A target repurposing approach identifies N-myristoyltransferase as a new candidate drug target in filarial nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC50 values of 2.5-10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis

Comparison of Costs, Re-Intervention Rates, and Length of Hospital Stay for Three Uterus Sparing Interventions for Uterine Fibroids: A 2-Year Retrospective Claims Analysis

David Eisenstein,1 Ghadear H Shukr,2 John J Carlow,3 Laura Kemp,4 Steve Yu5 1Department of Urology, Trinity/IHA Medical Group, Detroit, MI, USA; 2Department of Obstetrics and Gynecology, University of South Florida Morsani College of Medicine, Tampa Bay, FL, USA; 3Discovery Statistics, Laguna Niguel, CA, USA; 4Kemp Clinical Consulting Co LLC, Carlsbad, CA, USA; 5Department of Urology, Hoag Hospital, Newport Beach, CA, USACorrespondence: John J Carlow, Discovery Statistics, 31434 West Nine Drive, Laguna Niguel, CA, 92677, USA, Tel/Fax +1 760-519-7395, Email [email protected]: To describe two-year post-operative outcomes, and healthcare utilization of three uterus-sparing interventions used to treat women with intramural and/or subserosal uterine fibroids.Subjects and Methods: This was a post-market, randomized, prospective, multi-center, longitudinal, interventional, and comparative clinical study to evaluate the costs and health outcomes of LAP-RFA vs the standard uterine conserving technologies (myomectomy and UAE) for the treatment of symptomatic uterine fibroids in women who desire uterine conservation. For this RCT study, 54 subjects were randomized on a 1:1 ratio across the three procedures and followed out to two years. Their results were compared to retrospective US insurance claims from the IBM MarketScan® Commercial Database from 2017– 2020 for 96,854 women who underwent a uterus-sparing procedure for fibroids.Results: Mean ambulatory surgical center costs and the mean out-patient hospital costs were lowest for LAP-RFA ($13,134 and $14,428) and highest for UAE ($28,214 and $19,131). The total two-year re-intervention rate of any subsequent procedure (AM, LM, LAP-RFA, or UAE) was lowest in AM group (0%) followed by LM (4.2%), LAP-RFA (11%), and UAE (33%). Mean peri-operative reintervention costs and the mean reintervention total costs were $2429 and $5939 for LAP-RFA, $2122 and $8368 for LM, $4410 and $11,942 for AM, and $8113 and $46,692 for UAE subjects. In the RCT study, the average length of hospital stay was significantly less for the LAP-RFA group subjects (8.2 hours) in contrast to both the laparoscopic myomectomy group subjects (16.0 hours) and the abdominal myomectomy group subjects (33.6 hours). Despite the small numbers, two-year reintervention rates followed a similar pattern as the IBM MarketScan data.Conclusion: In comparing these three non-invasive approaches, LAP-RFA was associated with the lowest peri-operative cost, and UAE was associated with the highest peri-operative cost. Further studies are needed to assess the cost, effectiveness, and subject satisfaction with each procedure.Keywords: health economics, leiomyomas, interventional, cost/burde

Genomes of the human filarial parasites Mansonella perstans and Mansonella ozzardi

Funding Information: Research funding was provided by New England Biolabs, Ipswich, MA 01938, USA to AS, ZL, LE, RM, CP, CC. FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) supported the field research in Brazil through a research grant to M.U.F (2013/12723-7) and a doctoral scholarship to NL (2013/26928-0). Acknowledgments Funding Information: Research funding was provided by New England Biolabs, Ipswich, MA 01938, USA to AS, ZL, LE, RM, CP, CC. FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) supported the field research in Brazil through a research grant to M.U.F (2013/12723-7) and a doctoral scholarship to NL (2013/26928-0). Publisher Copyright: Copyright © 2023 Sinha, Li, Poole, Morgan, Ettwiller, Lima, Ferreira, Fombad, Wanji and Carlow.The filarial parasites Mansonella ozzardi and Mansonella perstans, causative agents of mansonellosis, infect hundreds of millions of people worldwide, yet remain among the most understudied of the human filarial pathogens. M. ozzardi is highly prevalent in Latin American countries and Caribbean Islands, while M. perstans is predominantly found in sub-Saharan Africa as well as in a few areas in South America. In addition to the differences in their geographical distribution, the two parasites are transmitted by different insect vectors, as well as exhibit differences in their responses to commonly used anthelminthic drugs. The lack of genome information has hindered investigations into the biology and evolution of Mansonella parasites and understanding the molecular basis of the clinical differences between species. In the current study, high quality genomes of two independent clinical isolates of M. perstans from Cameroon and two M. ozzardi isolates one from Brazil and one from Venezuela are reported. The genomes are approximately 76 Mb in size, encode about 10,000 genes each, and are largely complete based on BUSCO scores of about 90%, similar to other completed filarial genomes. These sequences represent the first genomes from Mansonella parasites and enabled a comparative genomic analysis of the similarities and differences between Mansonella and other filarial parasites. Horizontal DNA transfers (HDT) from mitochondria (nuMTs) as well as transfers from genomes of endosymbiotic Wolbachia bacteria (nuWTs) to the host nuclear genome were identified and analyzed. Sequence comparisons and phylogenetic analysis of known targets of anti-filarial drugs diethylcarbamazine (DEC), ivermectin and mebendazole revealed that all known target genes were present in both species, except for the DEC target encoded by gon-2 gene, which is fragmented in genome assemblies from both M. ozzardi isolates. These new reference genome sequences will provide a valuable resource for further studies on biology, symbiosis, evolution and drug discovery.publishersversionpublishe

Weak selection and stability of localized distributions in Ostwald ripening

We support and generalize a weak selection rule predicted recently for the self-similar asymptotics of the distribution function (DF) in the zero-volume-fraction limit of Ostwald ripening (OR). An asymptotic perturbation theory is developed that, when combined with an exact invariance property of the system, yields the selection rule, predicts a power-law convergence towards the selected self-similar DF and agrees well with our numerical simulations for the interface- and diffusion-controlled OR.Comment: 4 pages, 2 figures, submitted to PR

Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence

Phase 1 Trial of AMA1-C1/Alhydrogel plus CPG 7909: An Asexual Blood-Stage Vaccine for Plasmodium falciparum Malaria

Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909.A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 15), 80 microg of AMA1-C1/Alhydrogel (n = 30), or 80 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 30).Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 microg or 80 microg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 microg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition.The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing.ClinicalTrials.gov NCT00344539

Strongyloides stercoralis age-1: A Potential Regulator of Infective Larval Development in a Parasitic Nematode

Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The ‘dauer hypothesis’ predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 µM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases

Glutamate-Gated Chloride Channels of Haemonchus contortus Restore Drug Sensitivity to Ivermectin Resistant Caenorhabditis elegans

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance