Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Training Outcomes and Satisfaction in Canadian MD/PhD and MD/MSc Programs: Findings from a National Survey.

PURPOSE: Despite the impact of physician-scientists on scientific discovery and translational medicine, several reports have signalled their declining workforce, reduced funding, and insufficient protected research time. Given the paucity of outcome data on Canadian MD/PhD programs, this study presents a national portrait of the sociodemographic characteristics, training trajectories, productivity, and satisfaction in trainees and alumni from Canadian MD/PhD and MD/MSc programs. METHODS: Quantitative data were collected in a national survey launched in 2021. Respondents included 74 MD/PhD alumni and 121 trainees across 12 Canadian MD/PhD and MD/MSc programs. RESULTS: Among MD/PhD alumni, 51% were independent practitioners/researchers while others underwent residency training. Most trainees (88%) were in MD/PhD programs. Significantly more alumni identified as men than did trainees. Significantly more alumni conducted clinical and health services research, while more trainees conducted basic science research. Average time to MD/PhD completion was 8 years, with no correlation to subsequent research outcomes. Self-reported research productivity was highest during MD/PhD training. Concerning training trajectories, most alumni completed residency, pursued additional training, and practised in Canada. Finally, regression models showed that trainees and alumni were satisfied with programs, with significant moderators in trainee models. CONCLUSION: Survey findings showed Canadian MD/PhD and MD/MSc programs recruit more diverse cohorts of trainees than before, provide productive research years, and graduate alumni who pursue training and academic employment in Canada. Both alumni and trainees are largely satisfied with these training programs. The need to collect in-depth longitudinal data on Canadian MD/PhD graduates to monitor diversity and success metrics is discussed.

Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States.

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target ("prime substrate"), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H•) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

Circadian cardiac NAD+ metabolism, from transcriptional regulation to healthy aging.

Nicotinamide adenine dinucleotide (NAD+) is a critical redox factor and coenzyme with rhythmic availability, and reduced NAD+ levels are a common factor in many disease states, including risk factors associated with aging. Recent studies have expanded on the role of circadian rhythms and the core clock factors that maintain them in the regulation of NAD+ levels in the heart. This has revealed that NAD+ pools and their use are tightly linked to cardiac function, but also heart failure. The convergence of these fields, namely, clock regulation, heart disease, and NAD+ metabolism present a complex network ripe with potential scientific and clinical discoveries, given the growing number of animal models, recently developed technology, and opportunity for safe and accessible precursor supplementation. This review seeks to briefly present known information on circadian rhythms in the heart, connect that research to our understanding of cardiac NAD+ metabolism, and finally discuss potential future experiments to better understand interventional opportunities in cardiovascular health regarding these subjects.

Sex differences in microvascular function and arterial hemodynamics in nondialysis chronic kidney disease.

Cardiovascular disease (CVD) is the leading cause of death in chronic kidney disease (CKD). Abnormal arterial hemodynamics contribute to CVD, a relationship that can be mediated by microvascular dysfunction. The purpose of this study was to investigate potential sex differences in arterial hemodynamics and microvascular dysfunction in patients with stages 3 to 4 CKD. Vascular function was assessed in 22 male (mean ± SD; age, 56 ± 13 yr) and 10 female (age, 63 ± 9 yr) patients. Arterial hemodynamics were acquired with combined tonometry and oscillometry. Skin blood flow was used as a model of microvascular function. Participants were instrumented with three microdialysis fibers for the delivery of 1) Ringer's solution; 2) superoxide dismutase mimetic, Tempol; and 3) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin. Blood flow was measured via laser-Doppler flowmetry during standardized local heating (42°C). Central pulse pressure (mean ± SE; 62 ± 9 vs. 46 ± 3 mmHg; P = 0.01) and augmentation index (36 ± 3 vs. 26 ± 3%; P = 0.03) were higher in females. There was a trend for higher central systolic pressures in females (146 ± 9 vs. 131 ± 3 mmHg; P = 0.06). Females reported higher forward (39 ± 4 vs. 29 ± 2 mmHg; P = 0.004) and reflected (27 ± 3 vs. 19 ± 1 mmHg; P < 0.001) wave amplitudes. Cutaneous vascular function was impaired in females compared with males (77 ± 3 vs. 89 ± 1%, P = 0.001). Microvascular function was improved following the delivery of Tempol and apocynin in females but not in males. Female patients with CKD had poorer central hemodynamics and reduced microvascular function compared with their male counterparts. Oxidative stress may contribute to lower microvascular function observed in females.NEW & NOTEWORTHY There are limited data regarding the physiological mechanisms of potential sex differences in central hemodynamics and vascular function in chronic kidney disease (CKD). We report that older female patients with nondialysis CKD have higher central pulse pressures compared with male patients with CKD. In addition, older females with CKD have lower microvascular function compared with their male counterparts, and oxidative stress contributes to the lower microvascular function in older female patients with CKD.

Neuromotor deficits and altered physiological responses to repeated exertional heat stroke exposures in mice.

Exertional heat stroke (EHS) is a life-threatening illness that can lead to negative health outcomes. Using a "severe" preclinical mouse model of EHS, we tested the hypotheses that one EHS exposure results in altered susceptibility to a subsequent EHS and reduced neuromotor performance. Female C57BL/6 mice underwent two protocols, 2 wk apart, either an EHS trial (EHS) or a sham exercise control trial (EXC). For EHS, mice ran in a forced running wheel at 37.5°C/40% relative humidity until loss of consciousness, followed by a slow cooling protocol (2 h recovery at 37.5°C). EXC mice exercised equally but in ∼22°C. Mice were randomized into three groups: 1) EXC-EXC (two consecutive EXC, n = 6, 2) EHS-EXC (EHS followed by EXC, n = 5), and 3) EHS-EHS (repeated EHS, n = 9). Mice underwent noninvasive neuromotor and behavioral tests during recovery and isolated soleus force measurements at the end of recovery. At the first EHS, mice reached average peak core temperatures (Tc,max) of 42.4°C, (46% mortality). On the second EHS, average Tc,max was reduced by ∼0.7°C (P < 0.05; mortality 18%). After the first EHS, both EHS-EX and EHS-EHS showed significant reductions in maximum strength (24 h and 1 wk post). After the second EHS, strength, horizontal rotation, hindlimb tone, suspended hindlimb splay, trunk curl, and provoked biting continued to decline in the EHS-EHS group. In conclusion, exposure to a second EHS after 2 wk leads to increased exercise times in the heat, symptom limitation at a lower Tc,max, and greater deficits in neuromotor and behavioral function during recovery.

Continuous probing of cold complex molecules with infrared frequency comb spectroscopy.

For more than half a century, high-resolution infrared spectroscopy has played a crucial role in probing molecular structure and dynamics. Such studies have so far been largely restricted to relatively small and simple systems, because at room temperature even molecules of modest size already occupy many millions of rotational/vibrational states, yielding highly congested spectra that are difficult to assign. Targeting more complex molecules requires methods that can record broadband infrared spectra (that is, spanning multiple vibrational bands) with both high resolution and high sensitivity. However, infrared spectroscopic techniques have hitherto been limited either by narrow bandwidth and long acquisition time, or by low sensitivity and resolution. Cavity-enhanced direct frequency comb spectroscopy (CE-DFCS) combines the inherent broad bandwidth and high resolution of an optical frequency comb with the high detection sensitivity provided by a high-finesse enhancement cavity, but it still suffers from spectral congestion. Here we show that this problem can be overcome by using buffer gas cooling to produce continuous, cold samples of molecules that are then subjected to CE-DFCS. This integration allows us to acquire a rotationally resolved direct absorption spectrum in the C-H stretching region of nitromethane, a model system that challenges our understanding of large-amplitude vibrational motion. We have also used this technique on several large organic molecules that are of fundamental spectroscopic and astrochemical relevance, including naphthalene, adamantane and hexamethylenetetramine. These findings establish the value of our approach for studying much larger and more complex molecules than have been probed so far, enabling complex molecules and their kinetics to be studied with orders-of-magnitude improvements in efficiency, spectral resolution and specificity.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity.

Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients' data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.

A phenomenological exploration of the impact of COVID-19 on the medical education community.

INTRODUCTION: The COVID-19 pandemic has caused unprecedented stress to the medical education community, potentially worsening problems like burnout and work-life imbalance that its members have long been grappling with. However, the collective struggle sparked by the pandemic could generate the critical reflection necessary for transforming professional values and practices for the better. In this hermeneutic phenomenological study, we explore how the community is adapting-and even reconceptualising-their personal and professional roles amidst the COVID-19 crisis. METHOD: Between April and October 2020, we conducted 27 (17F, 10M) semi-structured interviews with medical trainees (8), physicians (8), graduate students (3) and PhD scientists (8) working in medical education in Canada, the United States and Switzerland. Data analysis involved a variety of strategies, including coding for van Manen's four lifeworld existentials, reflexive writing and multiple team meetings. RESULTS: Participants experienced grief related to the loss of long-established personal and professional structures and boundaries, relationships and plans for the future. However, experiences of grief were often conflicting. Some participants also experienced moments of relief, perceiving some losses as metaphorical permissions slips to slow down and focus on their well-being. In turn, many reflected on the opportunity they were being offered to re-imagine the nature of their work. DISCUSSION: Participants' experiences with grief, relief and opportunity resonate with Ratcliffe's account of grief as a process of relearning the world after a significant loss. The dismantling of prior life structures and possibilities incited in participants critical reflection on the nature of the medical education community's professional practices. Participants demonstrated their desire for more flexibility and autonomy in the workplace and a re-adjustment of the values and expectations inherent to their profession. On both individual and systems levels, the community must ensure that long-standing calls for wellness and work-life integration are realised-and persist-after the pandemic is over.

Circadian lipid synthesis in brown fat maintains murine body temperature during chronic cold.

Ambient temperature influences the molecular clock and lipid metabolism, but the impact of chronic cold exposure on circadian lipid metabolism in thermogenic brown adipose tissue (BAT) has not been studied. Here we show that during chronic cold exposure (1 wk at 4 °C), genes controlling de novo lipogenesis (DNL) including Srebp1, the master transcriptional regulator of DNL, acquired high-amplitude circadian rhythms in thermogenic BAT. These conditions activated mechanistic target of rapamycin 1 (mTORC1), an inducer of Srebp1 expression, and engaged circadian transcriptional repressors REV-ERBα and ß as rhythmic regulators of Srebp1 in BAT. SREBP was required in BAT for the thermogenic response to norepinephrine, and depletion of SREBP prevented maintenance of body temperature both during circadian cycles as well as during fasting of chronically cold mice. By contrast, deletion of REV-ERBα and ß in BAT allowed mice to maintain their body temperature in chronic cold. Thus, the environmental challenge of prolonged noncircadian exposure to cold temperature induces circadian induction of SREBP1 that drives fuel synthesis in BAT and is necessary to maintain circadian body temperature during chronic cold exposure. The requirement for BAT fatty acid synthesis has broad implications for adaptation to cold.

White shark genome reveals ancient elasmobranch adaptations associated with wound healing and the maintenance of genome stability.

The white shark (Carcharodon carcharias; Chondrichthyes, Elasmobranchii) is one of the most publicly recognized marine animals. Here we report the genome sequence of the white shark and comparative evolutionary genomic analyses to the chondrichthyans, whale shark (Elasmobranchii) and elephant shark (Holocephali), as well as various vertebrates. The 4.63-Gbp white shark genome contains 24,520 predicted genes, and has a repeat content of 58.5%. We provide evidence for a history of positive selection and gene-content enrichments regarding important genome stability-related genes and functional categories, particularly so for the two elasmobranchs. We hypothesize that the molecular adaptive emphasis on genome stability in white and whale sharks may reflect the combined selective pressure of large genome sizes, high repeat content, high long-interspersed element retrotransposon representation, large body size, and long lifespans, represented across these two species. Molecular adaptation for wound healing was also evident, with positive selection in key genes involved in the wound-healing process, as well as Gene Ontology enrichments in fundamental wound-healing pathways. Sharks, particularly apex predators such as the white shark, are believed to have an acute sense of smell. However, we found very few olfactory receptor genes, very few trace amine-associated receptors, and extremely low numbers of G protein-coupled receptors. We did however, identify 13 copies of vomeronasal type 2 (V2R) genes in white shark and 10 in whale shark; this, combined with the over 30 V2Rs reported previously for elephant shark, suggests this gene family may underlie the keen odorant reception of chondrichthyans.

The effect of dietary nitrate on exercise capacity in chronic kidney disease: a randomized controlled pilot study.

BACKGROUND: Chronic Kidney Disease (CKD) patients exhibit a reduced exercise capacity that impacts quality of life. Dietary nitrate supplementation has been shown to have favorable effects on exercise capacity in disease populations by reducing the oxygen cost of exercise. This study investigated whether dietary nitrates would acutely improve exercise capacity in CKD patients. METHODS AND RESULTS: In this randomized, double-blinded crossover study, 12 Stage 3-4 CKD patients (Mean ± SEM: Age, 60 ± 5yrs; eGFR, 50.3 ± 4.6 ml/min/1.73 m2) received an acute dose of 12.6 mmol of dietary nitrate in the form of concentrated beetroot juice (BRJ) and a nitrate depleted placebo (PLA). Skeletal muscle mitochondrial oxidative function was assessed using near-infrared spectroscopy. Cardiopulmonary exercise testing was performed on a cycle ergometer, with intensity increased by 25 W every 3 min until volitional fatigue. Plasma nitric oxide (NO) metabolites (NOm; nitrate, nitrite, low molecular weight S-nitrosothiols, and metal bound NO) were determined by gas-phase chemiluminescence. Plasma NOm values were significantly increased following BRJ (BRJ vs. PLA: 1074.4 ± 120.4 µM vs. 28.4 ± 6.6 µM, p < 0.001). Total work performed (44.4 ± 10.6 vs 39.6 ± 9.9 kJ, p = 0.03) and total exercise time (674 ± 85 vs 627 ± 86s, p = 0.04) were significantly greater following BRJ. Oxygen consumption at the ventilatory threshold was also improved by BRJ (0.90 ± 0.08 vs. 0.74 ± 0.06 L/min, p = 0.04). These changes occurred in the absence of improved skeletal muscle mitochondrial oxidative capacity (p = 0.52) and VO2peak (p = 0.35). CONCLUSIONS: Our findings demonstrate that inorganic nitrate can acutely improve exercise capacity in CKD patients. The effects of chronic nitrate supplementation on CKD related exercise intolerance should be investigated in future studies.

Overview Of The Canadian Clinician Investigator Trainees' Research Presented At The 2020 CSCI-CITAC Joint Meeting.

The 2020 Annual General Meeting (AGM) and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de Recherches Clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was the first meeting to be hosted virtually. The theme was "Navigating Uncertainty, Embracing Change and Empowering the Next Generation of Clinician-Scientists", and the meeting featured lectures and workshops that were designed to provide knowledge and skills for professional development of clinician investigator trainees. The opening remarks were given by Jason Berman (President of CSCI/SCRC), Tina Marvasti (President of CITAC/ACCFC) and Nicola Jones (University of Toronto Clinician Investigator Program Symposium Chair). Dr. Michael Strong, President of the Canadian Institutes of Health Research, delivered the keynote presentation titled "CIHR's COVID-19 Response and Strategic Planning". Dr. John Bell (University of Ottawa) received the CSCI Distinguished Scientist Award, Dr. Stanley Nattel (Université de Montréal) received the CSCI-RCPSC Henry Friesen Award (RCPSC; Royal College of Physicians and Surgeons of Canada) and Dr. Meghan Azad (University of Manitoba) received the CSCI Joe Doupe Young Investigator Award. Each scientist delivered talks on their award-winning research. The interactive workshops were "Developing Strategies to Maintain Wellness", "Understanding the Hidden Curriculum: Power and Privilege in Science and Medicine", "Hiring a Clinician Scientist Trainee: What Leaders Are Looking For" and "COVID-19: A Case Study for Pivoting Your Research". The AGM included presentations from clinician investigator trainees nationwide. Over 70 abstracts were showcased, most are summarized in this review, and six were selected for oral presentations.

