Insights for precision oncology from the integration of genomic and clinical data of 13,880 tumors from the 100,000 Genomes Cancer Programme.

The Cancer Programme of the 100,000 Genomes Project was an initiative to provide whole-genome sequencing (WGS) for patients with cancer, evaluating opportunities for precision cancer care within the UK National Healthcare System (NHS). Genomics England, alongside NHS England, analyzed WGS data from 13,880 solid tumors spanning 33 cancer types, integrating genomic data with real-world treatment and outcome data, within a secure Research Environment. Incidence of somatic mutations in genes recommended for standard-of-care testing varied across cancer types. For instance, in glioblastoma multiforme, small variants were present in 94% of cases and copy number aberrations in at least one gene in 58% of cases, while sarcoma demonstrated the highest occurrence of actionable structural variants (13%). Homologous recombination deficiency was identified in 40% of high-grade serous ovarian cancer cases with 30% linked to pathogenic germline variants, highlighting the value of combined somatic and germline analysis. The linkage of WGS and longitudinal life course clinical data allowed the assessment of treatment outcomes for patients stratified according to pangenomic markers. Our findings demonstrate the utility of linking genomic and real-world clinical data to enable survival analysis to identify cancer genes that affect prognosis and advance our understanding of how cancer genomics impacts patient outcomes.

Genome-wide Mapping of 5'-monophosphorylated Ends of Mammalian Nascent RNA Transcripts.

In eukaryotic cells, RNA biogenesis generally requires processing of the nascent transcript as it is being synthesized by RNA polymerase. These processing events include endonucleolytic cleavage, exonucleolytic trimming, and splicing of the growing nascent transcript. Endonucleolytic cleavage events that generate an exposed 5'-monophosphorylated (5'-PO4) end on the growing nascent transcript occur in the maturation of rRNAs, tRNAs, and mRNAs. These 5'-PO4 ends can be a target of further processing or be subjected to 5'-3' exonucleolytic digestion that may result in termination of transcription. Here, we describe how to identify 5'-PO4 ends of intermediates in nascent RNA metabolism. We capture these species via metabolic labeling with bromouridine followed by immunoprecipitation and specific ligation of 5'-PO4 RNA ends with the 3'-hydroxyl group of a 5' adaptor (5'-PO4 Bru-Seq) using RNA ligase I. These ligation events are localized at single nucleotide resolution via highthroughput sequencing, which identifies the position of 5'-PO4 groups precisely. This protocol successfully detects the 5'monophosphorylated ends of RNA processing intermediates during production of mature ribosomal, transfer, and micro RNAs. When combined with inhibition of the nuclear 5'-3' exonuclease Xrn2, 5'-PO4 Bru-Seq maps the 5' splice sites of debranched introns and mRNA and tRNA 3' end processing sites cleaved by CPSF73 and RNaseZ, respectively. Key features • Metabolic labeling for brief periods with bromouridine focuses the analysis of 5'-PO4 RNA ends on the population of nascent transcripts that are actively transcribed. • Detects 5'-PO4 RNA ends on nascent transcripts produced by all RNA polymerases. • Detects 5'-PO4 RNA ends at single nucleotide resolution.

Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome.

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A, and this occurred through suppression of IFN-responsive transcription factor activity. Moreover, we discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. Kinase inhibition altered splicing in pathway-specific ways and selectively affected IFN-responsive gene splicing in T21 cells. To further probe Mediator kinase function, we completed cytokine screens and untargeted metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways, including broad up-regulation of anti-inflammatory lipid mediators. Elevated levels of lipid mediators persisted at least 24hr after Mediator kinase inhibition, and many identified lipids serve as ligands for nuclear receptors (e.g. PPAR, LXR) or G-protein coupled receptors (GPCRs; e.g. FFAR4). Notably, ligand-dependent activation of these GPCRs or nuclear receptors will propagate anti-inflammatory signaling pathways and gene expression programs, and this mechanistic link suggests that metabolic changes caused by CDK8/CDK19 inhibition can durably and independently suppress pro-inflammatory IFN responses. Collectively, our results establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

Pre-mRNA splicing and its cotranscriptional connections.

Transcription of eukaryotic genes by RNA polymerase II (Pol II) yields RNA precursors containing introns that must be spliced out and the flanking exons ligated together. Splicing is catalyzed by a dynamic ribonucleoprotein complex called the spliceosome. Recent evidence has shown that a large fraction of splicing occurs cotranscriptionally as the RNA chain is extruded from Pol II at speeds of up to 5 kb/minute. Splicing is more efficient when it is tethered to the transcription elongation complex, and this linkage permits functional coupling of splicing with transcription. We discuss recent progress that has uncovered a network of connections that link splicing to transcript elongation and other cotranscriptional RNA processing events.

The genomic landscape of acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

Intrachromosomal amplification of chromosome 21 defines a subtype of high-risk childhood acute lymphoblastic leukemia (iAMP21-ALL) characterized by copy number changes and complex rearrangements of chromosome 21. The genomic basis of iAMP21-ALL and the pathogenic role of the region of amplification of chromosome 21 to leukemogenesis remains incompletely understood. In this study, using integrated whole genome and transcriptome sequencing of 124 patients with iAMP21-ALL, including rare cases arising in the context of constitutional chromosomal aberrations, we identified subgroups of iAMP21-ALL based on the patterns of copy number alteration and structural variation. This large data set enabled formal delineation of a 7.8 Mb common region of amplification harboring 71 genes, 43 of which were differentially expressed compared with non-iAMP21-ALL ones, including multiple genes implicated in the pathogenesis of acute leukemia (CHAF1B, DYRK1A, ERG, HMGN1, and RUNX1). Using multimodal single-cell genomic profiling, including single-cell whole genome sequencing of 2 cases, we documented clonal heterogeneity and genomic evolution, demonstrating that the acquisition of the iAMP21 chromosome is an early event that may undergo progressive amplification during disease ontogeny. We show that UV-mutational signatures and high mutation load are characteristic secondary genetic features. Although the genomic alterations of chromosome 21 are variable, these integrated genomic analyses and demonstration of an extended common minimal region of amplification broaden the definition of iAMP21-ALL for more precise diagnosis using cytogenetic or genomic methods to inform clinical management.

Reactive gene curation to support interpretation and reporting of a clinical genome test for rare disease: Experience from over 1,000 cases.

Current standards in clinical genetics recognize the need to establish the validity of gene-disease relationships as a first step in the interpretation of sequence variants. We describe our experience incorporating the ClinGen Gene-Disease Clinical Validity framework in our interpretation and reporting workflow for a clinical genome sequencing (cGS) test for individuals with rare and undiagnosed genetic diseases. This "reactive" gene curation is completed upon identification of candidate variants during active case analysis and within the test turn-around time by focusing on the most impactful evidence and taking advantage of the broad applicability of the framework to cover a wide range of disease areas. We demonstrate that reactive gene curation can be successfully implemented in support of cGS in a clinical laboratory environment, enabling robust clinical decision making and allowing all variants to be fully and appropriately considered and their clinical significance confidently interpreted.

Whole genome sequencing provides comprehensive genetic testing in childhood B-cell acute lymphoblastic leukaemia.

Childhood B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by recurrent genetic abnormalities that drive risk-directed treatment strategies. Using current techniques, accurate detection of such aberrations can be challenging, due to the rapidly expanding list of key genetic abnormalities. Whole genome sequencing (WGS) has the potential to improve genetic testing, but requires comprehensive validation. We performed WGS on 210 childhood B-ALL samples annotated with clinical and genetic data. We devised a molecular classification system to subtype these patients based on identification of key genetic changes in tumour-normal and tumour-only analyses. This approach detected 294 subtype-defining genetic abnormalities in 96% (202/210) patients. Novel genetic variants, including fusions involving genes in the MAP kinase pathway, were identified. WGS results were concordant with standard-of-care methods and whole transcriptome sequencing (WTS). We expanded the catalogue of genetic profiles that reliably classify PAX5alt and ETV6::RUNX1-like subtypes. Our novel bioinformatic pipeline improved detection of DUX4 rearrangements (DUX4-r): a good-risk B-ALL subtype with high survival rates. Overall, we have validated that WGS provides a standalone, reliable genetic test to detect all subtype-defining genetic abnormalities in B-ALL, accurately classifying patients for the risk-directed treatment stratification, while simultaneously performing as a research tool to identify novel disease biomarkers.

