Single-cell RNA sequencing reveals anti-tumor potency of CD56+ NK cells and CD8+ T cells in humanized mice via PD-1 and TIGIT co-targeting.

In solid tumors, the exhaustion of natural killer (NK) cells and cytotoxic T cells in the immunosuppressive tumor microenvironment poses challenges for effective tumor control. Conventional humanized mouse models of hepatocellular carcinoma patient-derived xenografts (HCC-PDX) encounter limitations in NK cell infiltration, hindering studies on NK cell immunobiology. Here, we introduce an improved humanized mouse model with restored NK cell reconstitution and infiltration in HCC-PDX, coupled with single-cell RNA sequencing (scRNA-seq) to identify potential anti-HCC treatments. A single administration of adeno-associated virus carrying human interleukin-15 reinstated persistent NK cell reconstitution and infiltration in HCC-PDX in humanized mice. scRNA-seq revealed NK cell and T cell subpopulations with heightened PDCD1 and TIGIT levels. Notably, combination therapy with anti-PD-1 and anti-TIGIT antibodies alleviated HCC burden in humanized mice, demonstrating NK cell-dependent efficacy. Bulk-RNA sequencing analysis also revealed significant alterations in the tumor transcriptome that may contribute to further resistance after combination therapy, warranting further investigations. As an emerging strategy, ongoing clinical trials with anti-PD-1 and anti-TIGIT antibodies provide limited data. The improved humanized mouse HCC-PDX model not only sheds light on the pivotal role of NK cells but also serves as a robust platform for evaluating safety and anti-tumor efficacy of combination therapies and other potential regimens, complementing clinical insights.

Albumin-Mediated Drug Uptake by Organic Anion Transporter 1/3 Is Real: Implications for the Prediction of Active Renal Secretion Clearance.

Modulation of the transport-mediated active uptake by human serum albumin (HSA) for highly protein-bound substrates has been reported and improved the in vitro-to-in vivo extrapolation (IVIVE) of hepatic clearance. However, evidence for the relevance of such a phenomenon in the case of renal transporters is sparse. In this study, transport of renal organic anion transporter 1 or 3 (OAT1/3) substrates into conditionally immortalized proximal tubular epithelial cells transduced with OAT1/3 was measured in the presence and absence of 1 and 4% HSA while keeping the unbound substrate concentration constant (based on measured fraction unbound, fu,inc). In the presence of 4% HSA, the unbound intrinsic active uptake clearance (CLint,u,active) of six highly protein-bound substrates increased substantially relative to the HSA-free control (3.5- to 122-fold for the OAT1 CLint,u,active, and up to 28-fold for the OAT3 CLint,u,active). The albumin-mediated uptake effect (fold increase in CLint,u,active) was more pronounced with highly bound substrates compared to no effect seen for weakly protein-bound substrates adefovir (OAT1-specific) and oseltamivir carboxylate (OAT3-specific). The relationship between OAT1/3 CLint,u,active and fu,inc agreed with the facilitated-dissociation model; a relationship was established between the albumin-mediated fold change in CLint,u,active and fu,inc for both the OAT1 and OAT3, with implications for IVIVE modeling. The relative activity factor and the relative expression factor based on global proteomic quantification of in vitro OAT1/3 expression were applied for IVIVE of renal clearance. The inclusion of HSA improved the bottom-up prediction of the level of OAT1/3-mediated secretion and renal clearance (CLsec and CLr), in contrast to the underprediction observed with the control (HSA-free) scenario. For the first time, this study confirmed the presence of the albumin-mediated uptake effect with renal OAT1/3 transporters; the extent of the effect was more pronounced for highly protein-bound substrates. We recommend the inclusion of HSA in routine in vitro OAT1/3 assays due to considerable improvements in the IVIVE of CLsec and CLr.

External auricle temperature enhances ear-based wearable accuracy during physiological strain monitoring in the heat.

Body core temperature (Tc) monitoring is crucial for minimizing heat injury risk. However, validated strategies are invasive and expensive. Although promising, aural canal temperature (Tac) is susceptible to environmental influences. This study investigated whether incorporation of external auricle temperature (Tea) into an ear-based Tc algorithm enhances its accuracy during multiple heat stress conditions. Twenty males (mean ± SD; age = 25 ± 3 years, BMI = 21.7 ± 1.8, body fat = 12 ± 3%, maximal aerobic capacity (VO2max) = 64 ± 7 ml/kg/min) donned an ear-based wearable and performed a passive heating (PAH), running (RUN) and brisk walking trial (WALK). PAH comprised of immersion in hot water (42.0 ± 0.3 °C). RUN (70 ± 3%VO2max) and WALK (50 ± 10%VO2max) were conducted in an environmental chamber (Tdb = 30.0 ± 0.2 °C, RH = 71 ± 2%). Several Tc models, developed using Tac, Tea and heart rate, were validated against gastrointestinal temperature. Inclusion of Tea as a model input improved the accuracy of the ear-based Tc algorithm. Our best performing model (Trf3) displayed good group prediction errors (mean bias error = - 0.02 ± 0.26 °C) but exhibited individual prediction errors (percentage target attainment ± 0.40 °C = 88%) that marginally exceeded our validity criterion. Therefore, Trf3 demonstrates potential utility for group-based Tc monitoring, with additional refinement needed to extend its applicability to personalized heat strain monitoring.

A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

Facing the Facts of Altered Plasma Protein Binding: Do Current Models Correctly Predict Changes in Fraction Unbound in Special Populations?

Accounting for variability in plasma protein binding of drugs is an essential input to physiologically-based pharmacokinetic (PBPK) models of special populations. Prediction of fraction unbound in plasma (fu) in such populations typically considers changes in plasma protein concentration while assuming that the binding affinity remains unchanged. A good correlation between predicted vs observed fu data reported for various drugs in a given special population is often used as a justification for such predictive methods. However, none of these analyses evaluated the prediction of the fold-change in fu in special populations relative to the reference population. This would be a more appropriate assessment of the predictivity, analogous to drug-drug interactions. In this study, predictive performance of the single protein binding model was assessed by predicting fu for alpha-1-acid glycoprotein and albumin bound drugs in hepatic impairment, renal impairment, paediatric, elderly, patients with inflammatory disease, and in different ethnic groups for a dataset of >200 drugs. For albumin models, the concordance correlation coefficients for predicted fu were >0.90 for 16 out of 17 populations with sub-groups, indicating strong agreement between predicted and observed values. In contrast, concordance correlation coefficients for predicted fold-change in fu for the same dataset were <0.38 for all populations and sub-groups. Trends were similar for alpha-1-acid glycoprotein models. Accordingly, the predictions of fu solely based on changes in protein concentrations in plasma cannot explain the observed values in some special populations. We recommend further consideration of the impact of changes in special populations to endogenous substances that competitively bind to plasma proteins, and changes in albumin structure due to posttranslational modifications. PBPK models of special populations for highly bound drugs should preferably use measured fu data to ensure reliable prediction of drug exposure or compare predicted unbound drug exposure between populations knowing that these will not be sensitive to changes in fu.

XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells.

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.

Up-regulation of SLC7A11/xCT creates a vulnerability to selenocystine-induced cytotoxicity.

The SLC7A11/xCT cystine and glutamate antiporter has emerged as an attractive target for cancer therapy due to its selective overexpression in multiple cancers and its role in preventing ferroptosis. Utilizing pharmacological and genetic approaches in hepatocellular carcinoma cell lines, we demonstrate that overexpression of SLC7A11 engenders hypersensitivity towards l-selenocystine, a naturally occurring diselenide that bears close structural similarity to l-cystine. We find that the abundance of SLC7A11 positively correlates with sensitivity to l-selenocystine, but surprisingly, not to Erastin, an inhibitor of SLC7A11 activity. Our data indicate that SLC7A11 acts as a transport channel for l-selenocystine, which preferentially incites acute oxidative stress and damage eventuating to cell death in cells that highly express SLC7A11. Hence, our findings raise the prospect of l-selenocystine administration as a novel strategy for targeting cancers that up-regulate SLC7A11 expression.

Vagal sensory neurons mediate the Bezold-Jarisch reflex and induce syncope.

Visceral sensory pathways mediate homeostatic reflexes, the dysfunction of which leads to many neurological disorders1. The Bezold-Jarisch reflex (BJR), first described2,3 in 1867, is a cardioinhibitory reflex that is speculated to be mediated by vagal sensory neurons (VSNs) that also triggers syncope. However, the molecular identity, anatomical organization, physiological characteristics and behavioural influence of cardiac VSNs remain mostly unknown. Here we leveraged single-cell RNA-sequencing data and HYBRiD tissue clearing4 to show that VSNs that express neuropeptide Y receptor Y2 (NPY2R) predominately connect the heart ventricular wall to the area postrema. Optogenetic activation of NPY2R VSNs elicits the classic triad of BJR responses-hypotension, bradycardia and suppressed respiration-and causes an animal to faint. Photostimulation during high-resolution echocardiography and laser Doppler flowmetry with behavioural observation revealed a range of phenotypes reflected in clinical syncope, including reduced cardiac output, cerebral hypoperfusion, pupil dilation and eye-roll. Large-scale Neuropixels brain recordings and machine-learning-based modelling showed that this manipulation causes the suppression of activity across a large distributed neuronal population that is not explained by changes in spontaneous behavioural movements. Additionally, bidirectional manipulation of the periventricular zone had a push-pull effect, with inhibition leading to longer syncope periods and activation inducing arousal. Finally, ablating NPY2R VSNs specifically abolished the BJR. Combined, these results demonstrate a genetically defined cardiac reflex that recapitulates characteristics of human syncope at physiological, behavioural and neural network levels.

General practitioners' perceptions of the roles of community pharmacists and their willingness to collaborate with pharmacists in primary care.

BACKGROUND: Community pharmacists (CPs) have the capacity to contribute to patient care given their expertise in medication and accessibility to residents in the community. However, multidisciplinary patient care programmes where CPs collaborate with general practitioners (GPs) in patient care is rare in Singapore despite increasing healthcare demand. OBJECTIVES: This study explores GPs' perceptions of CPs' current roles and GPs' ideas for and attitudes towards interprofessional collaboration. METHODS: Semi-structured qualitative interviews were conducted with 20 private GPs from August to December 2020 via an online video-chat platform. GPs were recruited from the Primacy Care Research Network (pcRn), primary care networks, and using snowballing strategies. All interviews were recorded, transcribed and coded thematically. RESULTS: Current working relationships between GPs and CPs appeared amicable but limited. GPs appreciate the existing roles of CPs: dispensing drugs not stocked in their practices and clarifying prescription details. Still, GPs appeared to rarely consider collaborative working. GPs acknowledged that CPs could enhance patient care with initiatives including medication reconciliation and advising on using medical devices. It was suggested that CPs could coordinate the purchase of drugs for primary care networks to improve GPs' inventory management, but less enthusiasm was expressed for clinical collaborations with CPs. Major concerns about GP-CP clinical collaborations included direct competition with GPs' own business interests, perceived low acceptability of pharmacy-led services by patients (citing extra time and cost), threat to continuity of care and the absence of a shared patient electronic health record system. Current funding mechanisms do not enable reimbursement of clinical services provided by CPs. Adoption of telemedicine technologies and governmental financial support were identified as possible enablers of GP-CP collaboration. CONCLUSIONS: GPs saw potential in CPs' increased involvement in patient care, but perceived multiple barriers. Strategies focusing on overcoming these barriers could enable GP-CP collaboration to enhance patient care.

Development of 4-Pyridoxic Acid PBPK Model to Support Biomarker-Informed Evaluation of OAT1/3 Inhibition and Effect of Chronic Kidney Disease.

Monitoring endogenous biomarkers is increasingly used to evaluate transporter-mediated drug-drug interactions (DDIs) in early drug development and may be applied to elucidate changes in transporter activity in disease. 4-pyridoxic acid (PDA) has been identified as the most sensitive plasma endogenous biomarker of renal organic anion transporters (OAT1/3). Increase in PDA baseline concentrations was observed after administration of probenecid, a strong clinical inhibitor of OAT1/3 and also in patients with chronic kidney disease (CKD). The aim of this study was to develop and verify a physiologically-based pharmacokinetic (PBPK) model of PDA, to predict the magnitude of probenecid DDI and predict the CKD-related changes in PDA baseline. The PBPK model for PDA was first developed in healthy population, building on from previous population pharmacokinetic modeling, and incorporating a mechanistic kidney model to consider OAT1/3-mediated renal secretion. Probenecid PBPK model was adapted from the Simcyp database and re-verified to capture its dose-dependent pharmacokinetics (n = 9 studies). The PBPK model successfully predicted the PDA plasma concentrations, area under the curve, and renal clearance in healthy subjects at baseline and after single/multiple probenecid doses. Prospective simulations in severe CKD predicted successfully the increase in PDA plasma concentration relative to healthy (within 2-fold of observed data) after accounting for 60% increase in fraction unbound in plasma and additional 50% decline in OAT1/3 activity beyond the decrease in glomerular filtration rate. The verified PDA PBPK model supports future robust evaluation of OAT1/3 DDI in drug development and increases our confidence in predicting exposure and renal secretion in patients with CKD.