The Potential Uses of Omega-3 Fatty Acids in Dermatology: A Review.

BACKGROUND: A growing interest exists in the relationship between diet and skin disease, with many recent studies identifying a role for omega-3 fatty acids (O3FAs) in various dermatological conditions. OBJECTIVE: Our objective was to identify the spectrum of uses for O3FA supplementation reported in literature and to evaluate the current level of evidence for its clinical application in skin disease prevention and management. METHODS: A search was conducted using Ovid MEDLINE for primary literature that examined O3FA intake and skin health. A manual search of reference lists was performed to identify additional articles for inclusion. RESULTS: A total of 38 studies met eligibility for review, reporting benefits for O3FA supplementation in the treatment of psoriasis, atopic dermatitis, acne, and skin ulcers. Additionally, a reduced incidence of skin cancer and a decrease in the severity of drug-associated mucocutaneous side effects were reported with O3FA supplementation. CONCLUSION: This review yielded many well-studied benefits of O3FA uses in dermatology. Given its high safety profile, low cost, and ease of supplementation, O3FA is a reasonable supplement that may benefit patients wishing to improve inflammatory skin conditions through diet. Areas of particular clinical interest where supplementation may be valuable include O3FAs for systemic UV photoprotection, as well as adjuvant treatment for acne to reduce both inflammatory lesion count and the severity of mucocutaneous side effects associated with isotretinoin use.

Effects of aerobic exercise on vascular function in nondialysis chronic kidney disease: a randomized controlled trial.

Endothelial dysfunction and arterial stiffness are nontraditional risk factors of chronic kidney disease (CKD)-related cardiovascular disease (CVD) that could be targeted with exercise. This study investigated the effect of moderate to vigorous aerobic exercise on vascular function in nondialysis CKD. In this randomized, controlled trial, 36 nondialysis patients with CKD (means ± SE, age: 58 ± 2 yr, estimated glomerular filtration rate: 44 ± 2 ml·min-1·1.73 m-2) were allocated to an exercise training (EXT) or control (CON) arm. The EXT group performed 3 × 45 min of supervised exercise per week at 60-85% heart rate reserve for 12 wk, whereas the CON group received routine care. Outcomes were assessed at 0 and 12 wk. The primary outcome, microvascular function, was assessed via cutaneous vasodilation during local heating measured by laser-Doppler flowmetry coupled with microdialysis. Participants were instrumented with two microdialysis fibers for the delivery of 1) Ringer solution and 2) the superoxide scavenger tempol. Conduit artery function was assessed via brachial artery flow-mediated dilation. Aortic pressure waveforms and pulse wave velocity were acquired with tonometry and oscillometry. Microvascular function improved after EXT (week 0 vs.week 12, EXT: 87 ± 2% vs. 91 ± 2% and CON: 86 ± 2% vs. 84 ± 3%, P = 0.03). At baseline, pharmacological delivery of tempol improved microvascular function (Ringer solution vs. tempol: 86 ± 1% vs. 90 ± 1%, P = 0.02) but was no longer effective after EXT (91 ± 2% vs. 87 ± 1%, P = 0.2), suggesting that an improved redox balance plays a role in EXT-related improvements. Brachial artery flow-mediated dilation was maintained after EXT (EXT: 2.6 ± 0.4% vs. 3.8 ± 0.8% and CON: 3.5 ± 0.6% vs. 2.3 ± 0.4%, P = 0.02). Central arterial hemodynamics and arterial stiffness were unchanged after EXT. Aerobic exercise improved microvascular function and maintained conduit artery function and should be considered as an adjunct therapy to reduce CVD risk in CKD.

