Spatial and seasonal variation of methanogenic community in a river-bay system in South China.

River-bay system is a transitional zone connecting land and ocean and an important natural source for methane emission. Methanogens play important roles in the global greenhouse gas budget and carbon cycle since they produce methane. The abundance and community assemblage of methanogens in such a dynamic system are not well understood. Here, we used quantitative PCR and high-throughput sequencing of the mcrA gene to investigate the abundance and community composition of methanogens in the Shenzhen River-Bay system, a typical subtropical river-bay system in Southern of China, during the wet and dry seasons. Results showed that mcrA gene abundance was significantly higher in the sediments of river than those of estuary, and was higher in wet season than dry season. Sequences of mcrA gene were mostly assigned to three orders, including Methanosarcinales, Methanomicrobiales, and Methanobacteriales. Specifically, Methanosarcina, Methanosaeta, and Methanobacterium were the most abundant and ubiquitous genera. Methanogenic communities generally clustered according to habitat (river vs. estuary), and salinity was the major factor driving the methanogenic community assemblage. Furthermore, the indicator groups for two habitats were identified. For example, Methanococcoides, Methanoculleus, and Methanogenium preferentially existed in estuarine sediments, whereas Methanomethylovorans, Methanolinea, Methanoregula, and Methanomassiliicoccales were more abundant in riverine sediments, indicating distinct ecological niches. Overall, these findings reveal the distribution patterns of methanogens and expand our understanding of methanogenic community assemblage in the river-bay system. Key Points • Abundance of methanogens was relatively higher in riverine sediments. • Methanogenic community in estuarine habitat separated from that in riverine habitat. • Salinity played a vital role in regulating methanogenic community assemblage.

Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes.

Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.

Intervention of pumpkin seed oil on metabolic disease revealed by metabonomics and transcript profile.

BACKGROUND: Understanding the metabolic and transcription basis of pumpkin seed oil (PSO) intervention on metabolic disease (MD) is essential to daily nutrition and health. RESULTS: This study analyzed the liver metabolic variations of Wistar rats fed normal diet (CON), high-fat diet (HFD) and high-fat plus PSO diet (PSO) to establish the relationship between the liver metabolite composition/transcript profile and the effects of PSO on MD. By using proton nuclear magnetic resonance spectroscopy together with multivariate data analysis, it was found that, compared with CON rats, HFD rats showed clear dysfunctions of choline metabolism, glucose metabolism and nucleotide and amino acid metabolism. Using quantitative real-time polymerase chain reaction (qPCR), it was found that, compared with HFD rats, PSO rats showed alleviated endoplasmic reticulum stress accompanied by lowered unfolded protein response. CONCLUSION: These findings provide useful information to understand the metabolic alterations triggered by MD and to evaluate the effects of PSO intervention. © 2016 Society of Chemical Industry.

The Way to Malignant Transformation: Can Epigenetic Alterations Be Used to Diagnose Early-Stage Head and Neck Cancer?

Identifying and treating tumors early is the key to secondary prevention in cancer control. At present, prevention of oral cancer is still challenging because the molecular drivers responsible for malignant transformation of the 11 clinically defined oral potentially malignant disorders are still unknown. In this review, we focused on studies that elucidate the epigenetic alterations demarcating malignant and nonmalignant epigenomes and prioritized findings from clinical samples. Head and neck included, the genomes of many cancer types are largely hypomethylated and accompanied by focal hypermethylation on certain specific regions. We revisited prior studies that demonstrated that sufficient uptake of folate, the primary dietary methyl donor, is associated with oral cancer reduction. As epigenetically driven phenotypic plasticity, a newly recognized hallmark of cancer, has been linked to tumor initiation, cell fate determination, and drug resistance, we discussed prior findings that might be associated with this hallmark, including gene clusters (11q13.3, 19q13.43, 20q11.2, 22q11-13) with great potential for oral cancer biomarkers, and successful examples in screening early-stage nasopharyngeal carcinoma. Although one-size-fits-all approaches have been shown to be ineffective in most cancer therapies, the rapid development of epigenome sequencing methods raises the possibility that this nonmutagenic approach may be an exception. Only time will tell.

