HBV DNA Integration into Telomerase or MLL4 Genes and TERT Promoter Point Mutation as Three Independent Signatures in Subgrouping HBV-Related HCC with Distinct Features.

Introduction: A set of genetic mutations to classify hepatocellular carcinoma (HCC) useful to clinical studies is an unmet need. Hepatitis B virus-related HCC (HBV-HCC) harbors a unique genetic mutation, namely, the HBV integration, among other somatic endogenous gene mutations. We explored a combination of HBV DNA integrations and common somatic mutations to classify HBV-HCC by using a capture-sequencing platform. Methods: A total of 153 HBV-HCCs after surgical resection were subjected to capture sequencing to identify HBV integrations and three common somatic mutations in genomes. Three mutually exclusive mutations, HBV DNA integration into the TERT promoter, HBV DNA integration into MLL4, or TERT promoter point mutation, were identified in HBV-HCC. Results: They were used to classify HBV-HCCs into four groups: G1 with HBV-TERT integration (25.5%); G2 with HBV-MLL4 integration (10.5%); G3 with TERT promoter mutation (30.1%); and G4 without these three mutations (34.0%). Clinically, G3 has the highest male-to-female ratio, cirrhosis rate, and associated with higher early recurrence and mortality after resection, but G4 has the best outcome. Transcriptomic analysis revealed a grouping different from the published ones and G2 with an active immune profile related to immune checkpoint inhibitor response. Analysis of integrated HBV DNA provided clues for HBV genotype and variants in carcinogenesis of different HCC subgroup. This new classification was also validated in another independent cohort. Conclusion: A simple and robust genetic classification was developed to aid in understanding HBV-HCC and in harmonizing clinical studies.

Hepatitis B Virus DNA Integration Drives Carcinogenesis and Provides a New Biomarker for HBV-related HCC.

Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection. However, HBV DNA is present in the genome of approximately 90% of HBV-related HCCs and is the most common somatic mutation. Whole genome sequencing of liver tissues from chronic hepatitis B patients showed integration occurring at random positions in human chromosomes; however, in the genomes of HBV-related HCC patients, there are integration hotspots. Both the enrichment of the HBV-integration proportion in HCC and the emergence of integration hotspots suggested a strong positive selection of HBV-integrated hepatocytes to progress to HCC. The activation of HBV integration hotspot genes, such as telomerase (TERT) or histone methyltransferase (MLL4/KMT2B), resembles insertional mutagenesis by oncogenic animal retroviruses. These candidate oncogenic genes might shed new light on HBV-related HCC biology and become targets for new cancer therapies. Finally, the HBV integrations in individual HCC contain unique sequences at the junctions, such as virus-host chimera DNA (vh-DNA) presumably being a signature molecule for individual HCC. HBV integration may thus provide a new cell-free tumor DNA biomarker to monitor residual HCC after curative therapies or to track the development of de novo HCC.

Analysis of the relationship between lifestyle habits and glycosylated hemoglobin control based on data from a Health Management Plan.

BACKGROUND/OBJECTIVES: Type 2 Diabetes mellitus (T2DM) is a hereditary disease that is also strongly dependent on environmental factors, lifestyles, and dietary habits. This study explored the relationship between lifestyle habits and glycosylated hemoglobin management in T2DM patients to provide empirical outcomes to improve T2DM management and patient health literacy. SUBJECTS/METHODS: This study enrolled 349 diabetic patients with more than 5 care visits to a Diabetes Mellitus care network under the Health Management Plan led by Taiwan Department of Health (DOH). Based on relevant literature, an Outpatient Record Form of Diabetes Mellitus Care was designed and lipid profile tests were conducted for data collection and analysis. RESULTS: When modeling the data, the results showed that the odds for HbA1c > 7.5% in T2DM patients duration over 10 years was 3.785 (P = 0.002) times that in patients with disease duration of fewer than 3 years. The odds of HbA1c > 7.5% in illiterate patients was 3.128 (P = 0.039) times that in patients with senior high school education or above. The odds of HbA1c > 7.5% in patients with other chronic illness was 2.207 (P = 0.019) times that in participants without chronic illness. Among 5 beneficial lifestyle habits, the odds of HbA1c > 7.5% in patients with 2 or 3 good habits were 3.243 (P = 0.003) and 3.424 (P = 0.001) times that in patients with more than 3 good habits, respectively. CONCLUSION: This empirical outcome shows that maintaining a good lifestyle improves T2DM management and patients' knowledge, motivation, and ability to use health information. Patients with longer disease duration, education, or good lifestyle habits had optimal HbA1c management than those in patients who did not. Thus, effective self-management and precaution in daily life and improved health literacy of diabetic patients are necessary to increase the quality of T2DM care.

The evaluation of genomic homozygosity for Xinjiang inbred population by SNP panels.

Xinjiang inbred cattle is a population which has been highly inbred for 45 years. However, the breed origin of this population cannot be traced back due to the lack of original records. To demonstrate the genetic background of Xinjiang inbred cattle, we analysed the worldwide genomic information of 16 cattle breeds using principal components analysis, and Admixture method. Furthermore, the shared SNP markers of Xinjiang inbred cattle, local Kazakh cattle, Holstein cattle, and Xinjiang Brown cattle were extracted to calculate population genetic parameters and genomic inbreeding indicators in order to evaluate the magnitude of inbreeding in each population. We also evaluated the relationship between inbreeding indicators and body size in the Xinjiang inbred population. Finally, the high frequency runs of homozygosity (ROH) regions for Xinjiang inbred cattle and local Kazakh population were selected for genes and QTL annotations. These results demonstrate that the ancestry proportions of inbreeding breed are similar to those of Kazakh cattle. The genomic homozygosity of Xinjiang inbred cattle is significantly higher than other populations; the inbreeding depression is observed in body size to a certain extent because body size decreased when corresponding homozygosity increased. Totally, six basic bio-pathways and 32 QTL regions that related to bovine economical traits were annotated. Our results provide the insights into breeding strategies, future protection, and utilization plan design for this special genetic material-Xinjiang inbred cattle.

Revisiting the Regenerative Therapeutic Advances Towards Erectile Dysfunction.

Erectile dysfunction (ED) is an inability to attain or maintain adequate penile erection for successful vaginal intercourse, leading to sexual and relationship dissatisfaction. To combat ED, various surgical and non-surgical approaches have been developed in the past to restore erectile functions. These therapeutic interventions exhibit significant impact in providing relief to patients; however, due to their associated adverse effects and lack of long-term efficacy, newer modalities such as regenerative therapeutics have gained attention due to their safe and prolonged efficacy. Stem cells and platelet-derived biomaterials contained in platelet-rich plasma (PRP) are thriving as some of the major therapeutic regenerative agents. In recent years, various preclinical and clinical studies have evaluated the individual, as well as combined of stem cells and PRP to restore erectile function. Being rich in growth factors, chemokines, and angiogenic factors, both stem cells and PRP play a crucial role in regenerating nerve cells, myelination of axons, homing and migration of progenitor cells, and anti-fibrosis and anti-apoptosis of damaged cavernous nerve in corporal tissues. Further, platelet-derived biomaterials have been proven to be a biological supplement for enhancing the proliferative and differentiation potential of stem cells towards neurogenic fate. Therefore, this article comprehensively analyzes the progresses of these regenerative therapies for ED.

Cell-Free Virus-Host Chimera DNA From Hepatitis B Virus Integration Sites as a Circulating Biomarker of Hepatocellular Cancer.

BACKGROUND AND AIMS: Early recurrence of hepatocellular carcinoma (HCC) after surgical resection compromises patient survival. Timely detection of HCC recurrence and its clonality is required to implement salvage therapies appropriately. This study examined the feasibility of virus-host chimera DNA (vh-DNA), generated from junctions of hepatitis B virus (HBV) integration in the HCC chromosome, as a circulating biomarker for this clinical setting. APPROACH AND RESULTS: HBV integration in 50 patients with HBV-related HCC was determined by the Hybridization capture-based next-generation sequencing (NGS) platform. For individual HCC, the vh-DNA was quantified by specific droplet digital PCR (ddPCR) assay in plasma samples collected before and 2 months after surgery. HBV integrations were identified in 44 out of 50 patients with HBV-related HCC. Tumor-specific ddPCR was developed to measure the corresponding vh-DNA copy number in baseline plasma from each patient immediately before surgery. vh-DNA was detected in 43 patients (97.7%), and the levels correlated with the tumor sizes (detection limit at 1.5 cm). Among the plasma collected at 2 months after surgery, 10 cases (23.3%) still contained the same signature vh-DNA detected at baseline, indicating the presence of residual tumor cells. Nine of them (90%) experienced HCC recurrence within 1 year, supporting vh-DNA as an independent risk factor in predicting early recurrence. Analysis of circulating vh-DNA at recurrence further helped identify the clonal origin. A total of 81.8% of recurrences came from original HCC clones sharing the same plasma vh-DNA, whereas 18.2% were from de novo HCC. CONCLUSIONS: vh-DNA was shown to be a circulating biomarker for detecting the tumor load in majority of patients with HBV-related HCC and aided in monitoring residual tumor and recurrence clonality after tumor resection.

Melatonin protects against paraquat-induced damage during in vitro maturation of bovine oocytes.

Paraquat (PQ), a broad-spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ-induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro-produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic-related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ-exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ-induced p38 mitogen-activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl-histone H3 lysine 4 (H3K4me3) and trimethyl-histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ-induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning.

Estimation of genomic inbreeding coefficients based on high-density SNP markers in Chinese Holstein cattle.

In livestock, inbreeding coefficient based on pedigree information is usually used to evaluate the level of inbreeding. Recently, with cost reduction of high-density SNP genotyping, it's possible to analyze real genomic inbreeding degree using genomic information. In this study, utilizing high-density SNP chip data, we analyzed the frequency and distribution of runs of homozygosity (ROH) in 2107 Chinese Holstein cattle in Beijing area, and calculated 2 genomic inbreeding coefficients, i.e., 1) the proportion of ROH length in the total length of autosomal genome (Froh), and 2) the percentage of homozygous SNPs (Fhom). Then we analyzed the correlation between 2 genomic inbreeding coefficients and the correlation between genomic and pedigree inbreeding coefficients. We totally detected 44 676 ROHs that mainly ranged from 1 to 10 Mb. Various lengths of ROHs existed in the genome. There were more short ROHs than long ROHs. ROHs aren't evenly distributed in chromosomes. The area with most ROHs is in the middle part of chromosome 10. Strong correlation (r > 0.90) existed between 2 kinds of genomic inbreeding coefficients, but the correlation between pedigree and genomic inbreeding coefficients were much lower (r < 0.50). Our finding suggests that pedigree completeness influences the correlation between genomic and pedigree inbreeding. Genomic inbreeding measures may reflect individuals' real inbreeding, which could be a useful tool to evaluate population inbreeding.

[Establishment of the detection method for two causative genes of cattle arachnomelia syndrome].

Arachnomelia syndrome (AS) is a recessive inherited disease in cattle. Although the arachnomelia phenotypes are virtually identical in Brown Swiss and Simmental cattle, the causative mutation are different, which are a 1 bp insertion c.363-364insG in the sulfite oxidase (SUOX) gene and a 2 bp deletion c.1224_1225delCA in the molybdenum cofactor syn-thesis step 1 (MOCS1) gene, respectively. In the current study, combining fluorescence PCR with capillary electrophoresis technology, an automatic fluorescence method was established, which could detect the two causative loci rapidly and cor-rectly with a single reaction. Samples from 51 Simmental bulls, 80 cows mated artificially using semen of Simmental bulls and their resulted 106 progeny, together with 55 Xinjiang Brown were collected and used for validation of the newly de-signed methods. Our results have laid a foundation for screening AS disease causing mutations in Chinese cattle.

[Factors analysis and genetic parameter estimation of female reproductive traits in pigs].

Reproductive performance of stock sows is one of the important factors of economic impact in pig farms. In this study, 8491 litter records from 2699 sows of Yorkshire, Landrace, and Duroc were analyzed using fixed model to determine the effect of parity, mating season, and breed on total number born (TNB), number healthy birth (NHB), litter birth weight (LWB), number weak birth (NWB), stillbirth, mummy fetus, and deform fetus by the least square analysis. Genetic parameters of the above traits were estimated by restricted maximum likelihood (REML) procedure. In addition, the effectiveness of pure-breeding and cross-breeding on litter performance were compared. The results showed that, parity, mating season, and breed had significant effect on TNB, NHB, and LWB(P < 0.001).The effects of parity and breed were significant on NWB(P < 0.001), while mating season had non-significant effect on NWB. Parity showed significant effect on stillbirth, while the effect of mating season and breed was not significant. Parity, mating season, and breed had no significant effect on mummy fetus and deform fetus. Landrace♂×Large White♀ showed the best litter performance, including TNB, NHB, and LWB. Moreover, LWB of Landrace depicted the highest heritability, while other traits were all bellow 0.2. The genetic correlation between TNB and NHB, NHB and LWB were higher than 0.96 in the three breeds. These results provided reference data for minimizing low-reproductive performance caused by non-infectious factors and improving sow reproductive performance in pig farms.

[Analysis of DNA methylation in different chicken tissues with MSAP].

With methylation sensitive amplified polymorphism (MSAP), the DNA methylation levels and patterns of CCGG sites in genomes was analyzed among four different tissues and between parents and offsprings from three groups of adult chicken, White Leghorn, White Plymouth Rock, and their F1 hybrids. The results indicated that the degree of methylation was approximate 29.7% in muscle, 27.5% in liver, 27.5% in heart, and 26.1% in kidney. There was significantly different in the level of methylation in the 3 different groups and in 4 different tissues (P<0.05). The fully-methylated sites were less than the hemi-methylated sites among the 4 tissues, which was different from that of plants. The two tissue-specific MSAP fragments were isolated, sequenced, and characterized, both of which were located in the coding regions. These results clearly demonstrated that there was difference in the methylation level among various tissues and different groups, which suggested that the genetic factor may have effect on the individual methylation level.

[Arachnomelia syndrome: a skeletal system malformation inherited disorder].

Arachnomelia syndrome (AS) is a lethal congenital malformation of skeleton in cattle, which proved to be an autosomal recessive inherited defect. This disease was mainly observed in European Brown Swiss and German Fleckvieh populations. This review focused on the discovery history, pathologic characteristics, mode of inheritance, and progresses on molecular mechanism of AS in both European Brown Swiss and German Fleckvieh populations. Moreover, through analyzing candidate genes in the mapping region related to bone development and using the methods of comparative genomics, this paper provides a starting point of identifying the causal gene(s) of AS and establishing detection method of the mutations.

[Identification of the complex vertebral malformation gene in Chinese Holstein and its association with dairy performance traits].

Complex vertebral malformation is caused by a single base mutation from G to T at the nucleotide position 559 in the bovine solute carrier family 35 member 3 (SLC35A3) gene exon 4 on Bos Taurus autosome (BTA) 3. The presence of the disease gene in Holstein dairy cattle has been reported in many countries. In this study, we examined 38 top Chinese Holstein sires in Beijing, four of which were identified as CVM carriers. Furthermore, 555 daughters of the four CV sires were examined and 44.0% of them were identified as heterozygotes at the mutation site. The association analysis between estimated breeding values (EBV) of dairy performance traits and the polymorphism showed that there were extremely significant differences between the carriers and the non-carriers (P<0.01). The EBVs of the five milk production traits of CVM carriers were significantly higher than those of non-carriers, and the lactation persistency and somatic cell score (SCS) were also higher in CVM carriers. Therefore, CVM gene seems to link with a QTL or gene associated with milk production traits on BTA3. It is recommended to cull the CVM carriers gradually for economical and breeding reasons.

The expression of osteoclastogenesis-associated factors and osteoblast response to osteolytic prostate cancer cells.

BACKGROUND: Prostate cancer (PCa) has a propensity to metastasize to bone. Tumor cells replace bone marrow and can elicit an osteoblastic, osteolytic, or mixed bone response. Our objective was to elucidate the mechanisms and key factors involved in promoting osteoclastogenesis in PCa bone metastasis. METHODS: We cultured osteoblast-like MC3T3-E1 cells with conditioned medium (CM) from PC-3 and C4-2B cells. MC3T3-E1 mineralization decreased in the presence of PC-3 CM, whereas C4-2B CM had no effect on mineralization. Using oligo arrays and validating by real-time PCR, we observed a decrease in the expression of mineralization-associated genes in MC3T3-E1 cells grown in the presence of PC-3 CM. In addition, PC-3 CM induced the expression of osteoclastogenesis-associated genes IGFBP-5, IL-6, MCP-1, and RANKL while decreasing OPG expression in MC3T3-E1 cells. Furthermore, CM from MC3T3-E1 cells cultured in the presence of PC-3 CM, in association with soluble RANKL, increased osteoclastogenesis in RAW 264.7 cells. Investigation of PCa metastases and xenografts by immunohistochemistry revealed that the osteoclastic factor IL-6 was expressed in the majority of PCa bone metastases and to a lesser extent in PCa soft tissue metastases. In vitro it was determined that soluble IL-6R (sIL-6R) was necessary for IL-6 to inhibit mineralization in MC3T3-E1 cells. RESULTS: PC-3 cells inhibit osteoblast activity and induce osteoblasts to produce osteoclastic factors that promote osteoclastogenesis, and one of these factors, IL-6, is highly expressed in PCa bone metastases. CONCLUSIONS: IL-6 may have an important role in promoting osteoclastogenesis in PCa bone metastasis through its' interaction with sIL-6R.

Differential expression of angiogenesis associated genes in prostate cancer bone, liver and lymph node metastases.

Our objective was to elucidate phenotypic differences between prostate cancer (PCa) liver, lymph node, and bone metastases. PCa metastases were obtained through a rapid tissue acquisition necropsy protocol. We grossly dissected metastatic foci from frozen samples and performed expression analyses using cDNA microarrays. Immunohistochemical analyses using a tissue microarray from thirty individuals with PCa metastases to lymph nodes, liver, and bone was used to confirm the gene expression changes associated with each metastatic site. Transcript alterations statistically-associated with bone metastases included increased expression of IBSP (Bone sialoprotein), F13A1 (factor XIII), and decreased expression of EFNA1 (ephrin-A1) and ANGPT2 (angiopoietin-2) when compared to liver and lymph node metastases. The metastasis-associated changes in proteins involved in coagulation and angiogenesis prompted further analysis of additional factors known to participate in the clotting cascade and blood vessel formation (angiopoitein-1, PAI-1, uPA, PAI-RBP-1 and hepsin). We also assessed tumor-associated microvessel density and distribution in liver, lymph node, and bone metastasis. Intense fibrin(ogen) and fibulin-1 staining was localized to epithelial cells at the periphery of metastatic tumors possibly to facilitate angiogenesis. The expression of hepsin, uPA, PAI-RBP1, PAI-1, and factor XIII may influence fibrinolysis and are regulated by the tumor microenvironment. The expression of angiopoietin-2 and apparent silencing of angiopoietin-1 in PCa bone, liver, and lymph node metastases may be critical for angiogenesis in this tumor type. In addition, the resulting tumor-associated microvessel density and distribution was significantly different between liver and bone metastasis possibly in response to the protein expression changes detailed above.