The effects of hip- vs. knee-dominant hamstring exercise on biceps femoris morphology, strength, and sprint performance: a randomized intervention trial protocol.

BACKGROUND: The hamstrings are an important muscle group that contribute to horizontal force during sprint acceleration and are also the most injured muscle group in running-based sports. Given the significant time loss associated with hamstrings injury and impaired sprinting performance following return to sport, identifying exercises that drive adaptations that are both protective of strain injury and beneficial to sprint performance is important for the strength and conditioning professional. This paper describes the study protocol investigating the effects of a 6-week training program using either the hip-dominant Romanian deadlift (RDL) or the knee-dominant Nordic hamstring exercise (NHE) on hamstring strain injury risk factors and sprint performance. METHODS: A permuted block randomized (1:1 allocation) intervention trial will be conducted involving young, physically-active men and women. A target sample size of 32 will be recruited and enrolled participants will undergo baseline testing involving extended-field-of-view ultrasound imaging and shear wave elastography of the biceps femoris long head muscle, maximal hamstrings strength testing in both the RDL and NHE, and on-field sprint performance and biomechanics. Participants will complete the 6-week training intervention using either the RDL or NHE, according to group allocation. Baseline testing will be repeated at the end of the 6-week intervention followed by 2 weeks of detraining and a final testing session. The primary outcome will be regional changes in fascicle length with secondary outcomes including pennation angle, muscle cross sectional area, hamstring strength, and maximal sprint performance and biomechanics. An exploratory aim will determine changes in shear wave velocity. DISCUSSION: Despite extensive research showing the benefits of the NHE on reducing hamstring strain injury risk, alternative exercises, such as the RDL, may offer similar or potentially even greater benefits. The findings of this study will aim to inform future researchers and practitioners investigating alternatives to the NHE, such as the RDL, in terms of their effectiveness in reducing rates of hamstring strain injury in larger scale prospective intervention studies. TRIAL REGISTRATION: The trial is prospectively registered on ClinicalTrials.gov (NCT05455346; July 15, 2022).

Benchmarking Refined and Unrefined AlphaFold2 Structures for Hit Discovery.

The recently developed AlphaFold2 (AF2) algorithm predicts proteins' 3D structures from amino acid sequences. The open AlphaFold protein structure database covers the complete human proteome. Using an industry-leading molecular docking method (Glide), we investigated the virtual screening performance of 37 common drug targets, each with an AF2 structure and known holo and apo structures from the DUD-E data set. In a subset of 27 targets where the AF2 structures are suitable for refinement, the AF2 structures show comparable early enrichment of known active compounds (avg. EF 1%: 13.0) to apo structures (avg. EF 1%: 11.4) while falling behind early enrichment of the holo structures (avg. EF 1%: 24.2). With an induced-fit protocol (IFD-MD), we can refine the AF2 structures using an aligned known binding ligand as the template to improve the performance in structure-based virtual screening (avg. EF 1%: 18.9). Glide-generated docking poses of known binding ligands can also be used as templates for IFD-MD, achieving similar improvements (avg. EF 1% 18.0). Thus, with proper preparation and refinement, AF2 structures show considerable promise for in silico hit identification.

Frail patients having vascular surgery during the early COVID-19 pandemic experienced high rates of adverse perioperative events and amputation.

BACKGROUND: Frailty predicts adverse perioperative outcomes and increased mortality in patients having vascular surgery. Frailty assessment is a potential tool to inform resource allocation, and shared decision-making about vascular surgery in the resource constrained COVID-19 pandemic environment. This cohort study describes the prevalence of frailty in patients having vascular surgery and the association between frailty, mortality and perioperative outcomes. METHODS: The COVID-19 Vascular Service in Australia (COVER-AU) prospective cohort study evaluates 30-day and six-month outcomes for consecutive patients having vascular surgery in 11 Australian vascular units, March-July 2020. The primary outcome was mortality, with secondary outcomes procedure-related outcomes and hospital utilization. Frailty was assessed using the nine-point visual Clinical Frailty Score, scores of 5 or more considered frail. RESULTS: Of the 917 patients enrolled, 203 were frail (22.1%). The 30 day and 6 month mortality was 2.0% (n = 20) and 5.9% (n = 35) respectively with no significant difference between frail and non-frail patients (OR 1.68, 95%CI 0.79-3.54). However, frail patients stayed longer in hospital, had more perioperative complications, and were more likely to be readmitted or have a reoperation when compared to non-frail patients. At 6 months, frail patients had twice the odds of major amputation compared to non-frail patients, after adjustment (OR 2.01; 95% CI 1.17-3.78), driven by a high rate of amputation during the period of reduced surgical activity. CONCLUSION: Our findings highlight that older, frail patients, experience potentially preventable adverse outcomes and there is a need for targeted interventions to optimize care, especially in times of healthcare stress.