Fruits are vehicles of drug-resistant pathogenic Candida tropicalis.

IMPORTANCE: Of 123 identified isolates from the fruit surface, C. tropicalis was the most frequently found species, followed by Meyerozyma caribbica and Candida krusei. All three fluconazole-resistant C. tropicalis were non-susceptible to voriconazole and belonged to the same predominant genotype of azole-resistant C. tropicalis causing candidemia in patients in Taiwan. Our findings provide evidence that fruit should be washed before eaten not only to remove chemicals but also potential drug-resistant pathogenic microbes, especially for immunocompromised individuals. To keep precious treatment options in patients, we not only continuously implement antimicrobial stewardship in hospitals but also reducing/stopping the use of agricultural fungicide classes used in human medicine.

Suppressive regulation of KSHV RTA with O-GlcNAcylation.

BACKGROUND: The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a molecular switch that initiates a productive replication of latent KSHV genomes. KSHV RTA (K-RTA) is composed of 691 amino acids with high Ser and Thr content (17.7%), but to what extent these Ser and Thr are modified in vivo has not been explored. METHODS: By using tandem mass spectrometric analysis of affinity-purified FLAG tagged K-RTA, we sought to identify Ser and Thr residues that are post-translationally modified in K-RTA. RESULTS: We found that K-RTA is an O-GlcNAcylated protein and Thr-366/Thr-367 is the primary motif with O-GlcNAcylation in vivo. The biological significance of O-GlcNAc modified Thr-366 and Thr-367 was assessed by site-specific amino acid substitution. Replacement of Thr with Ala at amino acid 366 or 367 caused a modest enhancement of K-RTA transactivation activity in a luciferase reporter assay and a cell model for KSHV reactivation. By using co-immunoprecipitation coupled with western blot analysis, we showed that the capacity of K-RTA in associating with endogenous PARP1 was significantly reduced in the Thr-366/Thr-367 O-GlcNAc mutants. PARP1 is a documented negative regulator of K-RTA that can be ascribed by the attachment of large negatively charged polymer onto K-RTA via PARP1's poly (ADP-ribose) polymerase activity. In agreement, shRNA-mediated depletion of O-GlcNAc transferase (OGT) in KSHV infected cells augmented viral reactivation and virus production that was accompanied by diminished K-RTA and PARP1 complexes. CONCLUSIONS: KSHV latent-lytic switch K-RTA is modified by cellular O-GlcNAcylation, which imposes a negative effect on K-RTA transactivation activity. This inhibitory effect involves OGT and PARP1, two nutritional sensors recently emerging as chromatin modifiers. Thus, we speculate that the activity of K-RTA on its target genes is continuously checked and modulated by OGT and PARP1 in response to cellular metabolic state.

Epstein-Barr virus DNase (BGLF5) induces genomic instability in human epithelial cells.

Epstein-Barr Virus (EBV) DNase (BGLF5) is an alkaline nuclease and has been suggested to be important in the viral life cycle. However, its effect on host cells remains unknown. Serological and histopathological studies implied that EBV DNase seems to be correlated with carcinogenesis. Therefore, we investigate the effect of EBV DNase on epithelial cells. Here, we report that expression of EBV DNase induces increased formation of micronucleus, an indicator of genomic instability, in human epithelial cells. We also demonstrate, using gammaH2AX formation and comet assay, that EBV DNase induces DNA damage. Furthermore, using host cell reactivation assay, we find that EBV DNase expression repressed damaged DNA repair in various epithelial cells. Western blot and quantitative PCR analyses reveal that expression of repair-related genes is reduced significantly in cells expressing EBV DNase. Host shut-off mutants eliminate shut-off expression of repair genes and repress damaged DNA repair, suggesting that shut-off function of BGLF5 contributes to repression of DNA repair. In addition, EBV DNase caused chromosomal aberrations and increased the microsatellite instability (MSI) and frequency of genetic mutation in human epithelial cells. Together, we propose that EBV DNase induces genomic instability in epithelial cells, which may be through induction of DNA damage and also repression of DNA repair, subsequently increases MSI and genetic mutations, and may contribute consequently to the carcinogenesis of human epithelial cells.

Diversity, metabolism and cultivation of archaea in mangrove ecosystems.

Mangroves comprise a globally significant intertidal ecosystem that contains a high diversity of microorganisms, including fungi, bacteria and archaea. Archaea is a major domain of life that plays important roles in biogeochemical cycles in these ecosystems. In this review, the potential roles of archaea in mangroves are briefly highlighted. Then, the diversity and metabolism of archaeal community of mangrove ecosystems across the world are summarized and Bathyarchaeota, Euryarchaeota, Thaumarchaeota, Woesearchaeota, and Lokiarchaeota are confirmed as the most abundant and ubiquitous archaeal groups. The metabolic potential of these archaeal groups indicates their important ecological function in carbon, nitrogen and sulfur cycling. Finally, some cultivation strategies that could be applied to uncultivated archaeal lineages from mangrove wetlands are suggested, including refinements to traditional cultivation methods based on genomic and transcriptomic information, and numerous innovative cultivation techniques such as single-cell isolation and high-throughput culturing (HTC). These cultivation strategies provide more opportunities to obtain previously uncultured archaea.

Toll­like receptor 4 activates the NLRP3 inflammasome pathway and periodontal inflammaging by inhibiting Bmi­1 expression.

Overproduction of pro­inflammatory cytokines in the aged, which is called inflammaging, leads to the deterioration of periodontitis. Toll­like receptor 4 (TLR4) plays a role in the regulation of cellular senescence, and its expression increases with age. However, there has been limited research into the molecular mechanisms underlying the onset of periodontal inflammaging, and the interplay between TLR4 and inflammaging. In the present study, wild­type and TLR4 gene knockout mice were used to investigate the activation of the TLR4 pathway in mouse periodontitis and the expression of the nucleotide­binding and oligomerization domain­like receptor 3 (NLRP3) inflammasome, an upstream immune checkpoint during the development of inflammaging. Activation of TLR4 in a mouse model of periodontitis enhanced the expression of a senescence­associated secretory phenotype (SASP), which boosted the inflammaging process. Conversely, TLR4 activation downregulated the expression of B cell­specific Moloney murine leukemia virus integration site 1 (Bmi­1) and promoted the priming of NLRP3 inflammasome, both of which are regulators of SASP. Treating gingival fibroblasts with Bmi­1 inhibitor PTC209, it was demonstrated that TLR4 activated the NLRP3 pathway and the inflammaging process by suppressing Bmi­1. In addition, there was a significant reduction in the expression of Bmi­1 expression in the gingiva of patients with periodontitis compared with healthy controls. In conclusion, the present study demonstrated that TLR4 acted by inhibiting Bmi­1 to enhance the NLRP3 pathway and SASP factors. This cascade of reactions may contribute to the senescence of the periodontium.

Identification of Prognostic Biomarkers Originating From the Tumor Stroma of Betel Quid-Associated Oral Cancer Tissues.

BACKGROUND: Partial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging. METHODS: Gene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan-Meier curves of estimated overall survival were compared for statistical difference using the log-rank test. RESULTS: Compared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types. CONCLUSIONS: As the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.

Discoidin Domain Receptor-1 (DDR1) is Involved in Angiolymphatic Invasion in Oral Cancer.

