Host-cell DNA methylation patterns during high-risk HPV-induced carcinogenesis reveal a heterogeneous nature of cervical pre-cancer

Cervical cancer development following a persistent infection with high-risk human papillomavirus (hrHPV) is driven by additional host-cell changes, such as altered DNA methylation. In previous studies, we have identified 12 methylated host genes associated with cervical cancer and pre-cancer (CIN2/3). This study systematically analyzed the onset and DNA methylation pattern of these genes during hrHPV-induced carcinogenesis using a longitudinal in vitro model of hrHPV-transformed cell lines (n = 14) and hrHPV-positive cervical scrapings (n = 113) covering various stages of cervical carcinogenesis. DNA methylation analysis was performed by quantitative methylation-specific PCR (qMSP) and relative qMSP values were used to analyze the data. The majority of genes displayed a comparable DNA methylation pattern in both cell lines and clinical specimens. DNA methylation onset occurred at early or late immortal passage, and DNA methylation levels gradually increased towards tumorigenic cells. Subsequently, we defined a so-called cancer-like methylation-high pattern based on the DNA methylation levels observed in cervical scrapings from women with cervical cancer. This cancer-like methylation-high pattern was observed in 72% (38/53) of CIN3 and 55% (11/20) of CIN2, whereas it was virtually absent in hrHPV-positive controls (1/26). In conclusion, hrHPV-induced carcinogenesis is characterized by early onset of DNA methylation, typically occurring at the pre-tumorigenic stage and with highest DNA methylation levels at the cancer stage. Host-cell DNA methylation patterns in cervical scrapings from women with CIN2 and CIN3 are heterogeneous, with a subset displaying a cancer-like methylation-high pattern, suggestive for a higher cancer risk.     

Pollination in Bruguiera gymnorrhiza (Rhizophoraceae) in Miyara River, Ishigaki Island, Japan, and Phangnga, Thailand

Abstract The bird species Hypsipetes amaurotis and Zosterops palpebrosa var. yonakuni , but no insects, were recorded regularly feeding on flower-nectar of Bruguiera gymnorrhiza in the Miyara River Biological Preserve, Ishigaki Island, Japan, at 7:00-9:15 a.m. during the investigation from December 20, 1987, to January 19, 1988. In contrast, Anthreptes malacensis foraged for nectar from 7:00 a.m. to 17:00 p.m. Oriolus chinensis visited the flowers mainly between 6:30 and 7:30 a.m. in Phangnga, Thailand, during the investigation from November 30 to December 19, 1986. Pollen grains were sprayed onto the birds'faces and bills and into the air. The highest mean volume of 0.24 pollen grains/ l /minute was triggered in calm air by the most frequent nectarivore between 6:45 and 7:00 a.m. at a height of 1.5m under a tree-branch carrying 145 fresh flowers in the Miyara River population, Japan. The highest mean volume of 0.27 pollen grains/ l /minute under the same conditions in the Phangnga population, Thailand. Bruguiera gymnorrhiza is bird-pollinated but with the possibility of some pollination indirectly by wind.     

Relevant assay to study the adhesion of Plasmodium falciparum-infected erythrocytes to the placental epithelium

In placental malaria, Plasmodium falciparum-infected erythrocytes adhere to the apical plasma membrane of the placental epithelium, triggering an impairment of placental function detrimental to the fetus. The design of anti-adhesion intervention strategies requires a detailed understanding of the mechanisms involved. However, most adhesion assays lack in vivo relevance and are hardly quantitative. Here, we describe a flow cytometry-based adhesion assay that is fully relevant by using apical epithelial plasma membrane vesicles as the adhesion matrix, and being applicable to infected erythrocytes directly isolated from patients. Adhesion is measured both as the percentage of pathogens bound to epithelial membrane vesicles as well as the mean number of vesicles bound per infected erythrocytes. We show that adhesins alternative to those currently identified could be involved. This demonstrates the power of this assay to advance our understanding of epithelial adhesion of infected erythrocytes and in the design of intervention strategies.     

The dynamics of major fibrolytic microbes and enzyme activity in the rumen in response to short- and long-term feeding of Sapindus rarak saponins

