The Chemopotential Effect of Annona muricata Leaves against Azoxymethane-Induced Colonic Aberrant Crypt Foci in Rats and the Apoptotic Effect of Acetogenin Annomuricin E in HT-29 Cells: A Bioassay-Guided Approach

Annona muricata has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of A. muricata leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome c from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of A. muricata leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the anticancer activity of A. muricata leaves.     

Virosome-formulated Plasmodium falciparum AMA-1 & CSP derived peptides as malaria vaccine: randomized phase 1b trial in semi-immune adults & children

Background

This trial was conducted to evaluate the safety and immunogenicity of two virosome formulated malaria peptidomimetics derived from Plasmodium falciparum AMA-1 and CSP in malaria semi-immune adults and children.

Methods

The design was a prospective randomized, double-blind, controlled, age-deescalating study with two immunizations. 10 adults and 40 children (aged 5–9 years) living in a malaria endemic area were immunized with PEV3B or virosomal influenza vaccine Inflexal®V on day 0 and 90.

Results

No serious or severe adverse events (AEs) related to the vaccines were observed. The only local solicited AE reported was pain at injection site, which affected more children in the Inflexal®V group compared to the PEV3B group (p = 0.014). In the PEV3B group, IgG ELISA endpoint titers specific for the AMA-1 and CSP peptide antigens were significantly higher for most time points compared to the Inflexal®V control group. Across all time points after first immunization the average ratio of endpoint titers to baseline values in PEV3B subjects ranged from 4 to 15 in adults and from 4 to 66 in children. As an exploratory outcome, we found that the incidence rate of clinical malaria episodes in children vaccinees was half the rate of the control children between study days 30 and 365 (0.0035 episodes per day at risk for PEV3B vs. 0.0069 for Inflexal®V; RR  = 0.50 [95%-CI: 0.29–0.88], p = 0.02).

Conclusion

These findings provide a strong basis for the further development of multivalent virosomal malaria peptide vaccines.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00513669","term_id":"NCT00513669"}}NCT00513669     

Elucidating the chemical and biochemical applications of Citrus sinensis-mediated silver nanocrystal

Abstract

Synthesis of nanoparticles using biodegradable source is safer and echo-friendly. Here, we describe the synthesis of polycrystalline silver nanocrystals using Citrus sinensis acting as both reducing and capping agents. After exposing the silver ions to orange extract, rapid reduction of silver ions led to the formation of stable silver nanocrystals due to the reducing and stabilizing properties of orange fruit juice. The synthesized silver nanocrystals were characterized using various analytical techniques like UV–vis spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscopy (TEM). The biochemical activity of the synthesized nanocrystals was studied in the light of affinity to bovine serum albumin using several biophysical methods like absorbance, fluorescence and circular dichroism spectroscopy. Cytotoxic activity of these nanocrystals was also studied against Hep-2 cell line using fluorescence microscopy. It was also found that the synthesized nanocrystals can sense mercuric ion down to 50?µM in the presence of a number of cations. Furthermore, we established that the silver nanoparticles can effectively catalyse the reduction of methylene blue by ascorbic acid. The present study will enrich our knowledge on the chemical and biochemical activities of green-synthesized silver nanocrystals.     

Association of Single Nucleotide Polymorphisms in Wnt Signaling Pathway Genes with Breast Cancer in Saudi Patients

Breast cancer is a complex heterogeneous disease involving genetic and epigenetic alterations in genes encoding proteins that are components of various signaling pathways. Candidate gene approach have identified association of genetic variants in the Wnt signaling pathway genes and increased susceptibility to several diseases including breast cancer. Due to the rarity of somatic mutations in key genes of Wnt pathway, we investigated the association of genetic variants in these genes with predisposition to breast cancers. We performed a case-control study to identify risk variants by examining 15 SNPs located in 8 genes associated with Wnt signaling. Genotypic analysis of individual locus showed statistically significant association of five SNPs located in β-catenin, AXIN2, DKK3, SFRP3 and TCF7L2 with breast cancers. Increased risk was observed only with the SNP in β-catenin while the other four SNPs conferred protection against breast cancers. Majority of these associations persisted after stratification of the cases based on estrogen receptor status and age of on-set of breast cancer. The rs7775 SNP in exon 6 of SFRP3 gene that codes for either arginine or glycine exhibited very strong association with breast cancer, even after Bonferroni''s correction. Apart from these five variants, rs3923086 in AXIN2 and rs3763511 in DKK4 that did not show any association in the overall population were significantly associated with early on-set and estrogen receptor negative breast cancers, respectively. This is the first study to utilize pathway based approach to identify association of risk variants in the Wnt signaling pathway genes with breast cancers. Confirmation of our findings in larger populations of different ethnicities would provide evidence for the role of Wnt pathway as well as screening markers for early detection of breast carcinomas.     

Parental Weight Perceptions: A Cause for Concern in the Prevention and Management of Childhood Obesity in the United Arab Emirates

Parental participation is a key factor in the prevention and management of childhood obesity, thus parental recognition of weight problems is essential. We estimated parental perceptions and their determinants in the Emirati population. We invited 1541 students (grade 1–12; 50% boys) and their parents, but only 1440 (6–19 years) and their parents consented. Of these, 945 Emirati nationals provided data for analysis. Anthropometric and demographic variables were measured by standard methods. CDC BMI percentile charts for age and sex were used to classify children’s weight. Parental perception of their children’s weight status (underweight, normal, and overweight/obese) was recorded. Logistic regression analyses were used to identify independent predictors of parental perceptions of children’s weight status. Of all parents, 33.8% misclassified their children’s’ weight status; underestimating (27.4%) or overestimating (6.3%). Misclassification was highest among parents of overweight/obese children (63.5%) and underweight (55.1%) children. More importantly, parental perceptions of their children being overweight or obese, among truly overweight/obese children, i.e. correct identification of an overweight/obese child as such, were associated with the true child’s BMI percentile (CDC) with an OR of 1.313 (95% CI: 1.209–1.425; p<0.001) per percentile point, but not age, parental education, household income, and child’s sex. We conclude that the majority of parents of overweight/obese children either overestimated or, more commonly, underestimated children’s weight status. Predictors of accurate parental perception, in this population, include the true children’s BMI, but not age, household income, and sex. Thus, parents having an incorrect perception of their child’s weight status may ignore otherwise appropriate health messages.     

Metarhizium anisopliae Pathogenesis of Mosquito Larvae: A Verdict of Accidental Death

Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host.     

Adenovirus E1A proteins direct subcellular redistribution of Nek9, a NimA-related kinase

A monoclonal antibody raised against adenovirus E1A-associated cellular proteins recognized Nek9, a NimA-related protein kinase. Subcellular fractionation and immunofluorescence indicated that Nek9 was primarily cytoplasmic with a small portion located in the nucleus whereas E1A was primarily nuclear. Although co-immunoprecipitation experiments indicated that nuclear Nek9 interacted, directly or indirectly, with E1A, the major effect of E1A was to diminish the amount of Nek9 in the nucleus suggesting that E1A alters the subcellular distribution of Nek9 and that the interaction is transient. A Nek9 deletion mutant lacking a central RCC1-like domain interacted stably with E1A and accumulated in the nucleus in the presence of E1A, possibly representing an intermediate stage of the normally transient Nek9/E1A interaction. The interaction of Nek9 with E1A was dependent on the N-terminal sequences of E1A. Attempts to stably overexpress either Nek9 or the kinase-inactive mutant in various cell lines were unsuccessful; however, the presence of E1A allowed stable overexpression of both proteins. These results suggest that E1A disrupts a nuclear function of Nek9.     

心草中总黄酮含量的测定及其提取工艺研究

目的:测定心草中的总黄酮含量并确定其提取条件。方法:采用分光光度法测定、乙醇浸提法提取心草中的总黄酮。结果:浸提的最佳条件是:A3B1C3D2,即用20倍50%乙醇在70℃下进行6h,总黄酮含量为15.675%。平均加样回收率为97.67%。结论:心草中的总黄酮含量较高,此方法简便、快速、重现性好,可作为检测心草中黄酮含量的一种手段。     

Prevalence of Malaria Parasitemia and Purchase of Artemisinin-Based Combination Therapies (ACTs) among Drug Shop Clients in Two Regions in Tanzania with ACT Subsidies

Background

Throughout Africa, many people seek care for malaria in private-sector drug shops where diagnostic testing is often unavailable. Recently, subsidized artemisinin-based combination therapies (ACTs), a first-line medication for uncomplicated malaria, were made available in these drug shops in Tanzania. This study assessed the prevalence of malaria among and purchase of ACTs by drug shop clients in the setting of a national ACT subsidy program and sub-national drug shop accreditation program.

Method and Findings

A cross-sectional survey of drug shop clients was performed in two regions in Tanzania, one with a government drug shop accreditation program and one without, from March-May, 2012. Drug shops were randomly sampled from non-urban districts. Shop attendants were interviewed about their education, training, and accreditation status. Clients were interviewed about their symptoms and medication purchases, then underwent a limited physical examination and laboratory testing for malaria. Malaria prevalence and predictors of ACT purchase were assessed using univariate analysis and multiple logistic regression. Amongst 777 clients from 73 drug shops, the prevalence of laboratory-confirmed malaria was 12% (95% CI: 6–18%). Less than a third of clients with malaria had purchased ACTs, and less than a quarter of clients who purchased ACTs tested positive for malaria. Clients were more likely to have purchased ACTs if the participant was <5 years old (aOR: 6.6; 95% CI: 3.9–11.0) or the shop attendant had >5 years, experience (aOR: 2.8; 95% CI: 1.2–6.3). Having malaria was only a predictor of ACT purchase in the region with a drug shop accreditation program (aOR: 3.4; 95% CI: 1.5–7.4).

Conclusion

Malaria is common amongst persons presenting to drug shops with a complaint of fever. The low proportion of persons with malaria purchasing ACTs, and the high proportion of ACTs going to persons without malaria demonstrates a need to better target who receives ACTs in these drug shops.