Practical considerations in the management of inhaled prostacyclin therapy for pulmonary hypertension associated with interstitial lung disease (WHO group 3).

Pulmonary hypertension (PH), as a consequence of lung disease or hypoxia, has been classified as Group 3 PH by the World Symposium on Pulmonary Hypertension. The most common lung diseases associated with Group 3 PH are chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). PH in ILD (PH-ILD) is associated with reduced exercise capacity, greater supplemental oxygen needs, decreased quality of life, and earlier death compared to ILD alone. Several agents have been evaluated in clinical trials for the treatment of Group 3 PH, but only one treatment has been recently approved by the FDA as conclusively demonstrating efficacy for the treatment of pulmonary hypertension in this group. In the INCREASE study, treprostinil inhalation solution (Tyvaso) demonstrated significant clinical benefit for patients with PH-ILD. The inhaled route of administration may be associated with cough, throat irritation, pharyngolaryngeal pain and risk of bronchospasm and are important considerations upon initiation of therapy. Here we provide a practical review of inhaled prostacyclin therapy and suggestions for healthcare professionals to optimize the management and outcomes for the treatment of WHO Group 3, PH-ILD patients. Recommendations include up-to-date practical considerations pertaining to the entire care team and encompass patient education and communication, monitoring, titration methods and mitigation of side effects.

A Meta-Analysis: Coronary Artery Calcium Score and COVID-19 Prognosis.

Background: Multiple studies have investigated the correlations of mortality, mechanical ventilation, and intensive care unit (ICU) admissions with CAC scores. This analysis overviews the prognostic capability of CAC scoring in mortality, mechanical ventilation, and ICU admission for hospitalized COVID-19 patients. Methods: Online search was conducted on PubMed, Cochrane Library, and Scopus from inception to 22 November 2021 to identify studies involving CAC scores in relation to ICU admission, mechanical ventilation, and death rates. Results: A total of eight studies were analyzed. In the absence of CAC group compared with the presence of CAC score, there was an increase in mortality in the presence of CAC (RR 2.24, 95% CI, 1.41−3.56; p < 0.001). In the low CAC group and high CAC group, high CAC group had increase in mortality (RR 2.74; 95% CI, 1.94−3.86; p < 0.00001). There was no statistical difference in outcomes of mechanical ventilation and ICU admission between any of the groups. Conclusion: This meta-analysis strictly examined the outcomes of interest in death, mechanical ventilation, and ICU admission while comparing the CAC scores in patients with COVID-19. Given these findings, CAC scoring can aid in stratifying patients, thus allowing earlier interventions in rapidly developing illnesses.

Cardiac radioablation-A systematic review.

Failure of drugs and catheter ablation procedures for the treatment of ventricular arrhythmias is still extremely relevant. Recently, stereotactic body radiotherapy has been introduced to treat therapy refractory patients. In this systematic review (International Prospective Register of Systematic Reviews, CRD42019133212), we aimed to summarize electrophysiological and histopathological effects of radioablation in animals, patients, and extracted and perfused hearts. A systematic search was performed in OVID MEDLINE, OVID Embase, the Cochrane Central Register of Controlled Trials, Web of Science, Google Scholar, ClinicalTrials.gov, and World Health Organization International Clinical Trials Registry Platform (WHO ICTRP) from inception to September 2019. Identified records were independently screened for eligibility by 2 reviewers. Risk of bias and methodological quality were assessed using the SYRCLE, ROBINS-I, or Murad tool and tailored to the different study designs. We included 13 preclinical and 10 clinical publications. Large heterogeneity in study designs prompted a narrative synthesis approach. Baseline, (pre-)procedural details, outcome, target tissue analyses, and safety data were extracted and summarized. In animal studies evaluating electrophysiological parameters, radioablation induced a reduction in voltage/potential amplitude or bidirectional block in target areas in 93.2% of animals. Atrioventricular block (first to third degree) was induced in 78.3% of animals, and in studies evaluating ventricular arrhythmia inducibility, 75% reduction was achieved. In patients, predominantly ventricular tachycardias were targeted with >85% reduction in arrhythmia episodes during follow-up with an encouraging short-term safety profile. Preclinical and clinical evidence on the efficacy and safety of radioablation is limited in both quantity and quality. The results of radioablation for therapy refractory patients with ventricular tachycardia are promising, but further research is needed.

Comparative molecular analysis of fecal microbiota of bobcats (Lynx rufus) and domestic cats (Felis catus).

The goal of this study was to explore and describe fecal microbiota of captive and wild bobcats (Lynx rufus) and compare the results to those of domestic cats (Felis catus). Fecal samples from 27 bobcats (8 wild, 19 zoo-kept) were used for novel bacterial deoxyribonucleic acid (DNA) identification using next-generation sequencing of the V4 region of the bacterial 16S ribosomal ribonucleic acid (rRNA) gene, analyzed by Illumina sequencing, and then compared to data obtained from a colony of 10 domestic cats. In this study, the microbiota of both species was dominated by Firmicutes, followed by Proteobacteria, Actinobacteria, Bacteroidetes, and Verrucomicrobia. When compared, fecal samples from bobcats harbored more Proteobacteria and Actinobacteria than fecal samples from domestic cats. There was a remarkable inter-bobcat variation in the relative abundances of the main bacterial genera. There were no significant differences, however, between the main phyla of the microbiota of the wild and domestic bobcats. Proteobacteria in wild bobcats (P = 0.079) and Firmicutes in zoo-kept bobcats (P = 0.079) approached significance. There were no differences in predominant genera between wild and captive bobcats. The results of this study showed that there are notable differences in fecal bacterial communities between domestic cats and both captive and wild bobcats. The lack of significant differences in bacterial communities between wild and zoo-kept bobcats suggests that the varied diet provided for these felids can result in a fecal microbiota resembling that generated by a wild diet.

L'objectif de la présente étude était d'explorer et de décrire le microbiote fécal de lynx (Lynx rufus) sauvages et captifs et de comparer les résultats à ceux de chats domestiques (Felis catus). Des échantillons de fèces provenant de 27 lynx (8 sauvages, 19 gardés en zoo) ont été utilisés pour identification d'ADN bactérien en utilisant le séquençage de prochaine génération de la région V4 du gène de l'ARNr 16S, analysé par séquençage Illumina, et par la suite comparé aux résultats obtenus d'une colonie de 10 chats domestiques. Dans cette étude, le microbiote des deux espèces était dominé par les Firmicutes, suivi des Proteobacteria, Actinobacteria, Bacteroidetes et Verrucomicobia. Lorsque comparés, les échantillons fécaux provenant des lynx avaient plus de Proteobacteria et d'Actionbacteria que les échantillons fécaux des chats domestiques. Il y avait une variation inter-lynx marquée dans l'abondance relative des principaux genres bactériens. Toutefois, il n'y avait pas de différence significative entre les principales lignées du microbiote des lynx sauvages et domestiques. Les Proteobacteria chez les lynx sauvages (P = 0,079) et les Firmicutes chez les lynx gardés en zoo (P = 0,079) s'approchaient d'une différence significative. Il n'y avait pas de différence des genres prédominants entre les lynx sauvages et en captivité. Les résultats de cette étude ont montré qu'il y avait des différences notables dans les communautés bactériennes fécales entre les chats domestiques et les lynx sauvages et en captivité. L'absence de différence significative dans les communautés bactériennes entre les lynx sauvages et ceux en zoo suggère que la diète variée fournies à ces derniers félins peut résulter en un microbiote fécal qui ressemble à celui généré par une diète sauvage.(Traduit par Docteur Serge Messier).

Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing.

The ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of an in vitro diagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy.

Identification of individuals by trait prediction using whole-genome sequencing data.

Prediction of human physical traits and demographic information from genomic data challenges privacy and data deidentification in personalized medicine. To explore the current capabilities of phenotype-based genomic identification, we applied whole-genome sequencing, detailed phenotyping, and statistical modeling to predict biometric traits in a cohort of 1,061 participants of diverse ancestry. Individually, for a large fraction of the traits, their predictive accuracy beyond ancestry and demographic information is limited. However, we have developed a maximum entropy algorithm that integrates multiple predictions to determine which genomic samples and phenotype measurements originate from the same person. Using this algorithm, we have reidentified an average of >8 of 10 held-out individuals in an ethnically mixed cohort and an average of 5 of either 10 African Americans or 10 Europeans. This work challenges current conceptions of personal privacy and may have far-reaching ethical and legal implications.

