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Background Patients with high-risk prostate cancer are at increased risk of lymph node metastasis and are thought to benefit from whole pelvis radiotherapy (WPRT). There has been recent interest in the use of hypofractionated radiotherapy in treating prostate cancer. However, toxicity and cancer outcomes associated with hypofractionated WPRT are unclear at this time. This phase II study aims to investigate the impact in quality of life associated with hypofractionated WPRT compared to conventionally fractionated WPRT. Methods Fifty-eight patients with unfavourable intermediate-, high- or very high-risk prostate cancer will be randomized in a 1:1 ratio between high-dose-rate brachytherapy (HDR-BT) + conventionally fractionated (45 Gy in 25 fractions) WPRT vs. HDR-BT + hypofractionated (25 Gy in 5 fractions) WPRT. Randomization will be performed with a permuted block design without stratification. The primary endpoint is late bowel toxicity and the secondary endpoints include acute and late urinary and sexual toxicity, acute bowel toxicity, biochemical failure-, androgen deprivation therapy-, metastasis- and prostate cancer-free survival of the hypofractionated arm compared to the conventionally fractionated arm. Discussion To our knowledge, this is the first study to compare hypofractionated WPRT to conventionally fractionated WPRT with HDR-BT boost. Hypofractionated WPRT is a more attractive and convenient treatment approach, and may become the new standard of care if demonstrated to be well-tolerated and effective. Trial registration This trial was prospectively registered in ClinicalTrials.gov as NCT04197141 on December 12, 2019.

Packed red blood cell (PRBC) transfusions are used to treat anemia in patients with cervical cancer undergoing radiotherapy (RT) owing to concerns of hypoxia-induced radioresistance. In the absence of high-quality evidence informing transfusion practices for patients receiving external beam RT (EBRT) and brachytherapy, various arbitrary hemoglobin target levels are used worldwide. To develop consensus statements to guide PRBC transfusion practices in patients with cervical cancer receiving curative-intent RT with EBRT and brachytherapy. This international Delphi consensus study was completed between November 1, 2019, and July 31, 2020. A total of 63 international clinical experts in gynecologic radiation oncology were invited; 39 (62%) accepted and consented to participate. Consensus building was achieved using a 3-round anonymous Delphi consensus method. Participants rated their agreement or disagreement with statements using a 5-point Likert scale. An a priori threshold of 75% or more was required for consensus. The preplanned primary outcome of this study was to assess hemoglobin transfusion thresholds and targets for both EBRT and brachytherapy by expert consensus. Response rates of 100% (39 of 39), 92% (36 of 39), and 97% (35 of 36) were achieved for the first, second, and third rounds of surveys, respectively. Twenty-three experts (59%) practiced in Canada, 11 (28%) in the United States, 3 (8%) in South America, 1 (3%) in Europe, and 1 (3%) in Asia. Consensus was reached for 44 of 103 statements (43%), which were combined to form the final 27-statement consensus guideline. No specific hemoglobin transfusion threshold was agreed on by consensus for EBRT or brachytherapy. By consensus (89% [31 of 35]), a hemoglobin transfusion target for patients who receive a PRBC transfusion should be 9 g/dL or more and less than 12 g/dL. This study presents the first international expert consensus guideline informing PRBC transfusion practices for patients with cervical cancer undergoing EBRT and brachytherapy. A minimum hemoglobin transfusion target of 9 g/dL was endorsed to balance tumor radiosensitivity with appropriate use of a scarce resource. Randomized clinical trials are required to evaluate the optimal transfusion threshold and target that maximize clinical benefit in this patient population.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells. Human pluripotent cells (hPSCs) in standard culture are similar to mouse epiblast cells, but heterogeneity within hPSC cultures complicates comparisons. Here the authors show that a subpopulation of hPSCs enriched for self-renewal capacity have distinct cell cycle, metabolic, DNA methylation, and ATAC-seq profiles.

The objective of this study was to determine the influence of a total-mixed ration including unsalable carrots at 45% DM on the rumen microbiome; and the plasma, rumen and liver metabolomes. Carrots discarded at processing were investigated as an energy-dense substitute for barley grain in a conventional feedlot diet, and improved feed conversion efficiency by 25%. Here, rumen fluid was collected from 34 Merino lambs at slaughter (n = 16 control; n = 18 carrot) after a feeding period of 11-weeks. The V4 region of the 16S rRNA gene was sequenced to profile archaeal and bacterial microbe communities. Further, a comprehensive, targeted profile of known metabolites was constructed for blood plasma, rumen fluid and biopsied liver metabolites using a gas chromatography mass spectrometry (GC–MS) metabolomics approach. An in vitro batch culture was used to characterise ruminal fermentation including gas and methane (CH 4 ) production. In vivo rumen microbial community structure of carrot fed lambs was dissimilar ( P  < 0.01; PERMANOVA), and all measures of alpha diversity were greater ( P  < 0.01), compared to those fed the control diet. Unclassified genera in Bacteroidales (15.9 ± 6.74% relative abundance; RA) were more abundant ( P  < 0.01) in the rumen fluid of carrot-fed lambs, while unclassified taxa in the Succinivibrionaceae family (11.1 ± 3.85% RA) were greater ( P  < 0.01) in the control. The carrot diet improved in vitro ruminal fermentation evidenced as an 8% increase ( P  < 0.01) in DM digestibility and a 13.8% reduction ( P  = 0.01) in CH 4 on a mg/ g DM basis, while the control diet increased ( P  = 0.04) percentage of propionate within total VFA by 20%. Fourteen rumen fluid metabolites and 27 liver metabolites were influenced ( P  ≤ 0.05) by diet, while no effect ( P  ≥ 0.05) was observed in plasma metabolites. The carrot diet enriched (impact value = 0.13; P  = 0.01) the tyrosine metabolism pathway (acetoacetic acid, dopamine and pyruvate), while the control diet enriched (impact value = 0.42; P  ≤ 0.02) starch and sucrose metabolism (trehalose and glucose) in rumen fluid. This study demonstrated that feeding 45% DM unsalable carrots diversified bacterial communities in the rumen. These dietary changes influenced pathways of tyrosine degradation, such that previous improvements in feed conversion efficiency in lambs could be explained.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-β-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H 2 S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

Establishing circadian and wake-dependent changes in the human metabolome are critical for understanding and treating human diseases due to circadian misalignment or extended wake. Here, we assessed endogenous circadian rhythms and wake-dependent changes in plasma metabolites in 13 participants (4 females) studied during 40-hours of wakefulness. Four-hourly plasma samples were analyzed by hydrophilic interaction liquid chromatography (HILIC)-LC-MS for 1,740 metabolite signals. Group-averaged (relative to DLMO) and individual participant metabolite profiles were fitted with a combined cosinor and linear regression model. In group-level analyses, 22% of metabolites were rhythmic and 8% were linear, whereas in individual-level analyses, 14% of profiles were rhythmic and 4% were linear. We observed metabolites that were significant at the group-level but not significant in a single individual, and metabolites that were significant in approximately half of individuals but not group-significant. Of the group-rhythmic and group-linear metabolites, only 7% and 12% were also significantly rhythmic or linear, respectively, in ≥50% of participants. Owing to large inter-individual variation in rhythm timing and the magnitude and direction of linear change, acrophase and slope estimates also differed between group- and individual-level analyses. These preliminary findings have important implications for biomarker development and understanding of sleep and circadian regulation of metabolism.

Membrane contact sites between organelles are critical for the transfer of biomolecules. Lipid droplets store fatty acids and form contacts with mitochondria, which regulate fatty acid oxidation and adenosine triphosphate production. Protein compartmentalization at lipid droplet-mitochondria contact sites and their effects on biological processes are poorly described. Using proximity-dependent biotinylation methods, we identify 71 proteins at lipid droplet-mitochondria contact sites, including a multimeric complex containing extended synaptotagmin (ESYT) 1, ESYT2, and VAMP Associated Protein B and C (VAPB). High resolution imaging confirms localization of this complex at the interface of lipid droplet-mitochondria-endoplasmic reticulum where it likely transfers fatty acids to enable β-oxidation. Deletion of ESYT1, ESYT2 or VAPB limits lipid droplet-derived fatty acid oxidation, resulting in depletion of tricarboxylic acid cycle metabolites, remodeling of the cellular lipidome, and induction of lipotoxic stress. These findings were recapitulated in Esyt1 and Esyt2 deficient mice. Our study uncovers a fundamental mechanism that is required for lipid droplet-derived fatty acid oxidation and cellular lipid homeostasis, with implications for metabolic diseases and survival. Protein-mediated transport is implicated in trafficking fatty acids at contact sites of lipid droplets and mitochondria. Here, the authors use proteomics to catalogue the proteins at this contact site and report a mechanism of fatty acid transfer that regulates fatty acid oxidation and lipid homeostasis.

In the original publication [...]

During mitochondrial damage, information is relayed between the mitochondria and nucleus to coordinate precise responses to preserve cellular health. One such pathway is the mitochondrial integrated stress response (mtISR), which is known to be activated by mitochondrial DNA (mtDNA) damage. However, the causal molecular signals responsible for activation of the mtISR remain mostly unknown. A gene often associated with mtDNA mutations/deletions is Polg1 , which encodes the mitochondrial DNA Polymerase γ (PolG). Here, we describe an inducible, tissue specific model of PolG mutation, which in muscle specific animals leads to rapid development of mitochondrial dysfunction and muscular degeneration in male animals from ~5 months of age. Detailed molecular profiling demonstrated robust activation of the mtISR in muscles from these animals. This was accompanied by striking alterations to enzymes in the mitochondrial folate cycle that was likely driven by a specific depletion in the folate cycle metabolite 5,10 methenyl-THF, strongly implying imbalanced folate intermediates as a previously unrecognised pathology linking the mtISR and mitochondrial disease. Bond et al. show that inducible PolG mutation in muscle causes mtDNA damage and muscle wasting. This is driven by the integrated stress response (ISR) and reduction in folate intermediates, linking impaired folate metabolism with ISR/disease induction.