A guide to the BRAIN Initiative Cell Census Network data ecosystem.

Characterizing cellular diversity at different levels of biological organization and across data modalities is a prerequisite to understanding the function of cell types in the brain. Classification of neurons is also essential to manipulate cell types in controlled ways and to understand their variation and vulnerability in brain disorders. The BRAIN Initiative Cell Census Network (BICCN) is an integrated network of data-generating centers, data archives, and data standards developers, with the goal of systematic multimodal brain cell type profiling and characterization. Emphasis of the BICCN is on the whole mouse brain with demonstration of prototype feasibility for human and nonhuman primate (NHP) brains. Here, we provide a guide to the cellular and spatial approaches employed by the BICCN, and to accessing and using these data and extensive resources, including the BRAIN Cell Data Center (BCDC), which serves to manage and integrate data across the ecosystem. We illustrate the power of the BICCN data ecosystem through vignettes highlighting several BICCN analysis and visualization tools. Finally, we present emerging standards that have been developed or adopted toward Findable, Accessible, Interoperable, and Reusable (FAIR) neuroscience. The combined BICCN ecosystem provides a comprehensive resource for the exploration and analysis of cell types in the brain.

Mechanistic Middle-Out Physiologically Based Toxicokinetic Modeling of Transporter-Dependent Disposition of Perfluorooctanoic Acid in Humans.

Perfluorooctanoic acid (PFOA) is an environmental toxicant exhibiting a years-long biological half-life (t1/2) in humans and is linked with adverse health effects. However, limited understanding of its toxicokinetics (TK) has obstructed the necessary risk assessment. Here, we constructed the first middle-out physiologically based toxicokinetic (PBTK) model to mechanistically explain the persistence of PFOA in humans. In vitro transporter kinetics were thoroughly characterized and scaled up to in vivo clearances using quantitative proteomics-based in vitro-to-in vivo extrapolation. These data and physicochemical parameters of PFOA were used to parameterize our model. We uncovered a novel uptake transporter for PFOA, highly likely to be monocarboxylate transporter 1 which is ubiquitously expressed in body tissues and may mediate broad tissue penetration. Our model was able to recapitulate clinical data from a phase I dose-escalation trial and divergent half-lives from clinical trial and biomonitoring studies. Simulations and sensitivity analyses confirmed the importance of renal transporters in driving extensive PFOA reabsorption, reducing its clearance and augmenting its t1/2. Crucially, the inclusion of a hypothetical, saturable renal basolateral efflux transporter provided the first unified explanation for the divergent t1/2 of PFOA reported in clinical (116 days) versus biomonitoring studies (1.3-3.9 years). Efforts are underway to build PBTK models for other perfluoroalkyl substances using similar workflows to assess their TK profiles and facilitate risk assessments.

Functional MRI reveals brain-wide actions of thalamically-initiated oscillatory activities on associative memory consolidation.

As a key oscillatory activity in the brain, thalamic spindle activities are long believed to support memory consolidation. However, their propagation characteristics and causal actions at systems level remain unclear. Using functional MRI (fMRI) and electrophysiology recordings in male rats, we found that optogenetically-evoked somatosensory thalamic spindle-like activities targeted numerous sensorimotor (cortex, thalamus, brainstem and basal ganglia) and non-sensorimotor limbic regions (cortex, amygdala, and hippocampus) in a stimulation frequency- and length-dependent manner. Thalamic stimulation at slow spindle frequency (8 Hz) and long spindle length (3 s) evoked the most robust brain-wide cross-modal activities. Behaviorally, evoking these global cross-modal activities during memory consolidation improved visual-somatosensory associative memory performance. More importantly, parallel visual fMRI experiments uncovered response potentiation in brain-wide sensorimotor and limbic integrative regions, especially superior colliculus, periaqueductal gray, and insular, retrosplenial and frontal cortices. Our study directly reveals that thalamic spindle activities propagate in a spatiotemporally specific manner and that they consolidate associative memory by strengthening multi-target memory representation.

Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis.

Cancer cells undergo transcriptional reprogramming to drive tumor progression and metastasis. Using cancer cell lines and patient-derived tumor organoids, we demonstrate that loss of the negative elongation factor (NELF) complex inhibits breast cancer development through downregulating epithelial-mesenchymal transition (EMT) and stemness-associated genes. Quantitative multiplexed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (qPLEX-RIME) further reveals a significant rewiring of NELF-E-associated chromatin partners as a function of EMT and a co-option of NELF-E with the key EMT transcription factor SLUG. Accordingly, loss of NELF-E leads to impaired SLUG binding on chromatin. Through integrative transcriptomic and genomic analyses, we identify the histone acetyltransferase, KAT2B, as a key functional target of NELF-E-SLUG. Genetic and pharmacological inactivation of KAT2B ameliorate the expression of EMT markers, phenocopying NELF ablation. Elevated expression of NELF-E and KAT2B is associated with poorer prognosis in breast cancer patients, highlighting the clinical relevance of our findings. Taken together, we uncover a crucial role of the NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis.

Brain Data Standards - A method for building data-driven cell-type ontologies.

Large-scale single-cell 'omics profiling is being used to define a complete catalogue of brain cell types, something that traditional methods struggle with due to the diversity and complexity of the brain. But this poses a problem: How do we organise such a catalogue - providing a standard way to refer to the cell types discovered, linking their classification and properties to supporting data? Cell ontologies provide a partial solution to these problems, but no existing ontology schemas support the definition of cell types by direct reference to supporting data, classification of cell types using classifications derived directly from data, or links from cell types to marker sets along with confidence scores. Here we describe a generally applicable schema that solves these problems and its application in a semi-automated pipeline to build a data-linked extension to the Cell Ontology representing cell types in the Primary Motor Cortex of humans, mice and marmosets. The methods and resulting ontology are designed to be scalable and applicable to similar whole-brain atlases currently in preparation.

Inpatient rehabilitation wheelchair management quality improvement project: Implications for patients with spinal cord injury.

Context/Objective: Evaluate hospital fleet wheelchair (WC) requests submitted by physical therapists (PT) for patients with spinal cord injury (SCI) to trial and use during inpatient rehabilitation.Design: Quality improvement project secondary analysis of delivery process and WC trials.Setting: Urban inpatient rehabilitation hospital.Participants: Internal review of 4,371 WC requests narrowed to 750 patients with SCI.Interventions: PTs submitted WC requests between March 25, 2017, and September 30, 2019.Outcome Measures: WC delivery timeframe, level of SCI, type of WC.Results: PTs requested power (28%), and manual WC bases: standard (19.1%), tilt (18.9%), ultralight rigid (18.9%), ultralight folding (13.5%), and recliner (1.6%) respectively. Patients received fleet WCs 49.9% of the time within specified urgency timeframes. A Chi-Square test showed a significant association between WC request urgency and fulfillment within established timeframes (χ2 = 19.68, P < 0.001, n = 750). Broken down by urgency level: 60.0% low (n = 12), 56.2% medium (n = 244), and 39.9% high (n = 118) received their WC within established timeframes. Patients with cervical level SCI (n = 162) had the highest mean wait time of 8.28 days for power WCs. The second highest wait time was 6.29 days (SD 6.6) for manual ultralight rigid WCs (n = 34).Conclusion: Inpatient fleet WC delivery is critical to patients with SCI. Variation occurs by WC type requested and by the level of injury. Gaps exist in providing appropriate WCs in facility timeframe guidelines by the level of urgency that is within 24 h for high, 3-5 days for medium, and 5-7 days for low.

Ontology Development Kit: a toolkit for building, maintaining and standardizing biomedical ontologies.