A Novel System Control for Quality Control of Diagnostic Tests Based on Next-Generation Sequencing.

BACKGROUND: We describe a novel system control (SC) implemented in an automated AmpliSeq™-based next-generation sequencing (NGS)2 run that simultaneously acts as (a) an external positive/sensitivity control, (b) a spike-in QC for DNA extraction, and (c) a nontemplate control to detect exogenous DNA contamination. METHODS: Plasmids carrying wild-type tobacco mosaic virus sequence and a sequence with three designed mutations were synthesized and mixed, such that the mutations are present at 5% variant frequency in the mixture designated as SC. SC was used as a stand-alone sample and spiked into each sample in each run. A cell line-derived reference material, in both a formalin-fixed paraffin-embedded (FFPE) sample and genomic DNA (gDNA), was sequenced in the same runs. RESULTS: By interpolation, 100 fg SC spiked in FFPE sample produced sequencing coverage equivalent to approximately 3 fg in the gDNA. In the SC-only sample, all three designed mutations were recovered around 5% as expected, while no significant reads of human genome were present. In samples with a common PCR inhibitor, coverage for both SC and target amplicons were eliminated. An inverse relationship between the coverage of SC and DNA input was observed. In clinical samples, the ratio of SC to the median coverage of sample can be used to indicate insufficient DNA input. CONCLUSIONS: The SC is an elegant and comprehensive QC concept for NGS-based diagnostic tests.

Comparison of microarray-predicted closest genomes to sequencing for poliovirus vaccine strain similarity and influenza A phylogeny.

We evaluate sequence data from the PathChip high-density hybridization array for epidemiological interpretation of detected pathogens. For influenza A, we derive similar relative outbreak clustering in phylogenetic trees from PathChip-derived compared to classical Sanger-derived sequences. For a positive polio detection, recent infection could be excluded based on vaccine strain similarity.

The lipid peroxidation product 4-hydroxy-trans-2-nonenal causes protein synthesis in cardiac myocytes via activated mTORC1-p70S6K-RPS6 signaling.

Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 µM for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value.

Detection of chromosomal breakpoints in patients with developmental delay and speech disorders.

Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7-11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide (GNAQ), RNA-binding protein, fox-1 homolog (RBFOX3), unc-5 homolog D (C.elegans) (UNC5D), transmembrane protein 47 (TMEM47), and X-linked inhibitor of apoptosis (XIAP). Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).

Pathogen chip for respiratory tract infections.

Determining the viral etiology of respiratory tract infections (RTI) has been limited for the most part to specific primer PCR-based methods due to their increased sensitivity and specificity compared to other methods, such as tissue culture. However, specific primer approaches have limited the ability to fully understand the diversity of infecting pathogens. A pathogen chip system (PathChip), developed at the Genome Institute of Singapore (GIS), using a random-tagged PCR coupled to a chip with over 170,000 probes, has the potential to recognize all known human viral pathogens. We tested 290 nasal wash specimens from Filipino children <2 years of age with respiratory tract infections using culture and 3 PCR methods-EraGen, Luminex, and the GIS PathChip. The PathChip had good diagnostic accuracy, ranging from 85.9% (95% confidence interval [CI], 81.3 to 89.7%) for rhinovirus/enteroviruses to 98.6% (95% CI, 96.5 to 99.6%) for PIV 2, compared to the other methods and additionally identified a number of viruses not detected by these methods.

Post-translational modification of serine/threonine kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at lysine residue 97 directly inhibits kinase activity.

Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 µm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE.

CTCF-mediated functional chromatin interactome in pluripotent cells.

Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. However, little is known about CTCF-associated higher-order chromatin structures at a global scale. Here we applied chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, we identified 1,480 cis- and 336 trans-interacting loci with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive cross-talk between promoters and regulatory elements. This highly complex nuclear organization offers insights toward the unifying principles that govern genome plasticity and function.

Transcriptional consequences of genomic structural aberrations in breast cancer.

Using a long-span, paired-end deep sequencing strategy, we have comprehensively identified cancer genome rearrangements in eight breast cancer genomes. Herein, we show that 40%-54% of these structural genomic rearrangements result in different forms of fusion transcripts and that 44% are potentially translated. We find that single segmental tandem duplication spanning several genes is a major source of the fusion gene transcripts in both cell lines and primary tumors involving adjacent genes placed in the reverse-order position by the duplication event. Certain other structural mutations, however, tend to attenuate gene expression. From these candidate gene fusions, we have found a fusion transcript (RPS6KB1-VMP1) recurrently expressed in ∼30% of breast cancers associated with potential clinical consequences. This gene fusion is caused by tandem duplication on 17q23 and appears to be an indicator of local genomic instability altering the expression of oncogenic components such as MIR21 and RPS6KB1.

Multiplexed detection and differentiation of the DNA strains for influenza A (H1N1 2009) using a silicon-based microfluidic system.

Pandemic influenza by the swine-origin influenza virus (H1N1 2009) has attracted considerable concern worldwide. A convenient and accurate diagnostic approach that can be deployed at the point of care, such as in a doctor's office or at an airport, is critical for disease control. Here we report the development of a silicon-based microfluidic system for subtype differentiation of the novel H1N1 2009 strain vs. the seasonal influenza A (FluA) strain. The proposed system included two functional modules: a multiplexed PCR module for amplification of nucleic acid targets and a multiplexed silicon nanowire (SiNW) module for sequence determination. The PCR module consisted of a microfluidic PCR chamber and an electrical controller to perform a multiplexed protocol that simultaneously enriched specific segments of both H1N1 and FluA strains (if present), with 10(4)-10(5) amplification efficiency. The PCR amplicon was subsequently denatured and transferred to the SiNW sensing module for a label-free, multiplexed detection. A control SiNW was implemented, for the first time, in order to eliminate background interference. The detection module demonstrated a 10× change in the magnitude of differential current when the target DNA was injected. Overall, the system achieved a sensitivity of 20-30 fg/µl for H1N1 and seasonal FluA nucleic acids in a 10 µl sample. The low sample consumption, high sensitivity and high specificity render it a potential point-of-care (POC) platform to help doctors reach a yes/no decision for infectious diseases.

Oseltamivir ring prophylaxis for containment of 2009 H1N1 influenza outbreaks.

BACKGROUND: From June 22 through June 25, 2009, four outbreaks of infection with the pandemic influenza A (H1N1) virus occurred in Singapore military camps. We report the efficacy of ring chemoprophylaxis (geographically targeted containment by means of prophylaxis) with oseltamivir to control outbreaks of 2009 H1N1 influenza in semiclosed environments. METHODS: All personnel with suspected infection were tested and clinically isolated if infection was confirmed. In addition, we administered postexposure ring chemoprophylaxis with oseltamivir and segregated the affected military units to contain the spread of the virus. All personnel were screened three times weekly both for virologic infection, by means of nasopharyngeal swabs and reverse-transcriptase-polymerase-chain-reaction assay with sequencing, and for clinical symptoms, by means of questionnaires. RESULTS: A total of 1175 personnel were at risk across the four sites, with 1100 receiving oseltamivir prophylaxis. A total of 75 personnel (6.4%) were infected before the intervention, and 7 (0.6%) after the intervention. There was a significant reduction in the overall reproductive number (the number of new cases attributable to the index case), from 1.91 (95% credible interval, 1.50 to 2.36) before the intervention to 0.11 (95% credible interval, 0.05 to 0.20) after the intervention. Three of the four outbreaks showed a significant reduction in the rate of infection after the intervention. Molecular analysis revealed that all four outbreaks were derived from the New York lineage of the 2009 H1N1 virus and that cases within each outbreak were due to transmission rather than unrelated episodes of infection. Of the 816 personnel treated with oseltamivir who were surveyed, 63 (7.7%) reported mild, nonrespiratory side effects of the drug, with no severe adverse events. CONCLUSIONS: Oseltamivir ring chemoprophylaxis, together with prompt identification and isolation of infected personnel, was effective in reducing the impact of outbreaks of 2009 H1N1 influenza in semiclosed settings.