AIMS: To investigate the short- and long-term effects of an extract of Sapindus rarak saponins (SE) on the rumen fibrolytic enzyme activity and the major fibrolytic micro-organisms. METHODS AND RESULTS: Two feeding trials were conducted. In the short-term trial, four fistulated goats were fed a basal diet containing sugar cane tops and wheat pollard (65:35, w/w) and were supplemented for 7 days with SE at a level of 0.6 g kg(-1) body weight. Rumen liquor was taken before, during and after SE feeding. In the long-term trial, 28 sheep were fed the same basal diet as the goats and were supplemented for 105 days with 0.24, 0.48 and 0.72 g kg(-1) body mass of the extract. Rumen liquor was taken on days 98 and 100. Protozoal numbers were counted under the microscope. Cell wall degradation was determined by enzyme assays and the major fibrolytic micro-organisms were quantified by dot blot hybridization. Sapindus extract significantly depressed rumen xylanase activity in both trials and carboxymethylcellulase activity in the long-term trial (P < 0.01). Fibrobacter sp. were not affected by the SE in both trials, while ruminococci and the anaerobic fungi showed a short-term response to the application of saponins. Protozoal counts were decreased only in the long-term trial with sheep. CONCLUSION: These data suggest that there is an adaptation of Ruminococcus albus, Ruminococcus flavefaciens and Chytridiomycetes (fungi) to saponin when fed over a long period. The fact that no correlation between the cell wall degrading enzyme activities and the cell wall degrading micro-organisms was observed suggests that the organisms tracked in this experiment are not the only key players in ruminal cell wall degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Sapindus rarak saponins partially defaunate the rumen flora. Their negative effect on cell wall degradation, however, is not related to rumen organisms currently recognized as the major cell wall degrading species. The adaptation of microbes in the long-term feeding experiment suggests that the results from short-term trial on the ruminal microbial community have to be interpreted carefully.     

HIV-1 inhibits phagocytosis and inflammatory cytokine responses of human monocyte-derived macrophages to P. falciparum infected erythrocytes

HIV-1 infection increases the risk and severity of malaria by poorly defined mechanisms. We investigated the effect of HIV-1_Ba-L infection of monocyte-derived macrophages (MDM) on phagocytosis of opsonised P. falciparum infected erythrocytes (IE) and subsequent proinflammatory cytokine secretion. Compared to mock-infected MDM, HIV-1 infection significantly inhibited phagocytosis of IE (median (IQR) (10 (0–28) versus (34 (27–108); IE internalised/100 MDM; p = 0.001) and decreased secretion of IL-6 (1,116 (352–3,387) versus 1,552 (889–6,331); pg/mL; p = 0.0078) and IL-1β (16 (7–21) versus 33 (27–65); pg/mL; p = 0.0078). Thus inadequate phagocytosis and cytokine production may contribute to impaired control of malaria in HIV-1 infected individuals.     

Dependence of forage quality and methanogenic potential of tropical plants on their phenolic fractions as determined by principal component analysis

This study was conducted to elucidate relationships among various phenolic fractions in, and methane (CH₄) emissions from, tropical plants when incubated in ruminal fluid in vitro. As a second objective, principal component analysis (PCA) was tested for its utility in screening plants for their ability to reduce CH₄ formation at simultaneously acceptable nutritional quality. Leaves from 27 tropical plants were analyzed for their nutritional composition and various phenolic fractions. They were incubated in vitro using the Hohenheim gas test method. Variables measured after 24 h of incubation were total gas and CH₄ production, and pH, ammonia, bacterial and protozoal counts, as well as short-chain fatty acids in the incubation fluid. In vitro organic matter (OM) digestibility was computed by a standard equation. The data obtained was subjected to analysis of variance, correlation, regression and PCA. Among phenolic fractions, total phenols had the closest relationship with CH₄/digestible OM (r = ?0.84, P<0.001). The total tannin fraction contributed strongly to this effect (r = ?0.74, P<0.001) whereas the non-tannin phenol fraction was less important (r = ?0.45, P<0.05). Methane reduction by the influence of non-tannin phenols was not associated with a negative effect on protein degradation, while this was the case with tannins. Condensed (r = ?0.60, P<0.01) and hydrolysable tannins (r = ?0.60, P<0.01) contributed to the decrease in CH₄/digestible OM. The loading plot of PCA showed that dietary crude protein (CP) content and incubation fluid ammonia, total short-chain fatty acids, propionate, valerate, iso-butyrate, iso-valerate as well as in vitro OM digestibility were clustered. They had inverse directions to contents of fiber fractions and incubation fluid acetate proportion and acetate-to-propionate ratio. The methane-to-total-gas ratio had the opposite effect of the contents of any phenolic fraction. Plants possessing a favorable forage quality, based on the corresponding PCA score plot, were Carica papaya, Manihot esculenta, Morinda citrifolia, Sesbania grandiflora and Melia azedarach, whereas CH₄ mitigating plants included Swietenia mahagoni, Acacia villosa, Eugenia aquea, Myristica fragrans and Clidemia hirta. All phenolic fractions studied reduced CH₄ emissions from in vitro incubations with ruminal fluid and PCA seems useful to screen plants for high nutritional quality and low ruminal CH₄ formation. However, high forage quality seemed to be partially associated with high CH₄ emission. The search for plants rich in non-tannin phenols might be promising as these compounds appear to decrease CH₄ while they obviously have less negative effect on protein degradation as compared to the tannin fractions.