Quantifying the contribution of dietary protein to whole body protein kinetics: examination of the intrinsically labeled proteins method.

Intrinsically labeled dietary proteins have been used to trace various aspects of digestion and absorption, including quantifying the contribution of dietary protein to observed postprandial amino acid and protein kinetics in human subjects. Quantification of the rate of appearance in peripheral blood of an unlabeled (tracee) amino acid originating from an intrinsically labeled protein (exogenous Ra) requires the assumption that there is no dilution of the isotope enrichment of the protein-bound amino acid in the gastrointestinal tract or across the splanchnic bed. It must also be assumed that the effective volume of distribution into which the tracer and tracee appear can be reasonably estimated by a single value and that any recycling of the tracer is minimal and thus does not affect calculated rates. We have assessed these assumptions quantitatively using values from published studies. We conclude that the use of intrinsically labeled proteins as currently described to quantify exogenous Ra systematically underestimates the true value. When used with the tracer-determined rates of amino acid kinetics, underestimation of exogenous Ra from the intrinsically labeled protein method likely translates to incorrect conclusions regarding protein breakdown, including the effect of a protein meal and the anabolic impact of the speed of digestion and absorption of amino acids. Estimation of exogenous Ra from the bioavailability of ingested protein has some advantages as compared with the intrinsically labeled protein method. We therefore conclude that the bioavailability method for estimating exogenous Ra is preferable to the intrinsically labeled protein method.

Role of mitochondria-derived reactive oxygen species in microvascular dysfunction in chronic kidney disease.

Cardiovascular disease is the leading cause of mortality in chronic kidney disease (CKD). Mitochondrial dysfunction secondary to CKD is a potential source of oxidative stress that may impair vascular function. This study sought to determine if mitochondria-derived reactive oxygen species contribute to microvascular dysfunction in stage 3-5 CKD. Cutaneous vasodilation in response to local heating was assessed in 20 CKD patients [60 ± 13 yr; estimated glomerular filtration rate (eGFR) 46 ± 13 ml·kg-1·1.73 m-2] and 11 matched healthy participants (58 ± 2 yr; eGFR >90 ml·kg-1·1.73 m-2). Participants were instrumented with two microdialysis fibers for the delivery of 1) Ringer solution, and 2) the mitochondria- specific superoxide scavenger MitoTempo. Skin blood flow was measured via laser Doppler flowmetry during standardized local heating (42°C). Cutaneous vascular conductance (CVC) was calculated as a percentage of the maximum conductance achieved with sodium nitroprusside infusion at 43°C. Urinary isofuran/F2-isoprostane ratios were assessed by gas-chromatography mass spectroscopy. Isofuran-to-F2-isoprostane ratios were increased in CKD patients (3.08 ± 0.32 vs. 1.69 ± 0.12 arbitrary units; P < 0.01) indicative of mitochondria-derived oxidative stress. Cutaneous vasodilation was impaired in CKD compared with healthy controls (87 ± 1 vs. 92 ± 1%CVCmax; P < 0.01). Infusion of MitoTempo significantly increased the plateau phase CVC in CKD patients (CKD Ringer vs. CKD MitoTempo: 87 ± 1 vs. 93 ± 1%CVCmax; P < 0.01) to similar levels observed in healthy controls ( P = 0.9). These data provide in vivo evidence that mitochondria-derived reactive oxygen species contribute to microvascular dysfunction in CKD and suggest that mitochondrial dysfunction may be a potential therapeutic target to improve CKD-related vascular dysfunction.

Quality-of-life analysis of the MITO-8, MaNGO, BGOG-Ov1, AGO-Ovar2.16, ENGOT-Ov1, GCIG study comparing platinum-based versus non-platinum-based chemotherapy in patients with partially platinum-sensitive recurrent ovarian cancer.

