Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

Background Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin–piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. Methods Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0–3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. Results Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0–3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. Conclusions This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance

Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs

Potent acyl-CoA synthetase 10 inhibitors kill <i>Plasmodium falciparum</i> by disrupting triglyceride formation

Identifying how small molecules act to kill malaria parasites can lead to new chemically validated targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein&nbsp;stability

Selection at Work in Plasmodium Falciparum: Lessons From the Expanded Acyl CoA Synthetase Gene Family and in Vitro Artemisinin Resistance.

Approximately one third of the world’s population is at risk of contracting malaria. The World Health Organization estimates there were over 200 million news cases of malaria in 2015, resulting in nearly 500,000 deaths from this preventable disease. The majority of fatalities occur in Sub-Saharan Africa, where Plasmodium falciparum malaria causes severe disease in children under the age of five and pregnant women. In the last decade, increased anti-malaria interventions have resulted in substantial decreases in cases and fatalities. However, the recent emergence of artemisinin drug resistance in Southeast Asia threatens these gains, and the loss of another first-line antimalarial therapy would be a devastating setback. The first goal of this work was to identify genetic markers of artemisinin drug resistance. Identifying the genetic determinants and molecular mechanisms of artemisinin resistance is crucial for understanding the emergence of this phenomenon and tracking the spread of these drug resistant parasites. Over the course of four years, we used an in vitro drug resistance selection approach to generate three independent artemisinin-resistant lines. Here we characterize those lines, and present Pfcoronin, a kelch13-like protein, as a novel candidate marker for artemisinin resistance. This study identifies additional non-kelch13 molecular markers of artemisinin resistance, increases our understanding of how this resistance is acquired, and sheds light on the molecular mechanisms of artemisinin resistance in the parasite. In contrast to in vitro selection, natural selection of parasites occurs during natural infection. Investigation of specific genes under selection in the parasite will increase our understanding of biological processes that provide a fitness advantage, and potentially identify novel pathways for therapeutic development. Here, we focused on the acyl Co-A synthetase (ACS) gene family, previously shown to be under recent positive selection in P. falciparum. The signatures of recent positive selection identified in natural parasite populations suggest that particular ACS alleles may confer a selective advantage. Using molecular genetics approaches, we show distinct expression and localization patterns for individual ACS isoforms, and identify a growth defect in the ACS5 knockout line. Follow up studies characterize the fatty acid and metabolic profiles of individual ACS knockout lines, and point to a role for ACS5 in central carbon metabolism in P. falciparum. Our investigation of the ACS gene family and their role in P. falciparum growth and metabolism led us to hypothesize a link between ACS activity and central carbon metabolism. In the final chapter, we explore the basic fatty acid and glucose requirements for P. falciparum growth in vitro, and present a metabolic profile for these starved parasites. Under starvation conditions, we were able to demonstrate fatty acid oxidation activity in the parasite. This is an unexpected finding, as this pathway was not previously annotated in the genome. Taken together, these two projects tell a story of the selective pressures acting on P. falciparum parasites. Investigating in vitro selected artemisinin-resistant lines provides important insights into genetic markers and acquisition of resistance. Molecular and biochemical characterization of a gene family under natural selection in P. falciparum increases our understanding of important metabolic pathways that support parasite growth.Biological Sciences in Public Healt

Exogenous kisspeptin enhances seasonal reproductive function in male Siberian hamsters

Contains all raw data used in the Functional Ecology article "Exogenous kisspeptin enhances seasonal reproductive function in male Siberian hamsters (Phodopus sungorus)" by Allison Bailey, Sandra Legan, and Gregory Demas, including hamster body mass, food intake, estimated testis volume, final reproductive mass, and serum hormone concentrations

WIPO Panel 4. Collective Management of Copyright and Related Rights - Developing Countries and Regional Perspectives

Includes descriptive metadata provided by producer in MP4 file: "Law School - WIPO Conference Video - Collective Management of Copyright and Related Rights: Developing Countries and Regional Perspectives."Issues discussed: What are the main problems encountered by societies in developing countries? The management of national and international repertoire. The role of governments and/or judiciary in the establishment and running of collective management organizations. The integration of regional entities in the worldwide network of collective management organizations: the objectives of the Caribbean Copyright Link (CCL). The "Latinet" network and its successful development. The developments of collective management in Canada, Brazil, Chile, Europe and United States of America: insights and comparison.World Intellectual Property Organization; Vanderbilt Law School; International Confederation of Societies of Authors, Composers and Publishers; American Society of Composers, Authors and Publishers, Broadcast Music, Inc.; SESAC Inc.Law Schoo

A Non-Radioactive DAPI-based High-Throughput In Vitro Assay to Assess Plasmodium falciparum Responsiveness to Antimalarials—Increased Sensitivity of P. falciparum to Chloroquine in Senegal

The spread of Plasmodium falciparum drug resistance is outpacing new antimalarial development and compromising effective malaria treatment. Combination therapy is widely implemented to prolong the effectiveness of currently approved antimalarials. To maximize utility of available drugs, periodic monitoring of drug efficacy and gathering of accurate information regarding parasite-sensitivity changes are essential. We describe a high-throughput, non-radioactive, field-based assay to evaluate in vitro antimalarial drug sensitivity of P. falciparum isolates from 40 Senegalese patients. Compared with earlier years, we found a significant decrease in chloroquine in vitro and in genotypic resistances (> 50% and > 65%, respectively, in previous studies) with only 23% of isolates showing resistance. This is possibly caused by a withdrawal of chloroquine from Senegal in 2002. We also found a range of artemisinin responses. Prevalence of drug resistance is dynamic and varies by region. Therefore, the implementation of non-radioactive, robust, high-throughput antimalarial sensitivity assays is critical for defining region-specific prophylaxis and treatment guidelines