Deletion of the WD40 domain of LRRK2 in Zebrafish causes Parkinsonism-like loss of neurons and locomotive defect.

LRRK2 plays an important role in Parkinson's disease (PD), but its biological functions are largely unknown. Here, we cloned the homolog of human LRRK2, characterized its expression, and investigated its biological functions in zebrafish. The blockage of zebrafish LRRK2 (zLRRK2) protein by morpholinos caused embryonic lethality and severe developmental defects such as growth retardation and loss of neurons. In contrast, the deletion of the WD40 domain of zLRRK2 by morpholinos targeting splicing did not induce severe embryonic developmental defects; rather it caused Parkinsonism-like phenotypes, including loss of dopaminergic neurons in diencephalon and locomotion defects. These neurodegenerative and locomotion defects could be rescued by over-expressing zLRRK2 or hLRRK2 mRNA. The administration of L-dopa could also rescue the locomotion defects, but not the neurodegeneration. Taken together, our results demonstrate that zLRRK2 is an ortholog of hLRRK2 and that the deletion of WD40 domain of zLRRK2 provides a disease model for PD.

Large-scale evolutionary surveillance of the 2009 H1N1 influenza A virus using resequencing arrays.

In April 2009, a new influenza A (H1N1 2009) virus emerged that rapidly spread around the world. While current variants of this virus have caused widespread disease, particularly in vulnerable groups, there remains the possibility that future variants may cause increased virulence, drug resistance or vaccine escape. Early detection of these virus variants may offer the chance for increased containment and potentially prevention of the virus spread. We have developed and field-tested a resequencing kit that is capable of interrogating all eight segments of the 2009 influenza A(H1N1) virus genome and its variants, with added focus on critical regions such as drug-binding sites, structural components and mutation hotspots. The accompanying base-calling software (EvolSTAR) introduces novel methods that utilize neighbourhood hybridization intensity profiles and substitution bias of probes on the microarray for mutation confirmation and recovery of ambiguous base queries. Our results demonstrate that EvolSTAR is highly accurate and has a much improved call rate. The high throughput and short turn-around time from sample to sequence and analysis results (30 h for 24 samples) makes this kit an efficient large-scale evolutionary biosurveillance tool.

Deregulated direct targets of the hepatitis B virus (HBV) protein, HBx, identified through chromatin immunoprecipitation and expression microarray profiling.

The hepatitis B-X (HBx) protein is strongly associated with hepatocellular carcinoma. It is implicated not to directly cause cancer but to play a role in hepatocellular carcinoma as a co-factor. The oncogenic potential of HBx primarily lies in its interaction with transcriptional regulators resulting in aberrant gene expression and deregulated cellular pathways. Utilizing ultraviolet irradiation to simulate a tumor-initiating event, we integrated chip-based chromatin immunoprecipitation (ChIP-chip) with expression microarray profiling and identified 184 gene targets directly deregulated by HBx. One-hundred forty-four transcription factors interacting with HBx were computationally inferred. We experimentally validated that HBx interacts with some of the predicted transcription factors (pTF) as well as the promoters of the deregulated target genes of these pTFs. Significantly, we demonstrated that the pTF interacts with the promoters of the deregulated HBx target genes and that deregulation by HBx of these HBx target genes carrying the pTF consensus sequences can be reversed using pTF small interfering RNAs. The roles of these deregulated direct HBx target genes and their relevance in cancer was inferred via querying against biogroup/cancer-related microarray databases using web-based NextBio(TM) software. Six pathways, including the Jak-STAT pathway, were predicted to be significantly deregulated when HBx binds indirectly to direct target gene promoters. In conclusion, this study represents the first ever demonstration of the utilization of ChIP-chip to identify deregulated direct gene targets from indirect protein-DNA binding as well as transcriptional factors directly interacting with HBx. Increased knowledge of the gene/transcriptional factor targets of HBx will enhance our understanding of the role of HBx in hepatocellular carcinogenesis and facilitate the design of better strategies in combating hepatitis B virus-associated hepatocellular carcinoma.