Background: MITO-8 showed that prolonging platinum-free interval by introducing non-platinum-based chemotherapy (NPBC) does not improve prognosis of patients with partially platinum-sensitive recurrent ovarian cancer. Quality of life (QoL) was a secondary outcome. Patients and methods: Ovarian cancer patients recurring or progressing 6-12 months after previous platinum-based chemotherapy (PBC) were randomized to receive PBC or NPBC as first treatment. QoL was assessed at baseline, third and sixth cycles, with the EORTC C-30 and OV-28 questionnaires. Mean changes and best response were analysed. Progression-free survival, response rate, and toxicity are also reported for proper interpretation of data. All analyses were based on intention-to-treat. Results: Out of the 215 patients, 151 (70.2%) completed baseline questionnaire, balanced between the arms; thereafter, missing rate was higher in the NPBC arm. At mean change analysis, C30 scores were prevalently worse in the NPBC than PBC arm, statistical significance being attained for emotional functioning, global health status/QoL, fatigue, and dyspnoea (effect sizes ranging from 0.30 to 0.51). Conversely, as for OV28 scale, the other chemotherapy side-effects item was significantly worse with PBC at three and six cycles, with a larger effect size (0.70 and 0.54, respectively). At best response analysis, improvement of emotional functioning and pain and worsening of peripheral neuropathy and other chemotherapy side-effects were significantly more frequent in the PBC arm. Progression-free survival (median 9 versus 5 months, P = 0.001) and objective response rate (51.6% versus 19.4%, P = 0.0001) were significantly better with PBC. Allergy, blood cell count, alopecia, nausea, musculoskeletal, and neurological side-effects were more frequent and severe with PBC; hand-foot skin reaction, rash/desquamation, mucositis, and vascular events were more frequent with NPBC. Conclusion: MITO-8 QoL analysis shows that deterioration of some functioning and symptom scales is lower with PBC, with improvement of emotional functioning and pain, despite worsening of toxicity-related items. ClinicalTrials.gov: NCT00657878.

Phase-stabilized 100 mW frequency comb near 10 µm.

Long-wavelength mid-infrared (MIR) frequency combs with high power and flexible tunability are highly desired for molecular spectroscopy, including investigation of large molecules such as C60. We present a high power, phase-stabilized frequency comb near 10 µm, generated by a synchronously pumped, singly resonant optical parametric oscillator (OPO) based on AgGaSe2. The OPO can be continuously tuned from 8.4 to 9.5 µm, with a maximum average idler power of 100 mW at the center wavelength of 8.5 µm. Both the repetition rate (f rep) and the carrier-envelope offset frequency (f ceo) of the idler wave are phase-locked to microwave signals referenced to a Cs clock. We describe the detailed design and construction of the frequency comb, and discuss potential applications for precise and sensitive direct frequency comb spectroscopy.

Release of calcium ions from particulate monosodium titanates for dental mineralization applications.

PURPOSE: The calcium ion [Ca(II)] release from monosodium titanates (MST) complexed to calcium ions [Ca(II)], referred to as MST-Ca(II), was examined under varying incubation times, pH conditions, and ion equilibrium disruptions. METHODS: Sample supernatants were analyzed for Ca(II) using the QuantiChrom Calcium Assay Kit. RESULTS: No Ca(II) was detected in native MST (control) supernatants but was detected in MST-Ca(II) supernatants. At pH 7, Ca(II) release increased from 0 to 2.5 mg/dL over 3 days (P< 0.05 compared to MST control), remaining constant over the completed incubation times. At pH 5, 15 mg/dL of Ca(II) was immediately released with no further release. When the pH was modulated pH 4 to pH 9, Ca(II) concentration dropped from 25 mg/dL to ~0 mg/dL. Finally, when equilibrium was disrupted by partial replacement of the supernatant with sterile water, Ca(II) release was ongoing, reaching a cumulative total of 20 mg/dL over 35 days. CLINICAL SIGNIFICANCE: The current results suggest that particulate MST-Ca(II) complexes exhibit sustained release of calcium, and that release might be customized by conditions of pH and ionic strength. Thus, these complexes appear promising for biological applications where calcium-mediated mineralization or re-mineralization are desired.