Metabolic Profiles of Encapsulated Chondrocytes Exposed to Short-Term Simulated Microgravity.

The mechanism by which chondrocytes respond to reduced mechanical loading environments and the subsequent risk of developing osteoarthritis remains unclear. This is of particular concern for astronauts. In space the reduced joint loading forces during prolonged microgravity (10-6 g) exposure could lead to osteoarthritis (OA), compromising quality of life post-spaceflight. In this study, we encapsulated human chondrocytes in an agarose gel of similar stiffness to the pericellular matrix to mimic the cartilage microenvironment. We then exposed agarose-chondrocyte constructs to simulated microgravity (SM) using a rotating wall vessel (RWV) bioreactor to better assess the cartilage health risks associated with spaceflight. Global metabolomic profiling detected a total of 1205 metabolite features across all samples, with 497 significant metabolite features identified by ANOVA (FDR-corrected p-value < 0.05). Specific metabolic shifts detected in response to SM exposure resulted in clusters of co-regulated metabolites, as well as key metabolites identified by variable importance in projection scores. Microgravity-induced metabolic shifts in gel constructs and media were indicative of protein synthesis, energy metabolism, nucleotide metabolism, and oxidative catabolism. The microgravity associated-metabolic shifts were consistent with early osteoarthritic metabolomic profiles in human synovial fluid, which suggests that even short-term exposure to microgravity (or other reduced mechanical loading environments) may lead to the development of OA.

A Novel Diastolic Doppler Index Less Affected by Aortic Arch Anomalies Co-existing with Coarctation.

Severity assessment for coarctation of the aorta (CoA) is challenging due to concomitant morphological anomalies (complex CoA) and inaccurate Doppler-based indices. Promising diagnostic performance has been reported for the continuous flow pressure gradient (CFPG), but it has not been studied in complex CoA. Our objective was to characterize the effect of complex CoA and associated hemodynamics on CFPG in a clinical cohort. Retrospective analysis identified discrete juxtaductal (n = 25) and complex CoA (n = 43; transverse arch and/or isthmus hypoplasia) patients with arm-leg systolic blood pressure gradients (BPG) within 24 h of echocardiography for comparison to BPG by conventional Doppler indices (simplified Bernoulli equation and modified forms correcting for proximal kinetic energy and/or recovered pressure). Results were interpreted using the current CoA guideline (BPG ≥ 20 mmHg) to compare diagnostic performance indicators including receiver operating characteristic curves, sensitivity, specificity, and diagnostic accuracy, among others. Echocardiography Z-scored aortic diameters were applied with computational simulations from a preclinical CoA model to understand aspects of the CFPG driving performance differences. Diagnostic performance was substantially reduced from discrete to complex CoA for conventional Doppler indices calculated from patient data, and by hypoplasia and/or long segment stenosis in simulations. In contrast, diagnostic indicators for the CFPG only modestly dropped for complex vs discrete CoA. Simulations revealed differences in performance due to inclusion of the Doppler velocity index and diastolic pressure half-time in the CFPG calculation. CFPG is less affected by aortic arch anomalies co-existing with CoA when compared to conventional Doppler indices.

The Influence of Dietary Isothiocyanates on the Effectiveness of Mitomycin C and Bacillus Calmette-Guérin in Treating Nonmuscle-Invasive Bladder Cancer.