Similar to managing software packages, managing the ontology life cycle involves multiple complex workflows such as preparing releases, continuous quality control checking and dependency management. To manage these processes, a diverse set of tools is required, from command-line utilities to powerful ontology-engineering environmentsr. Particularly in the biomedical domain, which has developed a set of highly diverse yet inter-dependent ontologies, standardizing release practices and metadata and establishing shared quality standards are crucial to enable interoperability. The Ontology Development Kit (ODK) provides a set of standardized, customizable and automatically executable workflows, and packages all required tooling in a single Docker image. In this paper, we provide an overview of how the ODK works, show how it is used in practice and describe how we envision it driving standardization efforts in our community. Database URL: https://github.com/INCATools/ontology-development-kit.

Tetratricopeptide repeat domain 9A knockout induces social anxiety and impairs offense behaviors in female mice.

Objectives: The involvement of tetratricopeptide repeat domain 9A (TTC9A) in anxiety-like behaviors through estrogen action has been reported in female mice, this study further investigated its effects on social anxiety and aggressive behaviors. Materials and sMethods: Using female Ttc9a knockout (Ttc9a-/-) mice, the role of TTC9A in anxiety was investigated in non-social and social environments through home-cage emergence and social interaction tests, respectively, whereas aggressive behaviors were examined under the female intruder test. Results: We observed significant social behavioral deficits with pronounced social and non-social anxiogenic phenotypes in female Ttc9a-/- mice. When tested for aggressive-like behaviors, we found a reduction in offense in Ttc9a-/- animals, suggesting that TTC9A deficiency impairs the offense responses in female mice. Conclusion: Future study investigating mechanisms underlying the social anxiety-like behavioral changes in Ttc9a-/- mice may promote the understanding of social and anxiety disorders.

Efficacy of Isothermic Conditioning over Military-Based Heat Acclimatization and Interval Training in Tropical Native Males.

PURPOSE: We compared the effectiveness of three field-based training programs, namely military-based heat acclimatization (MHA), isothermic conditioning (IC) and interval training (IT), in inducing physiological adaptations in tropical natives. METHODS: Fifty-one untrained tropical native males (mean ± standard deviation: age, 25 ± 2 yr; body mass index, 23.6 ± 3.2 kg·m -2 ; body fat, 19% ± 5%; 2.4-km run time, 13.2 ± 0.9 min) donned the Full Battle Order attire (22 kg) and performed a treadmill route march heat stress test in an environmental chamber (dry bulb temperature, 29.9°C ± 0.5°C; relative humidity, 70% ± 3%). Heat stress tests were conducted before (PRE) and after (POST) a 2-wk training intervention consisting of either a MHA ( n = 17, 10 sessions of military-based heat acclimatization), IC ( n = 17, 10 sessions with target gastrointestinal temperature ( Tgi ) ≥ 38.5°C) or IT ( n = 17, six sessions of high-intensity interval training) program. Tgi , HR, mean weighted skin temperature ( Tsk ), physiological strain index (PSI) and thigh-predicted sweat sodium concentration ([Na + ]) were measured and analyzed by one-factor and two-factor mixed design ANOVA with a 0.05 level of significance. RESULTS: Field-based IC induced a greater thermal stimulus than MHA ( P = 0.029) and IT ( P < 0.001) during training. Reductions in mean exercise Tgi (-0.2°C [-0.3°C, 0.0°C]; P = 0.009) , PSI (-0.4 [-0.7, -0.1]; P = 0.015) and thigh-predicted sweat [Na + ] (-9 [-13, -5 mmol·L -1 ]; P < 0.001) were observed in IC but not MHA and IT (all P > 0.05). Resting HR (MHA, -4 bpm [-7, 0 bpm]; P = 0.025; IC, -7 bpm [-10, -4 bpm]; P < 0.001; IT, -4 bpm [-8, -1 bpm]; P = 0.008) and mean exercise HR (MHA, -4 [-8, 0 bpm]; P = 0.034; IC, -11 bpm [-15, -8 bpm]; P < 0.001, IT = -5 bpm [-9, -1 bpm]; P = 0.012) were lowered in all groups after training. Isothermic conditioning elicited a greater attenuation in mean exercise HR and thigh-predicted sweat [Na + ] relative to MHA (both P < 0.05). No between-group differences were observed when comparing MHA and IT (all P > 0.05). CONCLUSIONS: Isothermic conditioning induced a more complete heat-adapted phenotype relative to MHA and IT. Interval training may serve as a time efficient alternative to MHA.