Integrative genomic analysis of childhood acute lymphoblastic leukaemia lacking a genetic biomarker in the UKALL2003 clinical trial.

Incorporating genetics into risk-stratification for treatment of childhood B-progenitor acute lymphoblastic leukaemia (B-ALL) has contributed significantly to improved survival. In about 30% B-ALL (B-other-ALL) without well-established chromosomal changes, new genetic subtypes have recently emerged, yet their true prognostic relevance largely remains unclear. We integrated next generation sequencing (NGS): whole genome sequencing (WGS) (n = 157) and bespoke targeted NGS (t-NGS) (n = 175) (overlap n = 36), with existing genetic annotation in a representative cohort of 351 B-other-ALL patients from the childhood ALL trail, UKALL2003. PAX5alt was most frequently observed (n = 91), whereas PAX5 P80R mutations (n = 11) defined a distinct PAX5 subtype. DUX4-r subtype (n = 80) was defined by DUX4 rearrangements and/or ERG deletions. These patients had a low relapse rate and excellent survival. ETV6::RUNX1-like subtype (n = 21) was characterised by multiple abnormalities of ETV6 and IKZF1, with no reported relapses or deaths, indicating their excellent prognosis in this trial. An inferior outcome for patients with ABL-class fusions (n = 25) was confirmed. Integration of NGS into genomic profiling of B-other-ALL within a single childhood ALL trial, UKALL2003, has shown the added clinical value of NGS-based approaches, through improved accuracy in detection and classification into the range of risk stratifying genetic subtypes, while validating their prognostic significance.

Metabolic and Performance Responses to a Simulated Routine in Elite Artistic Swimmers.

The purpose of this investigation was to examine the interrelationship between time spent underwater (UW), movement frequency and accompanying blood acid base balance response. Elite artistic swimmers (n = 6) participated in the investigation and were all familiar with the testing procedures. All athletes completed the same choreographed artistic swimming routine. The routine was videoed and the number of movements during each 'lap' of the routine counted. Fingertip capillary blood samples were collected prior to the routine 60 sec post routine for pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), bicarbonate (HCO3-) and potassium (K+). and lactate (La) concentration (mmol/L). On a separate day an incremental exercise test to exhaustion was performed on a cycle ergometer for determination of maximal oxygen uptake (VO2max). Over half the routine was performed underwater (56 ± 4%). Aside from pCO2 (-1.07 ± 12.29%, p = 0.686), there were significant changes in all variables measured from the BG analysis. VO2peak was significantly correlated to total UW of the routine (r = -0.93; p = 0.007). as well as ∆PO2 r = 0.47 and ∆HCO3 r = 0.51. There was also a significant correlation between total UW and post routine pCO2 (r = 0.86; p = 0.030). There was also a significant correlation between total movements during the routine and post pO2 (r = -0.83; p = 0.044). These data show UW in combination with movement rate during a AS routine imfluence the metabolic response to the exercise. In addition, VO2max represents an important performance variable influencing AS performance.

Nucleotide-level linkage of transcriptional elongation and polyadenylation.

Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

Xrn2 substrate mapping identifies torpedo loading sites and extensive premature termination of RNA pol II transcription.

The exonuclease torpedo Xrn2 loads onto nascent RNA 5'-PO4 ends and chases down pol II to promote termination downstream from polyA sites. We report that Xrn2 is recruited to preinitiation complexes and "travels" to 3' ends of genes. Mapping of 5'-PO4 ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are approximately two to 20 bases downstream from where CPSF73 cleaves at polyA sites and histone 3' ends. We propose that processing of all mRNA 3' ends comprises cleavage and limited 5'-3' trimming by CPSF73, followed by handoff to Xrn2. A similar handoff occurs at tRNA 3' ends, where cotranscriptional RNase Z cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3' flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3' flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and antisense nascent RNA within a few bases of the TSS, where 5'-PO4 ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminates prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.