The discoidin domain receptor-1 (DDR1) is a non-integrin collagen receptor recently implicated in the collective cell migration of other cancer types. Previously, we identified an elevated expression of DDR1 in oral squamous cell carcinoma (OSCC) cells. Through the data mining of a microarray dataset composed of matched tumor-normal tissues from forty OSCC patients, we distilled overexpressed genes statistically associated with angiolymphatic invasion, including DDR1, COL4A5, COL4A6 and PDPN. Dual immunohistochemical staining further confirmed the spatial locations of DDR1 and PDPN in OSCC tissues indicative of collective cancer cell invasion. An elevated DDR1 expression at both the transcription and protein level was observed by treating keratinocytes with collagen of fibrillar or basement membrane types. In addition, inhibition of DDR1 kinase activity in OSCC TW2.6 cells disrupted cell cohesiveness in a 2D culture, reduced spheroid invasion in a collagen gel matrix, and suppressed angiolymphatic invasion in xenograft tissues. Taken together, these results suggest that collagen deposition in the affected tissues followed by DDR1 overexpression could be central to OSCC tumor growth and angiolymphatic invasion. Thus, DDR1 inhibitors are potential therapeutic compounds in restraining oral cancer, which has not been previously explored.

Index of Cancer-Associated Fibroblasts Is Superior to the Epithelial-Mesenchymal Transition Score in Prognosis Prediction.

In many solid tumors, tissue of the mesenchymal subtype is frequently associated with epithelial-mesenchymal transition (EMT), strong stromal infiltration, and poor prognosis. Emerging evidence from tumor ecosystem studies has revealed that the two main components of tumor stroma, namely, infiltrated immune cells and cancer-associated fibroblasts (CAFs), also express certain typical EMT genes and are not distinguishable from intrinsic tumor EMT, where bulk tissue is concerned. Transcriptomic analysis of xenograft tissues provides a unique advantage in dissecting genes of tumor (human) or stroma (murine) origins. By transcriptomic analysis of xenograft tissues, we found that oral squamous cell carcinoma (OSCC) tumor cells with a high EMT score, the computed mesenchymal likelihood based on the expression signature of canonical EMT markers, are associated with elevated stromal contents featured with fibronectin 1 (Fn1) and transforming growth factor-ß (Tgfß) axis gene expression. In conjugation with meta-analysis of these genes in clinical OSCC datasets, we further extracted a four-gene index, comprising FN1, TGFB2, TGFBR2, and TGFBI, as an indicator of CAF abundance. The CAF index is more powerful than the EMT score in predicting survival outcomes, not only for oral cancer but also for the cancer genome atlas (TCGA) pan-cancer cohort comprising 9356 patients from 32 cancer subtypes. Collectively, our results suggest that a further distinction and integration of the EMT score with the CAF index will enhance prognosis prediction, thus paving the way for curative medicine in clinical oncology.

Dynamic lipid profile of hyperlipidemia mice.

Biomarkers of serum fatty acids in hyperlipidemia need to be elucidated. 90 SPF KM male mice were randomly divided into 18 groups (n=5/group), control groups, and high fat diet (HFD) groups at 9 time points. On day 7, 10, 15, 18, 21, 24, 28, 31, and 35, the mice were sacrificed; blood was collected into tubes from the eyes, serum samples for clinical biochemistry assays and gas chromatography-mass spectroscopy were attained after centrifugation, and the contents of serum fatty acids were detected with GC-MS. Sections of livers were taken and stored in formalin solution for histological assessments. No species differences existed in all these groups. The contents of C16:1, C18:1, C22:6 were significantly different between HFD groups and the corresponding controls; meanwhile, the proportion of fatty acids, especially the monounsaturated degree, the polyunsaturated degree, changed significantly and regularly (P<0.05). Thus the three unsaturated fatty acids C16:1, C18:1, C22:6 and the monounsaturated/polyunsaturated unsaturated degrees may be as potential biomarkers of hyperlipidemia.

Increased detection of coronary atherosclerosis on 320-slice computed tomographic angiography with burden of cardiovascular risk factors and complications in patients with type 2 diabetes.

AIMS: The cardiovascular risk factors and diabetic complications are related to coronary atherosclerosis. However, the evaluation of the prevalence of coronary atherosclerosis based on their accumulation remains to be determined. METHODS: 247 consecutive Chinese subjects with type 2 diabetes but without history of coronary heart disease (CHD) underwent 320-slice computed tomographic coronary angiography, including no coronary atherosclerosis, non-obstructive atherosclerosis (<50% stenosis) and obstructive atherosclerosis (≥50% stenosis). Conventional cardiovascular risk factors, albuminuria, renal dysfunction and diabetic retinopathy (DR) were determined. Framingham Risk Score (FRS) was used to assess the 10-year CHD risk. RESULTS: Increase in burden of cardiovascular risk factors and diabetic complications were significantly associated with the likelihood of being a higher coronary atherosclerosis category. In the analysis for trend through the categories of burden score or FRS stratification, the percentage of obstructive atherosclerosis was increased and the percentage of no atherosclerosis decreased as the burden score and FRS increased (all p<0.005), respectively. The areas under the receiver operator curve for the burden score versus FRS were greater at predicting coronary atherosclerosis and obstructive atherosclerosis (p=0.004 and p=0.002), respectively. CONCLUSIONS: The prevalence of coronary atherosclerosis was increased with the accumulation of cardiovascular risk factors and diabetic complications. The burden of these clinical and biochemical risk factors has increased ability for prediction of the presence and severity of coronary atherosclerosis over FRS in type 2 diabetic patients.

Identification and characterization of antioxidant peptides from enzymatic hydrolysates of duck meat.

The objective of this study was to prepare antioxidant peptides from duck meat hydrolysate (DMH) using Protamex. The DPPH(•) scavenging activity, hydroxyl radical ((•)OH) scavenging activity, and Fe(2+)-chelating ability of DMH were investigated. DMH was separated into three groups, MWCO-1 (69.57%), MWCO-2 (9.53%), and MWCO-3 (8.21%), by ultrafiltration. MWCO-3 exhibited the highest DPPH(•) scavenging activity (83.17 ± 0.73%) and was subsequently fractionated by using gel filtration chromatography to obtain fraction B (40.90%). Fraction B5 (6.71%) obtained from ion exchange chromatography exhibited the highest DPPH(•) scavenging activity (93.63 ± 0.13%) and contained seven peptides which were characterized by LC-MS/MS. Among these peptides, LQAEVEELRAALE showed the highest DPPH(•) scavenging activity (93.36 ± 0.53%) and Fe(2+)-chelating ability (87.13 ± 0.47%) and IEDPFDQDDWGAWKK exhibited the highest (•)OH scavenging activity (46.51 ± 0.16%). The results presented here indicated that DMH could serve as a suitable source of antioxidant peptides.

[Expression of peripheral blood gammadelta T cells, treg cells and cytokines IL-17 and TGF-beta1 in patients with allergic rhinitis].

OBJECTIVE: To investigate the expression of peripheral blood gammadelta T cells/CD4 CD25+ regulatory T cells(Treg) and cytokines interleukin 17 (IL-17) and transforming growth factor beta1 (TGF-beta1) in patients with allergic rhinitis. METHODS: From March 2012 to July 2012, 32 patients with allergic rhinitis (AR group) and 20 healthy control subjects (control group) were collected. The expression of peripheral blood gammadelta T cells/Treg cells were measured by flow cytometry and the levels of IL-17 and TGF-beta1 were evaluated by ELISA. SPSS 16.0 software was used to analyze the data. RESULTS: The percentages of gammadeltaT cells in AR group were (13.30 +/- 8.62)%, which was significantly higher (t = 5.18, P < 0.01) than those in control group (5.18 +/- 1.86)%. The percentages of Treg cells in AR group were (1.75 +/- 0.56)%, which were significantly lower (t = 7.46, P < 0. 01) than those in control group (4.76 +/- 1.74)%. The IL-17 levels in AR group were (668.55 +/- 45.15) pg/ml, which were also significantly higher (t = 8.97, P < 0.01) than those in control group (573.53 +/- 17.42) pg/ml. The TGF-beta1 levels in AR group were (0.34 +/- 0.04) pg/ml, which were also significantly lower (t = 9.51, P < 0.01) than those in control group (0.49 +/- 0.06) pg/ml. There was a negative correlation between the percentages of gammadelta T cells and Treg cells (r = -0.561, P < 0.01). There was a negative correlation between the percentages of gammadelta T cells and TGF-beta1 levels (r = -0.622, P < 0.01). A positive correlation was shown between the percentages of gammadelta T cells and IL-17 levels in AR (r = 0.469, P < 0.01). A positive correlation was shown between the percentages of Treg cells and TGF-beta1 levels in AR (r = 0.738, P < 0.01). There was no correlation between IL-17 levels and the percentages of Treg cells or TGF-beta1 levels (r value was -0.111, -0.196, all P > 0.05). CONCLUSION: There are imbalances of gammadelta T and Treg cells in peripheral blood of patients with allergic rhinitis. gammadelta T cells may be the main cell to produce IL-17, which may play an important role in allergic rhinitis.

[Geographic distribution and gene sequencing of Paragonimus westermani in some areas of Guangdong Province].

OBJECTIVE: To investigate the current distribution of Paragonimus westermani in Guangdong Province. METHOD: Snails and crabs collected from mountain streams in regional survey sites were dissected to detect cercarial and metacercarial infections of P. westermani. Domestic cats and dogs artificially infected with the collected metacercariae were also dissected to detect adult worms of P. westermani. The COI and ITS2 gene sequences of those adult worms were compared with those of known Paragonimus specimen deposited in the GenBank. RESULTS: All of the first intermediate hosts in five survey sites of Liangkou, Nankun, Mountain, Dadong, Muxi, Guowu, were identified as Semisulcospira libertina, whose cercariae infection rates were 0.33%, 0.15%, 0.058%, 0.10%, and 0.05%, respectively; the second intermediate hosts in above five sites were all identified as Sinopotamon denticulatum, whose metacercariae infection rates were 100%, 100%, 38.09%, 55.36%, and 65.26%, respectively. The numbers of metacercariae in the five sites were 79.4, 105.66, 9.16, 16.18, and 15.6 per positive crab, respectively, and 11.12, 7.87, 0.58, 0.69, and 0.85 per gram of crab, respectively. All the metacercariae were identical to those of P. westermani. Adult worms and eggs of P. westermani were found in both reservoir hosts of domestic cats and dogs infected artificially. By comparing the COI genes of five representative samples from each survey site with that of Paragonimus #AF219379.21, AF540958.1 from GenBank, we found out the homology to be 99%, 99%, 99%, 98%, and 99%, respectively. In addition, a comparison of the ITS2 gene sequences between the above five samples and Paragonimus #DQ836243.1, DQ351845.1, AB354217.1 from GenBank revealed 98%, 99%, 98%, 98%, and 98% gene homology, respectively. CONCLUSION: Two ultra-high and three high endemic areas of P. westermani are discovered in Guangdong Province. No obvious differences were found among the types of P. westermani in the above five endemic areas.

Ser-634 and Ser-636 of Kaposi's Sarcoma-Associated Herpesvirus RTA are Involved in Transactivation and are Potential Cdk9 Phosphorylation Sites.

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal ((527)KKRK(530)) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that (634)SPSP(637) motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full transcriptional potency of K-RTA.

Epstein-Barr virus (EBV) Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells.

Epstein-Barr virus (EBV) Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox)-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-ß-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1) an ideal environment for virus reactivation if EBV or KSHV coexists and (2) a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.