Subcellular localization analysis showed that ZNF268a and ZNF268b2 distributed diffusely throughout the cell, while ZNF268b1 mainly localized in the cytoplasm. Moreover, using a CAT reporter system fused to the Gal4 DNA binding domain of the ZNF268 gene, the ZNF268a and b2 activated the CAT reporter gene expression, while the KRAB domain, corresponding to the ZNF268b1 repressed the reporter gene expression. Taken together, our results showed that multiple ZNF268 splicing products encode multiple ZNF268 protein isoforms with different subcellular localization, and that the ZNF268 gene may function as a transcriptional activator in the growth and differentiation of cells in development and/or pathogenesis.

Human ZNF268 gene is a typical Kruppel-associated box/C2H2 zinc finger gene whose homolog has been found only in higher mammals and not in lower mammals such as mouse. Its expression profiles have suggested that it plays a role in the differentiation of blood cells during early human embryonic development and the pathogenesis of leukemia. To gain additional insight into the molecular mechanisms controlling the expression of the ZNF268 gene and to provide the necessary tools for further genetic studies of leukemia, we have mapped the 5′-end of the human ZNF268 mRNA by reverse transcription-PCR and primer extension assays. We then cloned the 5′-flanking genomic DNA containing the putative ZNF268 gene promoter and analyzed its function in several different human and mouse tissue culture cell lines. Interestingly, our studies show that the ZNF268 gene lacks a typical eukaryotic promoter that is present upstream of the transcription start site and directs a basal level of transcription. Instead, the functional promoter requires an essential element that is located within the first exon of the gene. Deletion and mutational analysis reveals the requirement for a cAMP response-element-binding protein (CREB)-binding site within this element for promoter function. Gel mobility shift and chromatin immunoprecipitation assays confirm that CREB-2 binds to the site in vitro and in vivo. Furthermore, overexpression of CREB-2 enhances the promoter activity. These results demonstrate that the human ZNF268 gene promoter is atypical and requires an intragenic element located within the first exon that mediates the effect of CREB for its activity.

The human ZNF268 gene was initially described as a gene associated with early human embryogenesis and was later implicated in human leukemia due to the identification of an alternatively splice form in leukemia patients. To systematically evaluate the correlation of ZNF268 with human hematological malignancy, expression of different alternatively spliced forms of ZNF268 mRNA in peripheral blood of 45 patients with hematological malignancies and 17 healthy donors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR. We demonstrated that presence of ZNF268a, ZNF268c, ZNF268f and ZNF268g were significantly different between the patients and healthy donors (P