A multi-centre, open label, randomised, parallel-group, superiority Trial to compare the efficacy of URsodeoxycholic acid with RIFampicin in the management of women with severe early onset Intrahepatic Cholestasis of pregnancy: the TURRIFIC randomised trial.

BACKGROUND: Severe early onset (less than 34 weeks gestation) intrahepatic cholestasis of pregnancy (ICP) affects 0.1% of pregnant women in Australia and is associated with a 3-fold increased risk of stillbirth, fetal hypoxia and compromise, spontaneous preterm birth, as well as increased frequencies of pre-eclampsia and gestational diabetes. ICP is often familial and overlaps with other cholestatic disorders. Treatment options for ICP are not well established, although there are limited data to support the use of ursodeoxycholic acid (UDCA) to relieve pruritus, the main symptom. Rifampicin, a widely used antibiotic including in pregnant women, is effective in reducing pruritus in non-pregnancy cholestasis and has been used as a supplement to UDCA in severe ICP. Many women with ICP are electively delivered preterm, although there are no randomised data to support this approach. METHODS: We have initiated an international multicentre randomised clinical trial to compare the clinical efficacy of rifampicin tablets (300 mg bd) with that of UDCA tablets (up to 2000 mg daily) in reducing pruritus in women with ICP, using visual pruritus scores as a measuring tool. DISCUSSION: Our study will be the first to examine the outcomes of treatment specifically in the severe early onset form of ICP, comparing "standard" UDCA therapy with rifampicin, and so be able to provide for the first-time high-quality evidence for use of rifampicin in severe ICP. It will also allow an assessment of feasibility of a future trial to test whether elective early delivery in severe ICP is beneficial. TRIAL IDENTIFIERS: Australian New Zealand Clinical Trials Registration Number (ANZCTR): 12618000332224p (29/08/2018). HREC No: HREC/18/WCHN/36. EudraCT number: 2018-004011-44. IRAS: 272398. NHMRC registration: APP1152418 and APP117853.

Meaningful Engagement to Save Lives - Working relationship of a service user organisation with police and mental health services.

WHAT IS KNOWN?: Police and mental health services benefit from meaningful service user engagement. Partnerships with organizations that are representative of community members-such as service users-are the most empowering model of collaboration. WHAT THIS PAPER ADDS?: Describes how a service user organization can effectively advocate for change in the policing and mental health systems through both mutual collaboration and external pressure. IMPLICATIONS FOR PRACTICE?: Methods of creating change that can save lives through partnerships with service user organizations can be applied by service user organizations, police and mental health services. The methods described have the potential to reduce deaths and injury as a result of police action or mental healthcare practices.

Ursodeoxycholic acid improves feto-placental and offspring metabolic outcomes in hypercholanemic pregnancy.

Perturbations in the intrauterine environment can result in lifelong consequences for metabolic health during postnatal life. Intrahepatic cholestasis of pregnancy (ICP) can predispose offspring to metabolic disease in adulthood, likely due to a combination of the effects of increased bile acids, maternal dyslipidemia and deranged maternal and fetal lipid homeostasis. Whereas ursodeoxycholic acid (UDCA) is a commonly used treatment for ICP, no studies have yet addressed whether it can also prevent the metabolic effects of ICP in the offspring and fetoplacental unit. We therefore analyzed the lipid profile of fetal serum from untreated ICP, UDCA-treated ICP and uncomplicated pregnancies and found that UDCA ameliorates ICP-associated fetal dyslipidemia. We then investigated the effects of UDCA in a mouse model of hypercholanemic pregnancy and showed that it induces hepatoprotective mechanisms in the fetal liver, reduces hepatic fatty acid synthase (Fas) expression and improves glucose tolerance in the adult offspring. Finally, we showed that ICP leads to epigenetic changes in pathways of relevance to the offspring phenotype. We therefore conclude that UDCA can be used as an intervention in pregnancy to reduce features of metabolic disease in the offspring of hypercholanemic mothers.

Water Networks and Correlated Motions in Mutant Isocitrate Dehydrogenase 1 (IDH1) Are Critical for Allosteric Inhibitor Binding and Activity.

Point mutations in human isocitrate dehydrogenase 1 (IDH1) can drive malignancies, including lower-grade gliomas and secondary glioblastomas, chondrosarcomas, and acute myeloid leukemias. These mutations, which usually affect residue R132, ablate the normal activity of catalyzing the NADP+-dependent oxidation of isocitrate to α-ketoglutarate (αKG) while also acquiring a neomorphic activity of reducing αKG to d-2-hydroxyglutarate (D2HG). Mutant IDH1 can be selectively therapeutically targeted due to structural differences that occur in the wild type (WT) versus mutant form of the enzyme, though the full mechanisms of this selectivity are still under investigation. Here we probe the mechanistic features of the neomorphic activity and selective small molecule inhibition through a new lens, employing WaterMap and molecular dynamics simulations. These tools identified a high-energy path of water molecules connecting the inhibitor binding site with the αKG and NADP+ binding sites in mutant IDH1. This water path aligns spatially with the α10 helix from WT IDH1 crystal structures. Mutating residues at the termini of this water path specifically disrupted inhibitor binding and/or D2HG production, revealing additional key residues to consider in optimizing druglike molecules against mutant IDH1. Taken together, our findings from molecular simulations and mutant enzyme kinetic assays provide insight into how disrupting water paths through enzyme active sites can impact not only inhibitor potency but also substrate recognition and activity.

Protein-Ligand Binding Free Energy Calculations with FEP.

Accurate and reliable calculation of protein-ligand binding free energy is of central importance in computational biophysics and structure-based drug design. Among the various methods to calculate protein-ligand binding affinities, alchemical free energy perturbation (FEP) calculations performed by way of explicitly solvated molecular dynamics simulations (FEP/MD) provide a thermodynamically rigorous and complete description of the binding event and should in turn yield highly accurate predictions. Although the original theory of FEP was proposed more than 60 years ago, subsequent applications of FEP to compute protein-ligand binding free energies in the context of drug discovery projects over much of that time period was sporadic and generally unsuccessful. This was mainly due to the limited accuracy of the available force fields, inadequate sampling of the protein-ligand conformational space, complexity of simulation set up and analysis, and the large computational resources required to pursue such calculations. Over the past few years, there have been advances in computing power, classical force field accuracy, enhanced sampling algorithms, and simulation setup. This has led to newer FEP implementations such as the FEP+ technology developed by Schrödinger Inc., which has enabled accurate and reliable calculations of protein-ligand binding free energies and positioned free energy calculations to play a guiding role in small-molecule drug discovery. In this chapter, we outline the methodological advances in FEP+, including the OPLS3 force fields, the REST2 (Replica Exchange with Solute Tempering) enhanced sampling, the incorporation of REST2 sampling with conventional FEP (Free Energy Perturbation) through FEP/REST, and the advanced simulation setup and data analysis. The validation of FEP+ method in retrospective studies and the prospective applications in drug discovery projects are also discussed. We then present the recent extension of FEP+ method to handle challenging perturbations, including core-hopping transformations, macrocycle modifications, and reversible covalent inhibitor optimization. The limitations and pitfalls of the current FEP+ methodology and the best practices in real applications are also examined.

Cancer cell killing by target antigen engagement with engineered complementary intracellular antibody single domains fused to pro-caspase3.

