Biology subjects

Ritchie, R.

Publications and source records attributed to Ritchie, R..

9 recordsLinked to original sources

bioRxiv2026

Comparative lipidomic profiling of the livestock pathogens Trypanosoma congolense and Trypanosoma brucei

African Animal Trypanosomosis (AAT) is a disease affecting domestic animals, in particular cattle, in sub-Saharan Africa, resulting in billion-dollar losses annually. New drugs to combat and control AAT are urgently required, yet few treatment candidates are currently on the horizon. This can be attributed, in part, to the relative challenges associated with culturing the clinically relevant parasite species in a laboratory environment. Particularly, effective culture of bloodstream form Trypanosoma congolense, the trypanosome species responsible for a large proportion of AAT disease in cattle, requires the use of goat serum, whilst T. brucei is typically cultured in FBS-supplemented culture. This constrains in vitro studies on biology, especially comparative analyses between AAT-causing species. The differing serum supplementation requirements of these two trypanosome species point to metabolic distinctions, which may be important considerations in developing experimental systems to enable the identification and design of novel, pan-species therapies. In this study, untargeted LC-MS lipidomics analyses were conducted to determine the relative lipidomic profiles of T. congolense and T. brucei bloodstream form parasites. Employing a new media formulation that permits effective in vitro culture of both species, it was possible to establish that their global lipidomic profiles are distinct. Notably, T. congolense exhibits a relatively low abundance of ether phospholipids compared to T. brucei, whilst also possessing an enrichment of long-chain polyunsaturated fatty acids (PUFAs). These observations indicate that there are significant differences in the ways these parasites synthesise and remodel their lipid complement, highlighting an evolutionary divergence between the species that likely carries implications for host-pathogen interactions as well as trypanosome membrane biology. Furthermore, this study demonstrates that fine-tuning fatty acid supplementation may aid in optimising a universal medium suited for multiple species of AAT parasites. SummaryMultiple species of protozoan parasites can cause African Animal Trypanosomosis (AAT) in livestock and other animals. However, AAT research has largely centred on a single species, Trypanosoma brucei, partially due to the comparative difficulties in sustaining the other economically important parasite species - Trypanosoma congolense and Trypanosoma vivax - in laboratory culture. In this work, we aimed to determine whether distinctions in use of lipids between T. brucei and T. congolense explains their differing in vitro culture requirements. Using a newly designed media formulation, it was possible to culture mammalian-infective forms of both parasite species under identical conditions, enabling direct comparison of their lipidome - a complete inventory of the different fats and lipids the cells contain. We demonstrate that the T. congolense lipidome significantly differs from that of T. brucei, and that T. congolense shows a preference for longer, more unsaturated lipids. These differences are likely to underlie species-specific differences observed during host infections. Furthermore, our work demonstrates that understanding the lipid biology of protozoan parasites aids in optimisation of laboratory culturing conditions, thereby facilitating further research into these understudied pathogens, including the development of new therapies.

microbiology↗

bioRxiv2026

DRP1 inhibition confers cardioprotection against doxorubicin while preserving anticancer efficacy

BackgroundAnthracyclines such as doxorubicin are effective chemotherapeutics but are limited by cardiotoxicity driven in part by mitochondrial dysfunction. Dysregulated mitochondrial dynamics, particularly excessive dynamin-related protein-1 (Drp1)-mediated fission, contribute to doxorubicin-induced cardiac injury and support selective survival of cancer cells. ObjectivesTo determine whether DRP1i2, a novel small molecule Drp1 inhibitor targeting a conserved domain shared between human and mouse, can function as a cardio-oncology therapeutic by reducing doxorubicin-induced cardiotoxicity while maintaining or enhancing anti-cancer efficacy. MethodsCardioprotective effects of DRP1i2 were evaluated in a murine model of chronic doxorubicin cardiotoxicity and in human induced pluripotent stem cell-derived cardiac microtissues exposed to acute doxorubicin injury. Anticancer activity was assessed across multiple cancer cell lines using 2D monolayers and 3D microtissues. ResultsIn vivo, DRP1i2 preserved left ventricular ejection fraction, reduced interstitial fibrosis and cardiomyocyte atrophy, and attenuated doxorubicin-induced myocardial proteomic remodelling. In human cardiac microtissues, DRP1i2 improved viability and restored contractile function despite persistent mitochondrial oxidative stress. DRP1i2 showed modest anticancer activity in MG63 osteosarcoma cells in both 2D and 3D systems and did not diminish doxorubicin efficacy in other cancer models (MDA-MB-231 breast, OVCAR3 ovarian, and A549 lung adenocarcinoma). Combined treatment further enhanced cytotoxicity selectively in MG63 cells. ConclusionsDRP1i2 exerts complementary cardioprotective and anticancer actions through modulation of shared mitochondrial pathways, identifying Drp1 as a druggable target in cardio-oncology. These findings support DRP1i2 as a first-in-class Drp1 inhibitor and highlight mitochondrial dynamics as a promising therapeutic axis to preserve anthracycline efficacy while reducing cardiotoxicity. Clinical PerspectivesExcessive Drp1-mediated mitochondrial fission links anthracycline cardiotoxicity with cancer cell survival. Inhibition with DRP1i2 preserved cardiac structure and function in a chronic doxorubicin cardiotoxicity model without compromising anti-cancer activity, representing mechanism-based cardioprotection, where the heart is protected by directly targeting the molecular processes driving injury. Translation will require pharmacologic profiling and testing in tumour-bearing and comorbid models, followed by early-phase trials to confirm safety and efficacy.

pharmacology and toxicology↗

bioRxiv2025

Genome-wide CRISPR base-editing screening defines drug response networks in Leishmania

We present a CRISPR/Cas9 cytosine base editing library for genome-wide loss-of-function screening in L. mexicana and apply it to dissect the genetics of Leishmania drug response. The results, accessible at https://www.LeishBASEeditDB.net, revealed novel response biomarkers for SbIII, miltefosine, amphotericin B, pentamidine, and the arylmethylaminosteroid 1c. We identified hundreds of loci linked to altered drug responses, including opposing effects among paralogs, cross-resistance, and collateral sensitivity. Among 41 validated candidates, we identified sterol defects in two novel amphotericin B markers, discovered a regulator of intracellular miltefosine transporter complex localization, and uncovered evidence that flagellar-associated defects reduce drug sensitivity. Parallel viability and motility screens revealed, for the first time, genome-wide fitness contributions of unique and multi-copy genes in Leishmania promastigotes. Our approach enables powerful reverse genetic screens across Leishmania species, advancing drug mechanism studies and guiding combination therapy designs. The library is available for others to screen a multitude of additional loss-of-function phenotypes.

microbiology↗

bioRxiv2025

Device encapsulated MSCs for adaptive secretome therapy to effectively target ischaemic heart injury

Effective long-term strategies to protect the ischaemic heart remain a significant challenge. Mesenchymal stem cells (MSCs) offer therapeutic potential primarily through their secretome, a bioactive factor-rich milieu with broad beneficial effects. However, existing delivery methods have not been shown to provide sustained benefits. Herein, we introduce an innovative approach for sustained MSC-secretome delivery for long-term cardioprotection. In a rat model of chronic myocardial infarction, Cymerus MSCs, derived from human induced pluripotent stem cells (iPSCs), were encapsulated in a Procyon immunoisolation device and implanted subcutaneously. A human-iPSC-derived engineered cardiac microtissue model was used to simulate ischaemia-reperfusion injury and assess cardioprotective effects in a human context. The MSC-loaded Procyon device significantly improved cardiac function and reduced adverse left ventricular remodelling over a 12-week period. These positive effects were consistent across both young and middle-aged, male and female rats, indicating broad applicability. The encapsulated MSCs remained viable and continuously released therapeutic secretome for 12 weeks. In vitro, the MSC secretome protected human engineered cardiac microtissues from simulated ischaemia-reperfusion injury, restoring contractile function, improving cell viability, and reducing oxidative stress. Proteomic analysis of MSCs revealed 179 unique cellular proteins post-implantation, linked to adaptive immune and inflammatory responses as well as wound healing. MSC secretome profiling revealed increased protein diversity associated with tissue repair and immune regulation, suggesting MSCs undergo an adaptive response to ischaemic conditions, enhancing their therapeutic potential. This translational study highlights a clinically viable, minimally invasive method for sustained cardioprotection, harnessing the MSC secretome to address a pivotal gap in current treatments for ischaemic heart disease.

pathology↗

bioRxiv2024

Title Longitudinal Observational (single cohort) Study on the Causes of Trypanocide Failure in cases of African Animal Trypanosomosis in Cattle Near Wildlife Protected Areas of Northern Tanzania

African animal trypanosomosis (AAT) in cattle is primarily managed through trypanocide administration and insecticide application. Trypanocides can be used for both treatment and prophylaxis, but failure is often reported; this may occur due to resistance, substandard drugs, or inappropriate administration. This study in Tanzania aims to quantify reasons for trypanocide failure. An observational year-long longitudinal study was conducted in high-risk AAT areas in Serengeti District from December 2019-October 2022. Purposive sampling targeted herds with high utilization of the prophylactic trypanocide isometamidium chloride (ISM). When a farmer administered a trypanocide (ISM, diminazine aceturate, homidium), the project veterinarian assessed administration and treatment outcomes were determined based on PCR results from blood samples. A multivariable mixed model was utilized to evaluate risk factors for prophylaxis failure. Quality analysis was performed on trypanocide samples using High Performance Liquid Chromatography. A total of 630 cattle from 21 farms were monitored for a year-long period. A total of 295 trypanocide administrations were reported, predominantly being ISM (56%) used for prophylaxis (87%). One-third of trypanocide administrations were not given adequately, and many trypanocides were given to animals that tested negative for trypanosome infections by PCR. Failures occurred in 7% (95% CI 3.0-14%) of curative treatments, and 44% (95% CI 35-42%) of prophylactic administrations. The brand of ISM was significantly associated with odds of prophylaxis failure (p = 0.011). On quality analysis, two ISM samples had no detectable ISM isomers, but the remainder of ISM and DA samples (n=46) fell within the range of acceptable levels. Drug counterfeiting, inadequate use of trypanocides, and resistance are all contributing to trypanocide failure, limiting effective AAT control and with implications for human disease risk. In order to curb trypanocide failure a multi-modal approach to managing the use of trypanocides is required to address all contributing factors. Author SummaryAfrican animal trypanosomosis (AAT) in cattle is commonly controlled through the use of medications called trypanocides. However, these interventions often fail to control AAT due to reasons such as drug resistance, poor-quality medicines, or incorrect administration. This study, conducted in Tanzania between December 2019 and October 2022, aimed to understand why trypanocides fail. Researchers focused particularly on cattle herds that used a preventive drug called isometamidium chloride (ISM). During the study, a veterinarian monitored how farmers administered trypanocides like ISM, diminazine aceturate (DA), or homidium, ensuring proper dosage and storage. Blood samples from cattle were tested to check if treatments were effective. The study found that one-third of trypanocide treatments were not given correctly, and many cattle treated with trypanocides were not actually infected. It was determined that 7% of treatments and 44% of preventive doses failed. The type of ISM brand used played a significant role in failure rates, with some brands performing worse than others. Additionally, tests revealed that some ISM samples lacked the active ingredients necessary for effectiveness. The study highlights that counterfeit drugs, improper use, and drug resistance all contribute to the problem, and a more comprehensive strategy is needed to tackle these issues.

microbiology↗

bioRxiv2023

Ovothiol is a redundant part of a complex thiol network in promastigote Leishmania mexicana

A gene encoding OvoA, a key enzyme involved in the biosynthesis of ovothiol, was excised form the genome of Leishmania mexicana promastigotes using CRISPR/cas mediated gene editing. The role of the enzyme in synthesising ovothiol was confirmed since both ovothiol A and ovothiol B were lost from the metabolome of the modified cells. The OvoA knockout line had similar growth kinetics to wild-type progenitor cells and, moreover, most of the changes in metabolism that accompanied the transition of log stage growth to stationary phase were mirrored in the KO line. Significant differences, however, were observed in the ratio of the reduced and oxidised forms of the other major low molecular weight thiols, glutathione and trypanothione, indicative of a role of these other thiols in maintaining reduced ovothiol and demonstrating an interconnected network of low molecular weight thiols in these cells. The OvoA knockout cells remained infective to macrophages where promastigotes transformed to amastigote forms in a manner similar to wild-type. The knockout line was tested for sensitivity to a range of current anti-leishmanial drugs and oxidative and nitrosative stresses. While generally the absence of ovothiol caused little or no change in sensitivity to these stress-inducing agents, enhanced sensitivity to amphotericin B was noted. Author summaryOvothiol is a low molecular weight histidine-derived thiol first described in sea urchin eggs, and later found in many organisms, including the protozoa of the order Kinetoplastida, that includes human pathogens such as the Leishmania species that cause leishmaniasis. Thiol metabolism in the Kinetoplastidae has been studied in some detail, particularly with regard to an unusual bis-glutathione, spermidine conjugate named trypanothione that takes on many of the roles performed by glutathione in most other organisms. Roles for ovothiol in Leishmania have not been previously defined, although potential roles in defence against oxidative stress have been hypothesised. A gene encoding the first enzyme of the pathway involved in ovothiol production, OvoA, was excised from the Leishmania mexicana genome. Its role in ovothiol synthesis was confirmed as ovothiol was absent from the mutants. Little changed, however, with respect to the phenotype of these cells, including their proliferation rate, their ability to infect macrophages or their sensitivity to a range of stress inducing agents. These included several leishmanicidal drugs, oxidative and nitrosative stresses. For amphotericin B, however, the Ovothiol lacking cells were more sensitive than wild-type indicating some role in defence against the impact of this drug.

biochemistry↗

bioRxiv2021

Kasturi Haldar, Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

microbiology↗

bioRxiv2021

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential drug sensitivity

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon drug sensitivity. Like T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly reduced in T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Comparative transcriptomics analysis showed higher levels of activity associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation and the succinate shunt in T. congolense. However, based on omics analysis and chemical inhibition, there does not appear to be significant levels of oxidative phosphorylation. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with pharmacological inhibitors, confirming a lack of essential electron transport chain activity in T. congolense, but increased sensitivity to inhibition of mitochondrial pyruvate import. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold greater EC50 against the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

microbiology↗

bioRxiv2019

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei the proteins that make up the kinetochore are highly divergent, with the inner kinetochore comprising at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2 and KKT3. We performed a phenotypic screen of T. brucei bloodstream forms with a Novartis kinase-focused inhibitor library, which identified a number of selective inhibitors with potent pan-kinetoplastid activity. Deconvolution of an amidobenzimidazole series using a selection of 37 T. brucei mutants that over-express known essential protein kinases identified CLK1 as the primary target. Biochemical studies show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with C215 in the ATP binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. We show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 to be essential for KKT2 function and for kinetochore assembly. We propose that CLK1 is part of a novel signalling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. This work highlights a novel drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

microbiology↗