The landscape of the methodology in drug repurposing using human genomic data: a systematic review.

The process of drug development is expensive and time-consuming. In contrast, drug repurposing can be introduced to clinical practice more quickly and at a reduced cost. Over the last decade, there has been a significant expansion of large biobanks that link genomic data to electronic health record data, public availability of various databases containing biological and clinical information and rapid development of novel methodologies and algorithms in integrating different sources of data. This review aims to provide a thorough summary of different strategies that utilize genomic data to seek drug-repositioning opportunities. We searched MEDLINE and EMBASE databases to identify eligible studies up until 1 May 2023, with a total of 102 studies finally included after two-step parallel screening. We summarized commonly used strategies for drug repurposing, including Mendelian randomization, multi-omic-based and network-based studies and illustrated each strategy with examples, as well as the data sources implemented. By leveraging existing knowledge and infrastructure to expedite the drug discovery process and reduce costs, drug repurposing potentially identifies new therapeutic uses for approved drugs in a more efficient and targeted manner. However, technical challenges when integrating different types of data and biased or incomplete understanding of drug interactions are important hindrances that cannot be disregarded in the pursuit of identifying novel therapeutic applications. This review offers an overview of drug repurposing methodologies, providing valuable insights and guiding future directions for advancing drug repurposing studies.

Integrated Metabolomic and transcriptomic analyses reveal deoxycholic acid promotes transmissible gastroenteritis virus infection by inhibiting phosphorylation of NF-κB and STAT3.

BACKGROUND: Acute diarrhea, dehydration and death in piglets are all symptoms of transmissible gastroenteritis virus (TGEV), which results in significant financial losses in the pig industry. It is important to understand the pathogenesis and identify new antiviral targets by revealing the metabolic interactions between TGEV and host cells. RESULTS: We performed metabolomic and transcriptomic analyses of swine testicular cells infected with TGEV. A total of 1339 differential metabolites and 206 differentially expressed genes were detected post TEGV infection. The differentially expressed genes were significantly enriched in the HIF-1 signaling pathway and PI3K-Akt signaling. Integrated analysis of differentially expressed genes and differential metabolites indicated that they were significantly enriched in the metabolic processes such as nucleotide metabolism, biosynthesis of cofactors and purine metabolism. In addition, the results showed that most of the detected metabolites involved in the bile secretion was downregulated during TGEV infection. Furthermore, exogenous addition of key metabolite deoxycholic acid (DCA) significantly enhanced TGEV replication by NF-κB and STAT3 signal pathways. CONCLUSIONS: We identified a significant metabolite, DCA, related to TGEV replication. It added TGEV replication in host cells by inhibiting phosphorylation of NF-κB and STAT3. This study provided novel insights into the metabolomic and transcriptomic alterations related to TGEV infection and revealed potential molecular and metabolic targets for the regulation of TGEV infection.

A fast and efficient approach for gene-based association studies of ordinal phenotypes.

Many human disease conditions need to be measured by ordinal phenotypes, so analysis of ordinal phenotypes is valuable in genome-wide association studies (GWAS). However, existing association methods for dichotomous or quantitative phenotypes are not appropriate to ordinal phenotypes. Therefore, based on an aggregated Cauchy association test, we propose a fast and efficient association method to test the association between genetic variants and an ordinal phenotype. To enrich association signals of rare variants, we first use the burden method to aggregate rare variants. Then we respectively test the significance of the aggregated rare variants and other common variants. Finally, the combination of transformed variant-level P values is taken as test statistic, that approximately follows Cauchy distribution under the null hypothesis. Extensive simulation studies and analysis of GAW19 show that our proposed method is powerful and computationally fast as a gene-based method. Especially, in the presence of an extremely low proportion of causal variants in a gene, our method has better performance.

Exploring the cross-cancer effect of smoking and its fingerprints in blood DNA methylation on multiple cancers: A Mendelian randomization study.

Aberrant smoking-related DNA methylation has been widely investigated as a carcinogenesis mechanism, but whether the cross-cancer epigenetic pathways exist remains unclear. We conducted two-sample Mendelian randomization (MR) analyses respectively on smoking behaviors (age of smoking initiation, smoking initiation, smoking cessation, and lifetime smoking index [LSI]) and smoking-related DNA methylation to investigate their effect on 15 site-specific cancers, based on a genome-wide association study (GWAS) of 1.2 million European individuals and an epigenome-WAS (EWAS) of 5907 blood samples of Europeans for smoking and 15 GWASs of European ancestry for multiple site-specific cancers. Significantly identified CpG sites were further used for colocalization analysis, and those with cross-cancer effect were validated by overlapping with tissue-specific eQTLs. In the genomic MR, smoking measurements of smoking initiation, smoking cessation and LSI were suggested to be casually associated with risk of seven types of site-specific cancers, among which cancers at lung, cervix and colorectum were provided with strong evidence. In the epigenetic MR, methylation at 75 CpG sites were reported to be significantly associated with increased risks of multiple cancers. Eight out of 75 CpG sites were observed with cross-cancer effect, among which cg06639488 (EFNA1), cg12101586 (CYP1A1) and cg14142171 (HLA-L) were validated by eQTLs at specific cancer sites, and cg07932199 (ATXN2) had strong evidence to be associated with cancers of lung (coefficient, 0.65, 95% confidence interval [CI], 0.31-1.00), colorectum (0.90 [0.61, 1.18]), breast (0.31 [0.20, 0.43]) and endometrium (0.98 [0.68, 1.27]). These findings highlight the potential practices targeting DNA methylation-involved cross-cancer pathways.

Dissecting the pathogenic effects of smoking and its hallmarks in blood DNA methylation on colorectal cancer risk.

BACKGROUND: Tobacco smoking is suggested as a risk factor for colorectal cancer (CRC), but the complex relationship and the potential pathway are not fully understood. METHODS: We performed two-sample Mendelian randomisation (MR) analyses with genetic instruments for smoking behaviours and related DNA methylation in blood and summary-level GWAS data of colorectal cancer to disentangle the relationship. Colocalization analyses and prospective gene-environment interaction analyses were also conducted as replication. RESULTS: Convincing evidence was identified for the pathogenic effect of smoking initiation on CRC risk and suggestive evidence was observed for the protective effect of smoking cessation in the univariable MR analyses. Multivariable MR analysis revealed that these associations were independent of other smoking phenotypes and alcohol drinking. Genetically predicted methylation at CpG site cg17823346 [ZMIZ1] were identified to decrease CRC risk; while genetically predicted methylation at cg02149899 would increase CRC risk. Colocalization and gene-environment interaction analyses added further evidence to the relationship between epigenetic modification at cg17823346 [ZMIZ1] as well as cg02149899 and CRC risk. DISCUSSION: Our study confirms the significant association between tobacco smoking, DNA methylation and CRC risk and yields a novel insight into the pathogenic effect of tobacco smoking on CRC risk.

An adaptive test based on principal components for detecting multiple phenotype associations using GWAS summary data.

Extensive evidence from genome-wide association studies (GWAS) has shown that jointly analyzing multiple phenotypes can improve the power of the association test compared to the traditional single variant versus single trait approach. Here we propose an adaptive test based on principal components (ATPC) that is powerful and efficient for discovering the association between a single variant and multiple traits. Our method only needs GWAS summary statistics that are often available. We first estimate the trait correlation matrix by LD score regression. Then, based on the correlation matrix, we construct a series of test statistics that contain different numbers of principal components. The ultimate test statistic combines the P values of these principal component-based statistics by using the aggregated Cauchy association test. The analytical P-value of the test statistic can be computed quickly without the permutation process, which is the notable feature of our proposed method. The extensive simulation studies demonstrate that ATPC can control the type I error rates and have powerful and robust performance compared to several existing tests in a wide range of simulation settings. The analysis of the lipids GWAS summary data from the Global Lipids Genetics Consortium shows that ATPC identifies 230 new SNPs that are missed by the original single trait association analysis. By searching the GWAS Catalog, some SNPs and mapped genes identified by ATPC are reported to be associated with lipid traits. Through further analysis for GWAS results, we also find some Gene Ontology terms and biological pathways related to lipids.

Injectable Black Phosphorus Nanosheets for Wireless Nongenetic Neural Stimulation.

Neurons can be modified to express light-sensitive proteins for enabling stimulation with a high spatial and temporal resolution, but such techniques require gene transfection and systematical implantation. Here, a black phosphorus nanosheet-based injectable strategy is described for wireless neural stimulation both in vitro and in vivo without cell modifications. These nanosheets, with minimal invasiveness, high biocompatibility, and biodegradability, are anchored on cell membranes as miniature near-infrared (NIR) light transducers to create local heating for neural activity excitation. Based on cultured multielectrode-array recording, in vivo electrophysiology analysis, and open field behavioral tests, it is demonstrated that remotely applied NIR illumination can reliably trigger spiking activity in cultured neurons and rat brains. Excitingly, reliable regulation of brain function to control animal behaviors is also described. Moreover, this approach has shown its potential for future clinical use by successful high-frequency stimulation in cells and animals in this proof-of-concept study. It is believed that this new method will offer a powerful alternative to other neural stimulation solutions and potentially be of independent value to the healthcare system.

Alcohol consumption, blood DNA methylation and breast cancer: a Mendelian randomisation study.

Alcohol intake is thought to be a risk factor for breast cancer, but the causal relationship and carcinogenic mechanisms are not clear. We performed an up-to-date meta-analysis of prospective studies to assess observational association, and then conducted MR analysis to make causal inference based on the genetic predisposition to alcohol consumption ("drinks per week") and pathological drinking behaviours ("alcohol use disorder" and "problematic alcohol use"), as well as genetically predicted DNA methylation at by alcohol-related CpG sites in blood. We found an observational dose-response association between alcohol intake and breast cancer incidence with an additional risk of 4% for per 10 g/day increase in alcohol consumption. Genetic predisposition to alcohol consumption ("drinks per week") was not causally associated with breast cancer incidence at the OR of 1.01 (95% CI 0.84, 1.23), but problematic alcohol use (PAU) was linked to a higher breast cancer risk at the OR of 1.76 (95% CI 1.04, 2.99) when conditioning on alcohol consumption. Epigenetic MR analysis identified four CpG sites, cg03260624 near CDC7 gene, cg10816169 near ZNF318 gene, cg03345232 near RIN3 gene, and cg26312998 near RP11-867G23.13 gene, where genetically predicted epigenetic modifications were associated with an increased breast cancer incidence risk. Our findings re-affirmed that alcohol consumption is of high risk for breast cancer incidence even at a very low dose, and the pathogenic effect of alcohol on breast cancer could be due to pathological drinking behaviour and epigenetic modification at several CpG sites, which could be potential intervention targets for breast cancer prevention.

Kinesin family member 2C promotes hepatocellular carcinoma growth and metastasis via activating MEK/ERK pathway.

The current work was intended to explore the function and mechanism of Kinesin family member 2C (KIF2C) in hepatocellular carcinoma (HCC). In this study, KIF2C expression was at a high level in HCC and indicated poor prognosis. Silencing KIF2C significantly suppressed the proliferation, migration, and invasion in HCC cells. Furthermore, silencing KIF2C markedly decreased the expression of Snail, Vimentin, p-MEK, and p-ERK, but increased E-cadherin expression in HCC cells. Moreover, we also found that MEK/ERK inhibitor U0126 could enhance the impact on cell proliferation, migration, and invasion induced by silencing KIF2C in HCC. On the contrary, MEK/ERK activator PAF could weaken the impact induced by silencing KIF2C in HCC. Thus, our findings indicate that KIF2C can promote the proliferation, migration, and invasion by activating MEK/ERK pathway in HCC.

Ultrasound Surveillance of Abnormal Cervical Lymph Nodes in Patients With Papillary Thyroid Carcinoma After Surgery.

OBJECTIVES: To document the changing trends of abnormal cervical lymph nodes (LNs) in patients with papillary thyroid carcinoma (PTC) who had serial follow-up ultrasound (US) scans after surgery and to determine how these node abnormalities progress in real time. METHODS: Ultrasound findings from 568 consecutive patients with PTC who were monitored postoperatively were reviewed. Abnormal LNs were classified as either suspicious or indeterminate according to the European Thyroid Association guidelines. Outcomes from US monitoring of the LNs were recorded and analyzed. RESULTS: Seventy-six (13.4%) of 568 patients were identified with abnormal LNs. Among them, 55 (72.4%) were initially found to have suspicious LNs, and the other 21 (27.6%) had indeterminate lesions. Of the 55 suspicious LNs, final scans showed that 38 (69.1%) lesions were still suspicious, whereas the remaining 17 (30.9%) nodes were shown to have resolved after a median follow-up of 36 months. Of the 21 indeterminate node abnormalities, final scans showed that 16 (76.2%) LNs remained indeterminate, whereas the other 5 (23.8%) nodes had developed into suspicious LNs after a median follow-up of 44 months. Loss of the fatty hilum and peripheral or diffusedly increased vascularity were more likely to be linked to persistent suspicious LNs (P = .02 and .04, respectively). Suspicious LNs with echogenic foci but a lack of other abnormal features were more frequently found to have resolved thereafter (P = .03). CONCLUSIONS: Abnormal LNs detected after PTC surgery can often remain indolent during US surveillance, and a small portion of the nodes would have resolved over time.

Bayesian interval mapping of count trait loci based on zero-inflated generalized Poisson regression model.

Count phenotypes with excessive zeros are often observed in the biological world. Researchers have studied many statistical methods for mapping the quantitative trait loci (QTLs) of zero-inflated count phenotypes. However, most of the existing methods consist of finding the approximate positions of the QTLs on the chromosome by genome-wide scanning. Additionally, most of the existing methods use the EM algorithm for parameter estimation. In this paper, we propose a Bayesian interval mapping scheme of QTLs for zero-inflated count data. The method takes advantage of a zero-inflated generalized Poisson (ZIGP) regression model to study the influence of QTLs on the zero-inflated count phenotype. The MCMC algorithm is used to estimate the effects and position parameters of QTLs. We use the Haldane map function to realize the conversion between recombination rate and map distance. Monte Carlo simulations are conducted to test the applicability and advantage of the proposed method. The effects of QTLs on the formation of mouse cholesterol gallstones were demonstrated by analyzing an F2 mouse data set.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Association analysis of rare and common variants with multiple traits based on variable reduction method.

Pleiotropy, the effect of one variant on multiple traits, is widespread in complex diseases. Joint analysis of multiple traits can improve statistical power to detect genetic variants and uncover the underlying genetic mechanism. Currently, a large number of existing methods target one common variant or only rare variants. Increasing evidence shows that complex diseases are caused by common and rare variants. Here we propose a region-based method to test both rare and common variant associated multiple traits based on variable reduction method (abbreviated as MULVR). However, in the presence of noise traits, the MULVR method may lose power, so we propose the MULVR-O method, which jointly analyses the optimal number of traits associated with genetic variants by the MULVR method, to guard against the effect of noise traits. Extensive simulation studies show that our proposed method (MULVR-O) is applied to not only multiple quantitative traits but also qualitative traits, and is more powerful than several other comparison methods in most scenarios. An application to the two genes (SHBG and CHRM3) and two phenotypes (systolic blood pressure and diastolic blood pressure) from the GAW19 dataset illustrates that our proposed methods (MULVR and MULVR-O) are feasible and efficient as a region-based method.

A characterization of structural proteins expressed by Bombyx mori bidensovirus.

Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5kb (viral DNA 1, VD1) and 6.0kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22-24nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism.

Quantitative Analysis of Hepatic Toxicity in Rats Induced by Inhalable Silica Nanoparticles Using Acoustic Radiation Force Imaging.

OBJECTIVES: The purposes of this study were to verify whether inhalable silicon dioxide (SiO2 ) nanoparticles could induce hepatic injury and to investigate the relationship between the exposure time and SiO2 nanoparticle dosage by using acoustic radiation force impulse imaging (ARFI). METHODS: A total of 72 rats were randomly separated into 9 groups with 8 in each: blank control group, 0.9% normal saline group, polyacrylate (PPE) group, 25%, 50%, and 100% SiO2 groups, and 25%, 50%, and 100% SiO2 /PPE groups with inhaled SiO2 nanoparticle concentrations similar to the SiO2 groups. After successful modeling and design, the hepatic shear wave velocity (SWV) values of the 9 groups were obtained on days 3, 7, 14, 21, and 28 by using ARFI, and the intragroup and intergroups differences in the SWVs were compared. The serum alanine aminotransferase (ALT) and aspartate aminotransferase were tested and compared on day 28. Hepatic tissues were collected for histologic observation on day 28. RESULTS: The pathologic results verified that inhalable SiO2 nanoparticles could induce hepatic injury. Compared with the control group, the hepatic SWV and serum ALT values in the SiO2 groups and SiO2 /PPE groups were elevated (P < .05). The dosage and exposure time of SiO2 played a key role in the elevation of the SWV in the SiO2 and SiO2 /PPE groups. The correlation between the ALT level and SWV was significant on day 28 (P < .05). CONCLUSIONS: Inhalable SiO2 nanoparticles and SiO2 /PPE were able to induce hepatic injury in rats. Using ARFI to evaluate hepatic toxicity induced by SiO2 nanoparticles was effective in this study.

Clinical application of rapid B-line score with lung ultrasonography in differentiating between pulmonary infection and pulmonary infection with acute left ventricular heart failure.

BACKGROUND: We have found that there are usually 2 causes of acute dyspnea in our emergency department: (1) pulmonary infection only and (2) pulmonary infection in the setting of acute left ventricular heart failure (LVHF). These conditions are sometimes difficult to differentiate. Lung ultrasonography (LUS) is easily performed at the bedside and provides accurate information for diagnosis. In this study, we propose a simple B-line score to allow rapid differential diagnosis between these 2 lung conditions. METHODS: A prospective, single-blind trial was conducted on 98 patients with acute dyspnea in the emergency department. Lung ultrasonography and transthoracic echocardiography were performed within 30 minutes after enrollment. The final clinical diagnosis was recorded for all patients. Using the Bedside Lung Ultrasound in Emergency protocol, we recorded the number of B lines at 4 standardized points. Based on the theory of Lichtenstein, scores of 1, 2, 3, and 4 were categorized by the number of B lines on a static screen (0 to <3, 3 to <6, 6 to <8, and ≥8, respectively). The B-line score of 4 Bedside Lung Ultrasound in Emergency protocol points was recorded, and the total B-line score was calculated. Receiver operating characteristic (ROC) curves were used to evaluate the accuracy of the rapid ultrasound measurements for the final clinical diagnosis. RESULTS: In our study, 27 patients were diagnosed with pulmonary infection and acute LVHF. The total number of B lines and the B-line score in patients with pulmonary infection in the setting of acute LVHF were 24.2±2.5 and 11.5±1.5, respectively, which were significantly higher than those in patients with pulmonary infection (12.5±6.4 and 7.2±1.9) (P=.000). In patients with pulmonary infection and acute LVHF, the effective diagnostic value of left ventricular ejection fraction and the total B-line score were similar (area under the ROC curve: 0.986 vs 0.962, P=.2607). The cutoff value of the total B-line score was 8, with a sensitivity of 80.7% and a specificity of 100%. A combination of LUS and echocardiography might improve the diagnostic accuracy (area under the ROC curve: 0.994; 95% confidence interval, 0.981-1.000; P=.000). CONCLUSIONS: This simple B-line score with LUS can help make a rapid differential diagnosis between pulmonary infection and pulmonary infection with acute LVHF. The diagnostic accuracy may be enhanced when used in conjunction with echocardiography.

Detecting association of rare and common variants by adaptive combination of P-values.