Whole-genome sequencing of chronic lymphocytic leukemia identifies subgroups with distinct biological and clinical features.

The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia.

The pausing zone and control of RNA polymerase II elongation by Spt5: Implications for the pause-release model.

The pause-release model of transcription proposes that 40-100 bases from the start site RNA Pol II pauses, followed by release into productive elongation. Pause release is facilitated by the PTEFb phosphorylation of the RNA Pol II elongation factor, Spt5. We mapped paused polymerases by eNET-seq and found frequent pausing in zones that extend ∼0.3-3 kb into genes even when PTEFb is inhibited. The fraction of paused polymerases or pausing propensity declines gradually over several kb and not abruptly as predicted for a discrete pause-release event. Spt5 depletion extends pausing zones, suggesting that it promotes the maturation of elongation complexes to a low-pausing state. The expression of mutants after Spt5 depletion showed that phosphomimetic substitutions in the CTR1 domain diminished pausing throughout genes. By contrast, mutants that prevent the phosphorylation of the Spt5 RNA-binding domain strengthened pausing. Thus, distinct Spt5 phospho-isoforms set the balance between pausing and elongation.

Effect of a polyphenol-rich dietary supplement containing Pinus massoniana bark extract on blood pressure in healthy adults: A parallel, randomized placebo-controlled trial.

OBJECTIVES: High blood pressure (BP) is a major risk factor for cardiovascular disease and prevalence rates continue to rise with ageing populations. Polypharmacy remains a burden among the ageing, thus alternative effective strategies are warranted. This study investigated the effects of a polyphenols rich dietary supplement containing Pinus massoniana bark extract (PMBE) for modulating BP in healthy Australian adults. DESIGN: This study is a secondary analysis of data from a double-blinded, placebo-controlled clinical trial. METHODS: Sixty-two healthy adults aged 55-75 years were randomized to receive 50 mL dietary supplement containing placebo (0 mg PMBE) or PMBE (1322 mg PMBE) daily for 12 weeks. Seated systolic BP (SBP) and diastolic (DBP) were measured at baseline, 6 weeks and 12 weeks. Effects of PMBE on modulating BP was also explored in this study stratified for SBP status (optimal v high) as well as by SBP medication status. Mixed effect regression modelling was employed involving fixed categorical effects for elapsed time, treatment assignment and their interaction as well as random subject-level intercept to account for within-subject correlations resulting from repeated measurements. Significant models were further examined by addition of covariates and power calculations were performed since this study was a secondary analysis. RESULTS: SBP significantly reduced (-3.29 mmHg, p = 0.028) after PMBE at 12 weeks compared to baseline. SBP in individuals with normal-high SBP (>120 mmHg) in the PMBE group reduced by - 6.46 mmHg (p = 0.001) at 12 weeks compared to baseline. No significant changes were reported for individuals with optimal (≤120 mmHg) SBP nor did DBP significantly change in either study groups. In individuals with non-medicated normal-high SBP, SBP significantly reduced by - 7.49 mmHg (p = 0.001) and DBP by - 3.06 mmHg (p = 0.011) at 12 weeks compared to baseline after PMBE. Cross-group comparisons were not statistically different. CONCLUSIONS: A polyphenol-rich dietary supplement derived from PMBE led to a clinically and statistically significant reduction in SBP in adults. Future studies to investigate the effects of PMBE-polyphenol supplementation on BP are warranted to confirm and explore optimal dose and impact on hypertension.

Antioxidant Effects of a Polyphenol-Rich Dietary Supplement Incorporating Pinus massoniana Bark Extract in Healthy Older Adults: A Two-Arm, Parallel Group, Randomized Placebo-Controlled Trial.

