Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

Frondoside A Suppressive Effects on Lung Cancer Survival,Tumor Growth,Angiogenesis, Invasion,and Metastasis

A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer.     

Species and Population Level Molecular Profiling Reveals Cryptic Recombination and Emergent Asymmetry in the Dimorphic Mating Locus of C. reinhardtii

Heteromorphic sex-determining regions or mating-type loci can contain large regions of non-recombining sequence where selection operates under different constraints than in freely recombining autosomal regions. Detailed studies of these non-recombining regions can provide insights into how genes are gained and lost, and how genetic isolation is maintained between mating haplotypes or sex chromosomes. The Chlamydomonas reinhardtii mating-type locus (MT) is a complex polygenic region characterized by sequence rearrangements and suppressed recombination between its two haplotypes, MT+ and MT−. We used new sequence information to redefine the genetic contents of MT and found repeated translocations from autosomes as well as sexually controlled expression patterns for several newly identified genes. We examined sequence diversity of MT genes from wild isolates of C. reinhardtii to investigate the impacts of recombination suppression. Our population data revealed two previously unreported types of genetic exchange in Chlamydomonas MT—gene conversion in the rearranged domains, and crossover exchanges in flanking domains—both of which contribute to maintenance of genetic homogeneity between haplotypes. To investigate the cause of blocked recombination in MT we assessed recombination rates in crosses where the parents were homozygous at MT. While normal recombination was restored in MT+×MT+ crosses, it was still suppressed in MT−×MT− crosses. These data revealed an underlying asymmetry in the two MT haplotypes and suggest that sequence rearrangements are insufficient to fully account for recombination suppression. Together our findings reveal new evolutionary dynamics for mating loci and have implications for the evolution of heteromorphic sex chromosomes and other non-recombining genomic regions.     

Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy

Background and Purpose

The targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer.

Methods

A fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature.

Results

The purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment.

Conclusion

These results suggest that this treatment could be useful as a targeted therapy for breast cancer.     

Neural Mechanisms Underlying Breathing Complexity

Breathing is maintained and controlled by a network of automatic neurons in the brainstem that generate respiratory rhythm and receive regulatory inputs. Breathing complexity therefore arises from respiratory central pattern generators modulated by peripheral and supra-spinal inputs. Very little is known on the brainstem neural substrates underlying breathing complexity in humans. We used both experimental and theoretical approaches to decipher these mechanisms in healthy humans and patients with chronic obstructive pulmonary disease (COPD). COPD is the most frequent chronic lung disease in the general population mainly due to tobacco smoke. In patients, airflow obstruction associated with hyperinflation and respiratory muscles weakness are key factors contributing to load-capacity imbalance and hence increased respiratory drive. Unexpectedly, we found that the patients breathed with a higher level of complexity during inspiration and expiration than controls. Using functional magnetic resonance imaging (fMRI), we scanned the brain of the participants to analyze the activity of two small regions involved in respiratory rhythmogenesis, the rostral ventro-lateral (VL) medulla (pre-Bötzinger complex) and the caudal VL pons (parafacial group). fMRI revealed in controls higher activity of the VL medulla suggesting active inspiration, while in patients higher activity of the VL pons suggesting active expiration. COPD patients reactivate the parafacial to sustain ventilation. These findings may be involved in the onset of respiratory failure when the neural network becomes overwhelmed by respiratory overload We show that central neural activity correlates with airflow complexity in healthy subjects and COPD patients, at rest and during inspiratory loading. We finally used a theoretical approach of respiratory rhythmogenesis that reproduces the kernel activity of neurons involved in the automatic breathing. The model reveals how a chaotic activity in neurons can contribute to chaos in airflow and reproduces key experimental fMRI findings.     

Omega-3 Eicosapentaenoic Acid Decreases CD133 Colon Cancer Stem-Like Cell Marker Expression While Increasing Sensitivity to Chemotherapy

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil.