Minnelide: A Novel Therapeutic That Promotes Apoptosis in Non-Small Cell Lung Carcinoma In Vivo

Background

Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.

Methods

Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA, APAF-1) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.

Results

In this study, we investigated in vitro and in vivo antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA) and up-regulated pro-apoptotic APAF-1 gene, in part, via attenuating the NF-κB signaling activity.

Conclusion

In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.     

Catestatin Increases the Expression of Anti-Apoptotic and Pro-Angiogenetic Factors in the Post-Ischemic Hypertrophied Heart of SHR

Background

In the presence of comorbidities the effectiveness of many cardioprotective strategies is blunted. The goal of this study was to assess in a hypertensive rat model if the early reperfusion with anti-hypertensive and pro-angiogenic Chromogranin A-derived peptide, Catestatin (CST:hCgA_352–372; CST-Post), protects the heart via Reperfusion-Injury-Salvage-Kinases (RISK)-pathway activation, limiting infarct-size and apoptosis, and promoting angiogenetic factors (e.g., hypoxia inducible factor, HIF-1α, and endothelial nitric oxide synthase, eNOS, expression).

Methods and Results

The effects of CST-Post on infarct-size, apoptosis and pro-angiogenetic factors were studied in isolated hearts of spontaneously hypertensive rats (SHR), which underwent the following protocols: (a) 30-min ischemia and 120-min reperfusion (I/R); (b) 30-min ischemia and 20-min reperfusion (I/R-short), both with and without CST-Post (75 nM for 20-min at the beginning of reperfusion). In unprotected Wistar-Kyoto hearts, used as normal counterpart, infarct-size resulted smaller than in SHR. CST-Post reduced significantly infarct-size and improved post-ischemic cardiac function in both strains. After 20-min reperfusion, CST-Post induced S-nitrosylation of calcium channels and phosphorylation of RISK-pathway in WKY and SHR hearts. Yet specific inhibitors of the RISK pathway blocked the CST-Post protective effects against infarct in the 120-min reperfusion groups. Moreover, apoptosis (evaluated by TUNEL, ARC and cleaved caspase) was reduced by CST-Post. Importantly, CST-Post increased expression of pro-angiogenetic factors (i.e., HIF-1α and eNOS expression) after two-hour reperfusion.

Conclusions

CST-Post limits reperfusion damages and reverses the hypertension-induced increase of I/R susceptibility. Moreover, CST-Post triggers antiapoptotic and pro-angiogenetic factors suggesting that CST-Post can be used as an anti-maladaptive remodeling treatment.     

Gene Expression Profiling in Leiomyosarcomas and Undifferentiated Pleomorphic Sarcomas: SRC as a New Diagnostic Marker

Background

Undifferentiated Pleomorphic Sarcoma (UPS) and high-grade Leiomyosarcoma (LMS) are soft tissue tumors with an aggressive clinical behavior, frequently developing local recurrence and distant metastases. Despite several gene expression studies involving soft tissue sarcomas, the potential to identify molecular markers has been limited, mostly due to small sample size, in-group heterogeneity and absence of detailed clinical data.

Materials and Methods

Gene expression profiling was performed for 22 LMS and 22 UPS obtained from untreated patients. To assess the relevance of the gene signature, a meta-analysis was performed using five published studies. Four genes (BAD, MYOCD, SRF and SRC) selected from the gene signature, meta-analysis and functional in silico analysis were further validated by quantitative PCR. In addition, protein-protein interaction analysis was applied to validate the data. SRC protein immunolabeling was assessed in 38 UPS and 52 LMS.

Results

We identified 587 differentially expressed genes between LMS and UPS, of which 193 corroborated with other studies. Cluster analysis of the data failed to discriminate LMS from UPS, although it did reveal a distinct molecular profile for retroperitoneal LMS, which was characterized by the over-expression of smooth muscle-specific genes. Significantly higher levels of expression for BAD, SRC, SRF, and MYOCD were confirmed in LMS when compared with UPS. SRC was the most value discriminator to distinguish both sarcomas and presented the highest number of interaction in the in silico protein-protein analysis. SRC protein labeling showed high specificity and a positive predictive value therefore making it a candidate for use as a diagnostic marker in LMS.

Conclusions

Retroperitoneal LMS presented a unique gene signature. SRC is a putative diagnostic marker to differentiate LMS from UPS.     

Mrassf1a-Pap,a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma

Objectives

RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma.

Methods

Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated.

Results

Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ∼3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%–100%).

Conclusions

Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.     

A Novel Simple Method for Determining CYP2D6 Gene Copy Number and Identifying Allele(s) with Duplication/Multiplication

Background

Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.

Methods

We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV) genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.

Results

The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.

Conclusions

Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.     

Deciphering the binding mode and mechanistic insights of pentadecylidenemalonate (1b) as activator of histone acetyltransferase PCAF

Histone acetyltransferases (HATs) is one among the conspicuous posttranslational modification in eukaryotic cells. p300/CBP Associated Factor (PCAF) and CREB-binding protein (CBP) are the two highly homologous HAT family which are vastly implicated in several diseases like cancer, diabetes, etc. Pentadecylidenemalonate, a simplified analog of anacardic acid, was reported as first mixed inhibitor/activator of HATs which inhibits p300/CBP and activates PCAF. It was appointed earlier as a valuable biological tool to understand the mechanism of lysine acetyltransferases due to its powerful apoptotic effect. In this study, pentadecylidenemalonate was taken for deciphering the binding mode, key interacting residues as well as mechanistic insights on PCAF and CBP as activator and inhibitor, respectively. This study is highly believed to help in rational design on antineoplastic drugs against PCAF.

Communicated by Ramaswamy H. Sarma     

Extracellular Vesicles from Leishmania-Infected Macrophages Confer an Anti-infection Cytokine-Production Profile to Na?ve Macrophages

Background

Extracellular vesicles (EVs) are structures with phospholipid bilayer membranes and 100–1000 nm diameters. These vesicles are released from cells upon activation of surface receptors and/or apoptosis. The production of EVs by dendritic cells, mast cells, macrophages, and B and T lymphocytes has been extensively reported in the literature. EVs may express MHC class II and other membrane surface molecules and carry antigens. The aim of this study was to investigate the role of EVs from Leishmania-infected macrophages as immune modulatory particles.

Methodology/Principal Findings

In this work it was shown that BALB/c mouse bone marrow-derived macrophages, either infected in vitro with Leishmania amazonensis or left uninfected, release comparable amounts of 50–300 nm-diameter extracellular vesicles (EVs). The EVs were characterized by flow cytometry and electron microscopy. The incubation of naïve macrophages with these EVs for 48 hours led to a statistically significant increase in the production of the cytokines IL-12, IL-1β, and TNF-α.

Conclusions/Significance

EVs derived from macrophages infected with L. amazonensis induce other macrophages, which in vivo could be bystander cells, to produce the proinflammatory cytokines IL-12, IL-1β and TNF-α. This could contribute both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection.     

Gene Expression Analysis of the Microdissected Trophoblast Layer of Human Placenta after the Spontaneous Onset of Labor

Background

Despite increasing evidence that human parturition is associated with alteration in gene expression in the uteroplacental unit, the precise mechanisms that elicit spontaneous term labor in humans remain unknown. Our goal in this study was to compare the mRNA expression pattern of the trophoblast layer of normal term placenta between women who had given natural birth (labor group) and those who had undergone an elective cesarean section without labor (non-labor group).

Methods

We collected placental tissue samples from six pregnant women after term vaginal deliveries (labor group) and from six pregnant women after scheduled Cesarean sections (non-labor group). Frozen sections were made immediately after placental delivery. Because the placenta is a heterogeneous tissue composed of several cell types, we used laser capture microdissection to separate the trophoblast layer from the rest of the placental tissues.

Results

A number of genes were differentially expressed in the trophoblast layer when the labor and non-labor groups were compared. The expression of SIRT1, KAP1, and CRH was significantly lower in the trophoblast layer of the labor group than of the non-labor group. The expression of IL-1b, NF-kB1 and TLR 8 in the labor group was significantly higher than that in the non-labor group.

Conclusions

Human term labor may be closely associated with inflammatory response. We suggest that downregulation of SIRT1, KAP1, and CRH gene expression in the trophoblast may play a key role in parturition and initiation of labor in pregnant human females.     

The expression and function of fatty acid transport protein-2 and -4 in the murine placenta

Background

The uptake and trans-placental trafficking of fatty acids from the maternal blood into the fetal circulation are essential for embryonic development, and involve several families of proteins. Fatty acid transport proteins (FATPs) uniquely transport fatty acids into cells. We surmised that placental FATPs are germane for fetal growth, and are regulated during hypoxic stress, which is associated with reduced fat supply to the fetus.

Methodology/Principal Findings

Using cultured primary term human trophoblasts we found that FATP2, FATP4 and FATP6 were highly expressed in trophoblasts. Hypoxia enhanced the expression of trophoblastic FATP2 and reduced the expression of FATP4, with no change in FATP6. We also found that Fatp2 and Fatp4 are expressed in the mouse amnion and placenta, respectively. Mice deficient in Fatp2 or Fatp4 did not deviate from normal Mendelian distribution, with both embryos and placentas exhibiting normal weight and morphology, triglyceride content, and expression of genes related to fatty acid mobilization.

Conclusions/Significance

We conclude that even though hypoxia regulates the expression of FATP2 and FATP4 in human trophoblasts, mouse Fatp2 and Fatp4 are not essential for intrauterine fetal growth.     

Aetiology of Pulmonary Symptoms in HIV-Infected Smear Negative Recurrent PTB Suspects in Kampala,Uganda: A Cross-Sectional Study

Introduction

Previously treated TB patients with pulmonary symptoms are often considered recurrent TB suspects in the resource-limited settings, where investigations are limited to microscopy and chest x-ray. Category II anti-TB drugs may be inappropriate and may expose patients to pill burden, drug toxicities and drug-drug interactions.

Objective

To determine the causes of pulmonary symptoms in HIV-infected smear negative recurrent pulmonary tuberculosis suspects at Mulago Hospital, Kampala.

Methods

Between March 2008 and December 2011, induced sputum samples of 178 consented HIV-infected smear negative recurrent TB suspects in Kampala were subjected to MGIT and LJ cultures for mycobacteria at TB Reference Laboratory, Kampala. Processed sputum samples were also tested by PCR to detect 18S rRNA gene of P.jirovecii and cultured for other bacteria.

Results

Bacteria, M. tuberculosis and Pneumocystis jirovecii were detected in 27%, 18% and 6.7% of patients respectively and 53.4% of the specimens had no microorganisms. S. pneumoniae, M. catarrhalis and H. influenzae were 100% susceptible to chloramphenicol and erythromycin but co-trimoxazole resistant.

Conclusion

At least 81.5% of participants had no microbiologically-confirmed TB. However our findings call for thorough investigation of HIV-infected smear negative recurrent TB suspects to guide cost effective treatment.     

Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia

Rationale

The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia.

Objectives

The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia.

Methods

To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage.

Measurements and Primary Results

SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality.

Conclusion

These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.     

Comparison of Vector Competence of Aedes mediovittatus and Aedes aegypti for Dengue Virus: Implications for Dengue Control in the Caribbean

Background

Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes.

Methods

To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1–4 per os at viral titers of 5–6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution.

Results

Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus.

Conclusions

This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.