Many tumour causing proteins, such as those expressed after chromosomal translocations or from point mutations, are intracellular and are not enzymes per se amenable to conventional drug targeting. We previously demonstrated an approach (Antibody-antigen Interaction Dependent Apoptosis (AIDA)) whereby a single anti-ß-galactosidase intracellular single chain Fv antibody fragment, fused to inactive procaspase-3, induced auto-activation of caspase-3 after binding to the tetrameric ß-galactosidase protein. We now demonstrate that co-expressing an anti-RAS heavy chain single VH domain, that binds to mutant RAS several thousand times more strongly than to wild type RAS, with a complementary light chain VL domain, caused programmed cell death (PCD) in mutant RAS expressing cells when each variable region is fused to procaspase-3. The effect requires binding of both anti-RAS variable region fragments and is RAS-specific, producing a tri-molecular complex that auto-activates the caspase pathway leading to cell death. AIDA can be generally applicable for any target protein inside cells by involving appropriate pairs of antigen-specific intracellular antibodies.

K-RasG12D Has a Potential Allosteric Small Molecule Binding Site.

KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771-785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-RasG12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-RasG12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind to four mutants in the P110 site, supporting our hypothesis that KAL-21404358 binds to the P110 site of K-RasG12D. This compound impaired the interaction of K-RasG12D with B-Raf and disrupted the RAF-MEK-ERK and PI3K-AKT signaling pathways. We synthesized additional compounds, based on the KAL-21404358 scaffold with more potent binding and greater aqueous solubility. In summary, these findings suggest that the P110 site is a potential site for binding of small molecule allosteric inhibitors of K-RasG12D.

Compulsory Use of the Backboard is Associated with Increased Frequency of Thoracolumbar Imaging.

BACKGROUND: Backboards have been shown to cause pain in uninjured patients. This may alter physical exam findings, leading emergency department (ED) providers to suspect a spinal injury when none exists resulting in additional imaging of the thoracolumbar spine. New York had previously employed a "Spinal Immobilization" protocol that included compulsory backboard application for all patients with suspected spinal injuries. In 2015, New York instituted a new "Spinal Motion Restriction" protocol that made backboard use optional for these patients. The objective of this study was to determine if this protocol change was associated with decreased backboard utilization and ED thoracolumbar spine imaging. METHODS: This was a retrospective before-and-after chart review of subjects transported by a single emergency medical services (EMS) agency to one of four EDs for emergency calls dispatched as motor vehicle collisions (MVC). EMS and ED data were included for all calls within a 6-month interval before and after the protocol change. The protocol change was implemented in the second half of 2015. Subject demographics, backboard use, and spine imaging were reviewed for the intervals January-June 2015 and January-June 2016. RESULTS: There were 818 subjects in the before period and 796 subjects in the after period. Subjects were similar in terms of gender, age and type of MVC in both periods. A backboard was utilized for 440 (54%) subjects in the before period and 92 (12%) subjects in the after period (p < 0.001). ED thoracic spine imaging was performed on 285 (35%) subjects in the before period, and 235 (30%) subjects in the after period (p = 0.02). ED lumbar spine imaging was performed for 335 (41%) subjects in the before period, and 281 (35%) subjects in the after period (p = 0.02). CONCLUSION: A shift from a spinal immobilization protocol to a spinal motion restriction protocol was associated with a decrease in backboard utilization by EMS providers and a decrease in thoracolumbar spine imaging by ED providers.

Intracellular immunization against HIV infection with an intracellular antibody that mimics HIV integrase binding to the cellular LEDGF protein.

Preventing the protein-protein interaction of the cellular chromatin binding protein Lens Epithelium-Derived Growth Factor (LEDGF) and human immunodeficiency virus (HIV) integrase is an important possible strategy for anti-viral treatment for AIDS. We have used Intracellular Antibody Capture technology to isolate a single VH antibody domain that binds to LEDGF. The crystal structure of the LEDGF-VH complex reveals that the single domain antibody mimics the effect of binding of HIV integrase to LEDGF which is crucial for HIV propagation. CD4-expressing T cell lines were constructed to constitutively express the LEDGF-binding VH and these cells showed interference with HIV viral replication, assayed by virus capsid protein p24 production. Therefore, pre-conditioning cells to express antibody fragments confers effective intracellular immunization for preventing chronic viral replication and can be a way to prevent HIV spread in infected patients. This raises the prospect that intracellular immunization strategies that focus on cellular components of viral integrase protein interactions can be used to combat the problems associated with latent HIV virus re-emergence in patients. New genome editing development, such as using CRISPR/cas9, offer the prospect intracellularly immunized T cells in HIV+ patients.

An expanded role for heterozygous mutations of ABCB4, ABCB11, ATP8B1, ABCC2 and TJP2 in intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) affects 1/140 UK pregnancies; with pruritus, hepatic impairment and elevated serum bile acids. Severe disease is complicated by spontaneous preterm delivery and stillbirth. Previous studies have reported mutations in hepatocellular transporters (ABCB4, ABCB11). High throughput sequencing in 147 patients was performed in the transporters ABCB4, ABCB11, ATP8B1, ABCC2 and tight junction protein 2 (TJP2). Twenty-six potentially damaging variants were identified with the following predicted protein changes: Twelve ABCB4 mutations - Arg47Gln, Met113Val, Glu161Gly, Thr175Ala, Glu528Glyfs*6, Arg590Gln, Ala601Ser, Glu884Ter, Gly722Ala, Tyr775Met (x2), Trp854Ter. Four potential ABCB11 mutations - Glu297Gly (x3) and a donor splice site mutation (intron 19). Five potential ATP8B1 mutations - Asn45Thr (x3), and two others, Glu114Gln and Lys203Glu. Two ABCC2 mutations - Glu1352Ala and a duplication (exons 24 and 25). Three potential mutations were identified in TJP2; Thr62Met (x2) and Thr626Ser. No patient harboured more than one mutation. All were heterozygous. An additional 545 cases were screened for the potential recurrent mutations of ATP8B1 (Asn45Thr) and TJP2 (Thr62Met) identifying three further occurrences of Asn45Thr. This study has expanded known mutations in ABCB4 and ABCB11 and identified roles in ICP for mutations in ATP8B1 and ABCC2. Possible novel mutations in TJP2 were also discovered.

Multivalent Small-Molecule Pan-RAS Inhibitors.

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Where is the client/patient voice in interprofessional healthcare team assessments? Findings from a one-day forum.

There is a growing interest in interprofessional care (IPC) as a way to provide better healthcare. However, it is difficult to evaluate this mode of healthcare delivery because identifying the appropriate measurement tool is a challenge, given the wide diversity in team composition and settings. Adding to this complexity is a key gap in the IPC evaluation research: the client/patient perspective. This perspective has generally not been included in the development of IPC healthcare team evaluations. The authors received a Canadian Institute for Health Research Planning Grant to host a one-day forum with 24 participants from across Canada representing health professions such as social work, medicine, occupational therapy, and physical therapy, in addition to researchers, client/patient advocates, and hospital administrators. The overarching goal of the forum was to create a demonstration project that supports the development of an IPC assessment tool for healthcare teams that includes clients/patients. Using a concept mapping methodology, participants discussed client/patient inclusion in IPC assessments, and through a consensus process, chose a demonstration project for further development.

Total synthesis of a biotinylated rocaglate: Selective targeting of the translation factors eIF4AI/II.

The total synthesis of a biotinylated derivative of methyl rocaglate is described. This compound was accessed from synthetic methyl rocaglate (2) via formation of the propargyl amide and subsequent click reaction with a biotin azide. Affinity purification revealed that biotinylated rocaglate (8) and methyl rocaglate (2) bind with high specificity to translation factors eIF4AI/II. This remarkable selectivity is in line with that found for the more complex rocaglate silvestrol (3).

The Most Crowded US Hospital Emergency Departments Did Not Adopt Effective Interventions To Improve Flow, 2007-10.

Emergency department (ED) crowding adversely affects patient care and outcomes. Despite national recommendations to address crowding, it persists in most US EDs today. Using nationally representative data, we evaluated the use of interventions to address crowding in US hospitals in the period 2007-10. We examined the relationship between crowding within an ED itself, measured as longer ED lengths-of-stay, and the number of interventions adopted. In our study period the average number of interventions adopted increased from 5.2 to 6.6, and seven of the seventeen studied interventions saw a significant increase in adoption. In general, more crowded EDs adopted greater numbers of interventions than less crowded EDs. However, in the most crowded quartile of EDs, a large proportion had not adopted effective interventions: 19 percent did not use bedside registration, and 94 percent did not use surgical schedule smoothing. Thus, while adoption of strategies to reduce ED crowding is increasing, many of the nation's most crowded EDs have not adopted proven interventions.

Barriers and facilitators to primary care for people with mental health and/or substance use issues: a qualitative study.

BACKGROUND: Mental health and/or substance use issues are associated with significant disparities in morbidity and mortality. The aim of this study was to identify the mechanisms underlying poor primary care access for this population. METHOD: This was a community-based participatory action qualitative study, in which 85 adults who self-identified as having a serious mental health and/or substance use issue and 17 service providers from various disciplines who worked with this population participated in a semi-structured interview. RESULTS: Client, service provider and health system barriers to access were identified. Client factors, including socioeconomic and psychological barriers, make it difficult for clients to access primary care, keep appointments, and/or prioritize their own health care. Provider factors, including knowledge and personal values related to mental health and substance use, determine the extent to which clients report their specific needs are met in the primary care setting. Health system factors, such as models of primary care delivery, determine the context within which both client and service provider factors operate. CONCLUSIONS: This study helps elucidate the mechanisms behind poor primary health care access among people with substance use and/or mental health issues. The results suggest that interdisciplinary, collaborative models of primary healthcare may improve accessibility and quality of care for this population, and that more education about mental health and substance use issues may be needed to support service providers in providing adequate care for their clients.

LMO2 at 25 years: a paradigm of chromosomal translocation proteins.

LMO2 was first discovered through proximity to frequently occurring chromosomal translocations in T cell acute lymphoblastic leukaemia (T-ALL). Subsequent studies on its role in tumours and in normal settings have highlighted LMO2 as an archetypical chromosomal translocation oncogene, activated by association with antigen receptor gene loci and a paradigm for translocation gene activation in T-ALL. The normal function of LMO2 in haematopoietic cell fate and angiogenesis suggests it is a master gene regulator exerting a dysfunctional control on differentiation following chromosomal translocations. Its importance in T cell neoplasia has been further emphasized by the recurrent findings of interstitial deletions of chromosome 11 near LMO2 and of LMO2 as a target of retroviral insertion gene activation during gene therapy trials for X chromosome-linked severe combined immuno-deficiency syndrome, both types of event leading to similar T cell leukaemia. The discovery of LMO2 in some B cell neoplasias and in some epithelial cancers suggests a more ubiquitous function as an oncogenic protein, and that the current development of novel inhibitors will be of great value in future cancer treatment. Further, the role of LMO2 in angiogenesis and in haematopoietic stem cells (HSCs) bodes well for targeting LMO2 in angiogenic disorders and in generating autologous induced HSCs for application in various clinical indications.

Use of (13)C stable isotope labelling for pathway and metabolic flux analysis in Leishmania parasites.

This protocol describes the combined use of metabolite profiling and stable isotope labelling to define pathways of central carbon metabolism in the protozoa parasite, Leishmania mexicana. Parasite stages are cultivated in standard or completely defined media and then rapidly transferred to chemically equivalent media containing a single (13)C-labelled nutrient. The incorporation of label can be followed over time or after establishment of isotopic equilibrium by harvesting parasites with rapid metabolic quenching. (13)C enrichment of multiple intracellular polar and apolar (lipidic) metabolites can be quantified using gas chromatography-mass spectrometry (GC-MS), while the uptake and secretion of (13)C-labelled metabolites can be measured by (13)C-NMR. Analysis of the mass isotopomer distribution of key metabolites provides information on pathway structure, while analysis of labelling kinetics can be used to infer metabolic fluxes. This protocol is exemplified using L. mexicana labelled with (13)C-U-glucose. The method can be used to measure perturbations in parasite metabolism induced by drug inhibition or genetic manipulation of enzyme levels and is broadly applicable to any cultured parasite stages.