Oxidative stress is a key physiological phenomenon underpinning the ageing process and plays a major developmental role in age-associated chronic diseases. This study investigated the antioxidant effects of a polyphenol-rich dietary supplement containing Pinus massoniana bark extract (PMBE) in healthy older adults. In a double-blinded, placebo-controlled clinical trial, participants were randomised (in a 1:1 ratio) to receive a 50 mL/day dietary supplement containing placebo (0 mg PMBE) or PMBE (1322 mg PMBE) for 12 weeks. The primary outcome was fasting plasma malondialdehyde (MDA) concentrations and secondary outcomes were plasma inflammatory markers. MDA concentrations significantly reduced following PMBE for 6 weeks (−1.19 nmol/mL, 95%CI −1.62, −0.75, p < 0.001) and 12 weeks (−1.35 nmol/mL, 95%CI −1.74, −0.96, p < 0.001) compared to baseline. MDA did not significantly change after the placebo. MDA levels at 6 and 12 weeks were significantly lower following PMBE compared to placebo (p < 0.001). At 12 weeks in the PMBE group, fibrinogen concentrations significantly reduced (−0.25 g/L, 95%CI −0.39, −0.11; p < 0.0001) and interleukin-6 significantly increased compared to placebo (0.30 pg/mL, 95%CI 0.02, 0.59; p < 0.05). PMBE in a polyphenol-rich dietary supplement reduced oxidative stress in healthy older adults. Further studies are warranted to investigate the antioxidant capacity of PMBE in conditions with heightened oxidative stress, such as osteoarthritis, hypertension, type 2 diabetes, or other lifestyle related diseases.

Acute Physiological Response to Different Sprint Training Protocols in Normobaric Hypoxia.

BACKGROUND: the purpose of this study was to examine acute physiological responses to and the performance effects of two sprint training protocols in normobaric hypoxic conditions. METHODS: Healthy competitive female (n = 2) and male (n = 5) kayakers (19 ± 2.1 years) performed four sprint training sessions on a kayak ergometer over a period of two weeks. Participants performed five sets of 12 × 5 s sprints or 3 × 20 s sprints in both normobaric normoxic (NOR, FiO2 = 20.9%) or normobaric hypoxic (HYP, FiO2 = 13.6%) conditions. The peak power output (PPO), rate of perceived exertion (RPE), and heart rate (HR) of each participant were monitored continuously. Their blood lactate concentrations ([BLa+]), in addition to their blood gas (mixed-venous partial pressure (p) of carbon dioxide (pCO2), O2 (pO2), and oxygen saturations (sO2)) were collected before and after exercise. RESULTS: A significantly greater RPE, HR, and [BLa+] response and a significant decrease in pCO2, pO2, and sO2 were observed in HYP conditions versus NOR ones, independent of the type of training session. The PPO of participants did not differ between sessions. Their RPE in HYP12 × 5 was greater compared to all other sessions. CONCLUSIONS: The HYP conditions elicited significantly greater physiological strain compared to NOR conditions and this was similar in both training sessions. Our results suggest that either sprint training protocol in HYP conditions may induce more positive training adaptations compared to sprint training in NOR conditions.

Whole-genome sequencing reveals host factors underlying critical COVID-19.

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2-4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease.

Menthol Mouth Rinsing Is More Than Just a Mouth Wash-Swilling of Menthol to Improve Physiological Performance.

Interventions that solely act on the central nervous system (CNS) are gaining considerable interest, particularly products consumed through the oral cavity. The oropharyngeal cavity contains a wide array of receptors that respond to sweet, bitter, and cold tastants, all of which have been shown to improve physiological performance. Of late, the ergogenic benefits of carbohydrate (CHO) and caffeine (CAF) mouth rinsings (MRs) have been widely studied; however, less is known about menthol (MEN). That the physiological state and environmental conditions impact the response each product has is increasingly recognized. While the effects of CHO and CAF MRs have been thoroughly studied in both hot and thermoneutral conditions, less is known about MEN as it has only been studied in hot environments. As such, this review summarizes the current knowledge regarding the MEN MR and exercise modality, frequency of the mouth rinse, and mouth rinse duration and compares two different types of study designs: time trials vs. time to exhaustion (TTE).