Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Seasonal decline of the tomato leafminer,Tuta absoluta,in the shifting landscape of a vegetable‐growing area

Insect pest populations exhibit seasonal dynamics in response to changes in resource availability or other environmental factors such as climatic conditions, natural enemies, and intra‐ or interspecific competition. Understanding such dynamics is critical for developing effective integrated pest management strategies. The objective of the present study was to identify factors driving the seasonal decline of the tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), in the shifting landscape of a vegetable‐growing area in Senegal. A set of 42 tomato fields was monitored for the number of T. absoluta adults caught in pheromone traps and for the incidence of larvae, during 5 months from June to November 2016 in the Niayes area (Senegal). The surface of solanaceous host crops, climatic conditions, and abundance of natural enemies were also monitored. A drastic decline in T. absoluta abundance was observed during the rainy season. The decrease in resource availability, especially tomato crops, in the surrounding landscape of monitored fields was the main factor affecting the population dynamics during the rainy season. However, alternative host crops such as eggplant and Ethiopian eggplant, can provide ‘reservoirs’ for residual populations of the pest. For applied purpose, it would be interesting to focus the management efforts on residual populations during the rainy season, to make more difficult the reconstitution of populations during the dry season.     

Pollen from archaeological layers and cultural landscape reconstruction: Case studies from the Bradano valley (Basilicata,southern Italy)

Abstract

The article aims at presenting some aspects of environmental reconstruction through pollen analysis from archaeological contexts. The anthropogenic pollen transport into archaeological sites is regarded as an interesting tool to improve knowledge on flora and vegetation in the area of influence of sites. The zoophilous plants can be found more easily than in the regional airborne pollen rain where anemophilous pollen is generally overrepresented. Moreover, pollen from archaeological contexts is mainly a result of the cultural landscape shaped by human activities. Two case studies from the Bradano Valley (Basilicata, southern Italy), rich in archaeological sites dating altogether from the Middle Bronze Age to the Medieval age, are reported. Difesa San Biagio and its surroundings is one of the biggest settlements of the area, settled in early times by Enotrians. Altojanni is an extended area mainly frequented in Hellenistic, Roman late Imperial and Medieval times. A very open landscape, and clear signs of plant exploitation and cultivation, breeding and settlements were present in the two sites. Though samples are disturbed and preservation problems are sometimes observed, the main characters of pollen spectra are recurrent. High percentages of Poaceae and Cichorioideae, together with coprophilous fungal spores, strongly suggest a long tradition of pastoral activities. These case study examples suggest that human activities would have produced a fairly xeric environment.     

Use of a Cholera Rapid Diagnostic Test during a Mass Vaccination Campaign in Response to an Epidemic in Guinea, 2012

Background

During the 2012 cholera outbreak in the Republic of Guinea, the Ministry of Health, supported by Médecins Sans Frontières - Operational Center Geneva, used the oral cholera vaccine Shanchol as a part of the emergency response. The rapid diagnostic test (RDT) Crystal VC, widely used during outbreaks, detects lipopolysaccharide antigens of Vibrio cholerae O1 and O139, both included in Shanchol. In the context of reactive use of a whole-cell cholera vaccine in a region where cholera cases have been reported, it is essential to know what proportion of vaccinated individuals would be reactive to the RDT and for how long after vaccination.

Methodology/Principal Findings

A total of 108 vaccinated individuals, selected systematically among all persons older than one year, were included at vaccination sites and 106 were included in the analysis. Stools samples of this cohort of vaccinated participants were collected and tested with the RDT every day until the test was negative for two consecutive visits or for a maximum of 7 days. A total of 94.3% of cholera vaccine recipients had a positive test after vaccination; all except one of these positive results were reactive only with the O139 antigen. The mean time to become negative in those with an initial positive result after vaccination was 3.8 days, standard deviation 1.1 days.

Conclusions/Significance

The RDT Crystal VC becomes positive in persons recently vaccinated against cholera, although almost exclusively to the O139 antigen. This reactivity largely disappeared within five days after vaccination. These results suggest that the test can be used normally as soon as 24 hours after vaccination in a context of O1 epidemics, which represent the vast majority of cases, and after a period of five days in areas where V. cholerae O139 is present. The reason why only O139 test line became positive remains to be investigated.     

RNA interference targeting nucleocapsid protein inhibits porcine reproductive and respiratory syndrome virus replication in Marc-145 cells

Porcine reproductive and respiratory syndrome (PRRS) is an important disease, which leads to severe economic losses in swine-producing areas of the world. However, current antiviral strategies cannot provide highly effective protection. In this study, three theoretically effective interference target sites (71–91, 144–164, 218–238) targeting the nucleocapsid (N) gene of PRRSV were designed and selected, and then three siRNA-expressing plasmids were constructed, respectively named p2.1-N71, p2.1-N144, and p2.1-N218. The recombinant siRNA-expressing plasmids were transfected into Marc-145 cells; then the cells were infected with PRRSV (JL07SW strain); finally, after incubation for 48 h, the antiviral activity of those siRNA-expressing plasmids in Marc-145 cells was assessed by cytopathic effects, virus titers, indirect immunofluorescence, and quantitative real-time PCR. Experimental results demonstrated that these three siRNA-expressing plasmids could effectively and significantly inhibit the replication of PRRSV by 93.2%, 83.6%, and 89.2% in Marc-145 cells, respectively. Among these three siRNA-expressing plasmids, p2.1-N71 was found to be most effective, while p2.1-N144 and p2.1-N218 displayed relatively weak inhibition of virus replication. The results indicated that siRNA-expressing plasmids targeting the N gene of PRRSV could significantly inhibit PRRSV replication in Marc-145 cells. Based on our experimental results and previous reports, the 71–91, 179–197, and 234–252 sites of the N gene are good choices to effectively inhibit the replication of PRRSV, and this RNA interference technique can be a potential anti-PRRSV strategy.     

Interactions between hepatitis B virus infection and exposure to aflatoxins in the development of hepatocellular carcinoma: a molecular epidemiological approach.

Aflatoxins and hepatitis B virus (HBV) are major risk factors for hepatocellular carcinoma (HCC) in high incidence areas for this cancer, namely southeast Asia and parts of Africa. There is evidence from both epidemiological studies and animal models that the two factors can act synergistically to increase the risk of HCC. The cellular and molecular mechanism of the interaction between these two factors is as yet undefined. However, one possible mechanism attested to by studies in HBV transgenic mice is that chronic liver injury alters the expression of specific carcinogen metabolising enzymes thus modulating the binding of aflatoxin to DNA in hepatocytes. The high levels of aflatoxin exposure which occur in many areas of the world where chronic HBV infection is endemic indicate that measures to reduce aflatoxin exposure would contribute to reducing HCC incidence. In preliminary studies, Guinea-Conakry have established baseline data for the implementation of a community-based intervention study to evaluate the effectiveness of improved post-harvest processing and storage of the groundnut crop, a major source of aflatoxins. Aflatoxin-albumin adducts were measured in 423 sera from individuals living in the four natural geographic zones of Guinea. More than 95% of the serum samples were positive for this biomarker and highest exposures were found in Lower Guinea where groundnuts are consumed as a dietary staple. Variations in mean levels between villages within a geographic region did not vary greatly. HBV infection was endemic in all regions with an overall prevalence of 16.7% chronic carriers. Thus in this population both HBV vaccination and reduction in aflatoxin exposure would be beneficial in decreasing morbidity and mortality from liver disease.     

The Abundance and Diversity of Legume-Nodulating Rhizobia in 28-Year-Old Plantations of Tropical, Subtropical, and Exotic Tree Species: a Case Study from the Forest Reserve of Bandia, Senegal

Several fast-growing and multipurpose tree species have been widely used in West Africa to both reverse the tendency of land degradation and restore soil productivity. Although beneficial effects have been reported on soil stabilization, there still remains a lack of information about their impact on soil microorganisms. Our investigation has been carried out in exotic and native tree plantations of 28 years and aimed to survey and compare the abundance and genetic diversity of natural legume-nodulating rhizobia (LNR). The study of LNR is supported by the phylogenetic analysis which clustered the isolates into three genera: Bradyrhizobium, Mesorhizobium, and Sinorhizobium. The results showed close positive correlations between the sizes of LNR populations estimated both in the dry and rainy seasons and the presence of legume tree hosts. There were significant increases in Rhizobium spp. population densities in response to planting with Acacia spp., and high genetic diversities and richness of genotypes were fittest in these tree plantations. This suggests that enrichment of soil Rhizobium spp. populations is host specific. The results indicated also that species of genera Mesorhizobium and Sinorhizobium were lacking in plantations of non-host species. By contrast, there was a widespread distribution of Bradyrhizobium spp. strains across the tree plantations, with no evident specialization in regard to plantation type. Finally, the study provides information about the LNR communities associated with a range of old tree plantations and some aspects of their relationships to soil factors, which may facilitate the management of man-made forest systems that target ecosystem rehabilitation and preservation of soil biota.