Genome-wide association studies (GWAS) can detect common variants associated with diseases. Next generation sequencing technology has made it possible to detect rare variants. Most of association tests, including burden tests and nonburden tests, mainly target rare variants by upweighting rare variant effects and downweighting common variant effects. But there is increasing evidence that complex diseases are caused by both common and rare variants. In this paper, we extend the ADA method (adaptive combination of P-values; Lin et al., 2014) for rare variants only and propose a RC-ADA method (common and rare variants by adaptive combination of P-values). Our proposed method combines the per-site P-values with the weights based on minor allele frequencies (MAFs). The RC-ADA is robust to directions of effects of causal variants and inclusion of a high proportion of neutral variants. The performance of the RC-ADA method is compared with several other association methods. Extensive simulation studies show that the RC-ADA method is more powerful than other association methods over a wide range of models.

Characteristics of clinical studies of summer acupoint herbal patching: a bibliometric analysis.

BACKGROUND: Summer acupoint herbal patching (SAHP) has been widely used in China for thousands of years. This bibliometric analysis aims to provide a comprehensive review of the characteristics of clinical studies on SAHP for any condition. METHODS: We included clinical studies such as randomized clinical trials (RCTs), controlled clinical studies (CCTs), case series (CSs), case reports (CRs), and cross-sectional studies on SAHP for any condition. Six databases were searched from date of inception to March 2015. Bibliometric information and study details such as study type, characteristics of participants, details of the intervention and comparison, and outcome were extracted and analyzed. RESULTS: A total of 937 clinical studies were identified and which were published between 1977 and 2015. This included 404 RCTs, 52 CCTs, 458 CSs, 19 CRs and 4 cross-sectional studies and involved 232,138 participants aged 2 to 90 years from two countries. Almost all studies were from China (936, 99.89%). The five conditions most commonly treated by SAHP were asthma (401, 42.80%), chronic bronchitis (146, 15.58%), allergic rhinitis (117, 12.49%), chronic obstructive pulmonary disease (73, 7.79%), and recurrent respiratory tract infection (42, 4.48%). Among 502 controlled studies, the majority compared SAHP alone with different controls (16 categories, 275 comparisons). The most commonly used controls were western medicine, placebo, traditional Chinese medicine, no treatment and non-pharmaceutical traditional Chinese therapies. Composite outcome measures were the most frequently reported outcome (512, 69.19%). CONCLUSION: A substantial amount of research on SAHP has been published in China and which predominantly focuses on respiratory conditions. The findings from this study can be used to inform further research by highlighting areas of greatest impact for SAHP.

Identification of RNF8 as a ubiquitin ligase involved in targeting the p12 subunit of DNA polymerase δ for degradation in response to DNA damage.

DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin-conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA damage response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage-induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8(-/-) mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage-induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways.

Cloning and immune characterization of the c-type lysozyme gene in red-spotted grouper, Epinephelus akaara.

Lysozyme is an important component of the innate immune response against pathogen infection. The gene coding for c-type lysozyme in red-spotted grouper Epinephelus akaara was cloned and designated EaClys. The complete cDNA contains a 432 bp open reading frame encoding a protein of 144 amino acids displaying 65-91% similarity with the amino acid sequences of human, mouse, chicken, and fish counterparts. Recombinant EaClys (rEaClys) was expressed in Escherichia coli, displayed antibacterial activity against Gram-positive and Gram-negative bacteria, and possessed bactericidal activity against Vibrio alginolyticus. EaClys mRNA was constitutively expressed in all tested E. akaara tissues, and its expression increased after pathogen challenge. Most notably, challenges with LPS, SGIV or V. alginolyticus upregulated EaClys mRNA expression in the head, kidney, and blood. Its expression peaked between 16 and 24 h after challenge before dropping back to the baseline level. By using recombinant cytokines as signaling pathway mimetics and blocking antibodies and chemical inhibitors as pathway inhibitors, we show that LPS-induced lysozyme release from macrophages is promoted by cytokines TNF-α and IL-1ß, and dependent on NF-κB pathway activation. These data suggest that EaClys is a constitutive and inducible acute-phase protein that is involved in the innate immune defense of E. akaara, and provide new clues about the molecular mechanisms that regulate innate immune responses in fish.