PURPOSE: Nonmuscle-invasive bladder cancer (NMIBC) has high recurrence rates and is often treated with mitomycin C (MMC) and bacillus Calmette-Guérin (BCG). Their efficacy relies on phase 2 enzyme metabolism and immune response activation, respectively. Dietary isothiocyanates, phytochemicals in cruciferous vegetables, are phase 2 enzyme inducers and immunomodulators, and may impact treatment outcomes. We investigated the modifying effects of cruciferous vegetable and isothiocyanate intake on recurrence risk following MMC or BCG treatment. MATERIALS AND METHODS: Self-reported cruciferous vegetable intake, estimated isothiocyanate intake, and urinary isothiocyanate metabolites were collected from 1158 patients with incident NMIBC in the prospective Be-Well Study. Hazard ratios (HRs) and 95% CIs were calculated from Cox proportional hazards regression models for risk of first recurrences, and random effects Cox shared frailty models for multiple recurrences. RESULTS: Over median follow-up of 23 months, 343 (30%) recurrences occurred. Receipt of MMC and BCG was associated with decreased risks of first recurrence (MMC: HR = 0.58; 95% CI: 0.46-0.73; BCG: HR = 0.66; 95% CI: 0.49-0.88) and multiple recurrences (MMC: HR = 0.55; 95% CI: 0.44-0.68; BCG: HR = 0.72; 95% CI: 0.55-0.95). Patients receiving BCG and having high intake (>2.4 servings/mo), but not low intake, of raw cruciferous vegetables had reduced risk of recurrence (HR: 0.56; 95% CI: 0.36-0.86; P for interaction = .02) and multiple recurrences (HR: 0.51; 95% CI: 0.34-0.77; P for interaction < .001). The inverse association between MMC receipt and recurrence risk was not modified. CONCLUSIONS: For NMIBC patients who receive induction BCG, increasing consumption of raw cruciferous vegetables could be a promising strategy to attenuate recurrence risk.

Metabolomic Profiling and Characterization of a Novel 3D Culture System for Studying Chondrocyte Mechanotransduction.

Articular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction, and PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels have been used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. Here, we demonstrate that primary chondrocytes cultured in alginate will form a pericellular matrix and display a phenotype similar to in vivo conditions. We found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism. Our data suggest that 3D preculture of chondrocytes in alginate before encapsulation in physiologically-stiff agarose leads to a pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This novel model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.

Starve a cold or feed a fever? Identifying cellular metabolic changes following infection and exposure to SARS-CoV-2.

Viral infections induce major shifts in cellular metabolism elicited by active viral replication and antiviral responses. For the virus, harnessing cellular metabolism and evading changes that limit replication are essential for productive viral replication. In contrast, the cellular response to infection disrupts metabolic pathways to prevent viral replication and promote an antiviral state in the host cell and neighboring bystander cells. This competition between the virus and cell results in measurable shifts in cellular metabolism that differ depending on the virus, cell type, and extracellular environment. The resulting metabolic shifts can be observed and analyzed using global metabolic profiling techniques to identify pathways that are critical for either viral replication or cellular defense. SARS-CoV-2 is a respiratory virus that can exhibit broad tissue tropism and diverse, yet inconsistent, symptomatology. While the factors that determine the presentation and severity of SARS-CoV-2 infection remain unclear, metabolic syndromes are associated with more severe manifestations of SARS-CoV-2 disease. Despite these observations a critical knowledge gap remains between cellular metabolic responses and SARS-CoV-2 infection. Using a well-established untargeted metabolomics analysis workflow, we compared SARS-CoV-2 infection of human lung carcinoma cells. We identified significant changes in metabolic pathways that correlate with either productive or non-productive viral infection. This information is critical for characterizing the factors that contribute to SARS-CoV-2 replication that could be targeted for therapeutic interventions to limit viral disease.

Aging alters the subchondral bone response 7 days after noninvasive traumatic joint injury in C57BL/6JN mice.

Posttraumatic osteoarthritis (PTOA) commonly develops following anterior cruciate ligament (ACL) injuries, affecting around 50% of individuals within 10-20 years. Recent studies have highlighted early changes in subchondral bone structure after ACL injury in adolescent or young adult mice, which could contribute to the development of PTOA. However, ACL injuries do not only occur early in life. Middle-aged and older patients also experience ACL injuries and PTOA, but whether the aged subchondral bone also responds rapidly to injury is unknown. This study utilized a noninvasive, single overload mouse injury model to assess subchondral bone microarchitecture, turnover, and material properties in both young adults (5 months) and early old age (22 months) female C57BL/6JN mice at 7 days after injury. Mice underwent either joint injury (i.e., produces ACL tears) or sham injury procedures on both the loaded and contralateral limbs, allowing evaluation of the impacts of injury versus loading. The subchondral bone response to ACL injury is distinct for young adult and aged mice. While 5-month mice show subchondral bone loss and increased bone resorption postinjury, 22-month mice did not show loss of bone structure and had lower bone resorption. Subchondral bone plate modulus increased with age, but not with injury. Both ages of mice showed several bone measures were altered in the contralateral limb, demonstrating the systemic skeletal response to joint injury. These data motivate further investigation to discern how osteochondral tissues differently respond to injury in aging, such that diagnostics and treatments can be refined for these demographics.

Unraveling sex-specific risks of knee osteoarthritis before menopause: Do sex differences start early in life?

OBJECTIVE: Sufficient evidence within the past two decades have shown that osteoarthritis (OA) has a sex-specific component. However, efforts to reveal the biological causes of this disparity have emerged more gradually. In this narrative review, we discuss anatomical differences within the knee, incidence of injuries in youth sports, and metabolic factors that present early in life (childhood and early adulthood) that can contribute to a higher risk of OA in females. DESIGN: We compiled clinical data from multiple tissues within the knee joint-since OA is a whole joint disorder-aiming to reveal relevant factors behind the sex differences from different perspectives. RESULTS: The data gathered in this review indicate that sex differences in articular cartilage, meniscus, and anterior cruciate ligament are detected as early as childhood and are not only explained by sex hormones. Aiming to unveil the biological causes of the uneven sex-specific risks for knee OA, we review the current knowledge of sex differences mostly in young, but also including old populations, from the perspective of (i) human anatomy in both healthy and pathological conditions, (ii) physical activity and response to injury, and (iii) metabolic signatures. CONCLUSIONS: We propose that to close the gap in health disparities, and specifically regarding OA, we should address sex-specific anatomic, biologic, and metabolic factors at early stages in life, as a way to prevent the higher severity and incidence of OA in women later in life.

Same-day discharge after percutaneous coronary procedures-Consensus statement of the working group of interventional cardiology (AGIK) of the Austrian Society of Cardiology.

INTRODUCTION: Percutaneous coronary intervention is a well-established revascularization strategy for patients with coronary artery disease. Recent technical advances such as radial access, third generation drug-eluting stents and highly effective antiplatelet therapy have substantially improved the safety profile of coronary procedures. Despite several practice guidelines and a clear patient preference of early hospital discharge, the percentage of coronary procedures performed in an outpatient setting in Austria remains low, mostly due to safety concerns. METHODS: The aim of this consensus statement is to provide a practical framework for the safe and effective implementation of coronary outpatient clinics in Austria. Based on a structured literature review and an in-depth analysis of available practice guidelines a consensus statement was developed and peer-reviewed within the working group of interventional cardiology (AGIK) of the Austrian Society of Cardiology. RESULTS: Based on the available literature same-day discharge coronary procedures show a favorable safety profile with no increase in the risk of major adverse events compared to an overnight stay. This document provides a detailed consensus in various clinical settings. The most important prerequisite for same-day discharge is, however, adequate selection of suitable patients and a structured peri-interventional and postinterventional management plan. CONCLUSION: Based on the data analysis this consensus document provides detailed practice guidelines for the safe operation of daycare cathlab programs in Austria.

Acute hypernatremia increases functional connectivity of NaCl sensing regions in the human brain: An fMRI pilot study.

Rodent studies demonstrated specialized sodium chloride (NaCl) sensing neurons in the circumventricular organs, which mediate changes in sympathetic nerve activity, arginine vasopressin, thirst, and blood pressure. However, the neural pathways involved in NaCl sensing in the human brain are incompletely understood. The purpose of this pilot study was to determine if acute hypernatremia alters the functional connectivity of NaCl-sensing regions of the brain in healthy young adults. Resting-state fMRI scans were acquired in 13 participants at baseline and during a 30 min hypertonic saline infusion (HSI). We used a seed-based approach to analyze the data, focusing on the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) as regions of interest (ROIs). Blood chemistry and perceived thirst were assessed pre- and post-infusion. As expected, serum sodium increased from pre- to post-infusion in the HSI group. The primary finding of this pilot study was that the functional connectivity between the SFO and a cluster within the OVLT increased from baseline to the late-phase of the HSI. Bidirectional connectivity changes were found with cortical regions, with some regions showing increased connectivity with sodium-sensing regions while others showed decreased connectivity. Furthermore, the functional connectivity between the SFO and the posterior cingulate cortex (a control ROI) did not change from baseline to the late-phase of the HSI. This finding indicates a distinct response within the NaCl sensing network in the human brain specifically related to acute hypernatremia that will need to be replicated in large-scale studies.

Same-day discharge after percutaneous coronary procedures-Structured review and comprehensive meta-analysis.

INTRODUCTION: Percutaneous coronary intervention is a well-established revascularization strategy for patients with coronary artery disease. The safety and feasibility of performing these procedures on a same-day discharge basis for selected patients has been studied in a large number of mostly nonrandomized trials. An up to date literature review should focus on trials with radial access, representing the current standard for coronary procedures in Austria and other European countries. METHODS: The aim of this consensus statement is to review the most recent evidence for the safety and feasibility of performing same-day discharge procedures in selected patients. A structured literature search was performed using prespecified search criteria, focusing on trials with radial access procedures. RESULTS: A total of 44 clinical trials and 4 large meta-analyses were retrieved, spanning 21 years of clinical evidence from 2001 to 2022. The outcome data from a wide range of clinical settings were unanimous in showing no negative effect on early (24 h) or late (30 day) major adverse events after same-day discharge coronary procedures. Based on nine prospective trials a comprehensive meta-analysis was compiled. Using 1­month major adverse events data the pooled odds ratio of same-day discharge versus overnight stay procedures was 0.66 (95% confidence interval, CI 0.35-01.24; p = 0.19; I2 0%), indicating a noninferiority in carefully selected patients. CONCLUSION: Outcome data from same-day discharge coronary intervention trials with radial access confirm the robust safety profile showing no increase in the risk of major adverse events compared to overnight stay.

Structure-property relationships in dicyanopyrazinoquinoxalines and their hydrogen-bonding-capable dihydropyrazinoquinoxalinedione derivatives.

Presented here is the design, synthesis, and study of a variety of novel hydrogen-bonding-capable π-conjugated N-heteroacenes, 1,4-dihydropyrazino[2,3-b]quinoxaline-2,3-diones (DPQDs). The DPQDs were accessed from the corresponding weakly hydrogen-bonding dicyanopyrazinoquinoxaline (DCPQ) suspensions with excess potassium hydroxide, resulting in moderate to good yields. Both families of compounds were analyzed by UV-vis and NMR spectroscopy, where the consequences of hydrogen bonding capability could be assessed through the structure-property studies. Conversion of the DCPQs into hydrogen-bonding capable DPQDs results in modulation of frontier MO energies, higher molar extinction coefficients, enhanced crystallinity, and on-average higher thermal stability (where in some cases the 5% weight loss temperature is increased by up to 100 °C). Single crystal X-ray diffraction data could be obtained for three DPQDs. One reveals pairwise hydrogen bonding in the solid state as well as a herringbone packing arrangement rendering it a promising candidate for additional studies in the context of organic optoelectronic devices.

Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry.

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.

Osteochondral fluid transport in an ex vivo system.

OBJECTIVE: Alterations to bone-to-cartilage fluid transport may contribute to the development of osteoarthritis (OA). Larger biological molecules in bone may transport from bone-to-cartilage (e.g., insulin, 5 kDa). However, many questions remain about fluid transport between these tissues. The objectives of this study were to (1) test for diffusion of 3 kDa molecular tracers from bone-to-cartilage and (2) assess potential differences in bone-to-cartilage fluid transport between different loading conditions. DESIGN: Osteochondral cores extracted from bovine femurs (N = 10 femurs, 10 cores/femur) were subjected to either no-load (i.e., pure diffusion), pre-load only, or cyclic compression (5 ± 2% or 10 ± 2% strain) in a two-chamber bioreactor. The bone was placed into the bone compartment followed by a 3 kDa dextran tracer, and tracer concentrations in the cartilage compartment were measured every 5 min for 120 min. Tracer concentrations were analyzed for differences in beginning, peak, and equilibrium concentrations, loading effects, and time-to-peak tracer concentration. RESULTS: Peak tracer concentration in the cartilage compartment was significantly higher compared to the beginning and equilibrium tracer concentrations. Cartilage-compartment tracer concentration and maximum fluorescent intensity were influenced by strain magnitude. No time-to-peak relationship was found between strain magnitudes and cartilage-compartment tracer concentration. CONCLUSION: This study shows that bone-to-cartilage fluid transport occurs with 3 kDa dextran molecules. These are larger molecules to move between bone and cartilage than previously reported. Further, these results demonstrate the potential impact of cyclic compression on osteochondral fluid transport. Determining the baseline osteochondral fluid transport in healthy tissues is crucial to elucidating the mechanisms OA pathology.

Intrathecal delivery of adipose-derived mesenchymal stem cells in traumatic spinal cord injury: Phase I trial.

Intrathecal delivery of autologous culture-expanded adipose tissue-derived mesenchymal stem cells (AD-MSC) could be utilized to treat traumatic spinal cord injury (SCI). This Phase I trial (ClinicalTrials.gov: NCT03308565) included 10 patients with American Spinal Injury Association Impairment Scale (AIS) grade A or B at the time of injury. The study's primary outcome was the safety profile, as captured by the nature and frequency of adverse events. Secondary outcomes included changes in sensory and motor scores, imaging, cerebrospinal fluid markers, and somatosensory evoked potentials. The manufacturing and delivery of the regimen were successful for all patients. The most commonly reported adverse events were headache and musculoskeletal pain, observed in 8 patients. No serious AEs were observed. At final follow-up, seven patients demonstrated improvement in AIS grade from the time of injection. In conclusion, the study met the primary endpoint, demonstrating that AD-MSC harvesting and administration were well-tolerated in patients with traumatic SCI.

Metabolomic Profiles and Pathways in Osteoarthritic Human Cartilage: A Comparative Analysis with Healthy Cartilage.

Osteoarthritis (OA) is a chronic joint disease with heterogenous metabolic pathology. To gain insight into OA-related metabolism, metabolite extracts from healthy (n = 11) and end-stage osteoarthritic cartilage (n = 35) were analyzed using liquid chromatography-mass spectrometry metabolomic profiling. Specific metabolites and metabolic pathways, including lipid and amino acid pathways, were differentially regulated in osteoarthritis-derived and healthy cartilage. The detected alterations in amino acids and lipids highlighted key differences in bioenergetic resources, matrix homeostasis, and mitochondrial alterations in OA-derived cartilage compared to healthy cartilage. Moreover, the metabolomic profiles of osteoarthritic cartilage separated into four distinct endotypes, highlighting the heterogenous nature of OA metabolism and the diverse landscape within the joint in patients. The results of this study demonstrate that human cartilage has distinct metabolomic profiles in healthy and end-stage OA patients. By taking a comprehensive approach to assess metabolic differences between healthy and osteoarthritic cartilage and within osteoarthritic cartilage alone, several metabolic pathways with distinct regulation patterns were detected. Additional investigation may lead to the identification of metabolites that may serve as valuable indicators of disease status or potential therapeutic targets.

Modifying Effects of Genetic Variations on the Association Between Dietary Isothiocyanate Exposure and Non-muscle Invasive Bladder Cancer Prognosis in the Be-Well Study.

SCOPE: Dietary isothiocyanate (ITC) exposure from cruciferous vegetable (CV) intake may improve non-muscle invasive bladder cancer (NMIBC) prognosis. This study aims to investigate whether genetic variations in key ITC-metabolizing/functioning genes modify the associations between dietary ITC exposure and NMIBC prognosis outcomes. METHODS AND RESULTS: In the Bladder Cancer Epidemiology, Wellness, and Lifestyle Study (Be-Well Study), a prospective cohort of 1472 incident NMIBC patients, dietary ITC exposure is assessed by self-reported CV intake and measured in plasma ITC-albumin adducts. Using Cox proportional hazards regression models, stratified by single nucleotide polymorphisms (SNPs) in nine key ITC-metabolizing/functioning genes, it is calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for recurrence and progression. The rs15561 in N-acetyltransferase 1 (NAT1) is alter the association between CV intake and progression risk. Multiple SNPs in nuclear factor E2-related factor 2 (NRF2) and nuclear factor kappa B (NFκB) are modify the associations between plasma ITC-albumin adduct level and progression risk (pint < 0.05). No significant association is observed with recurrence risk. Overall, >80% study participants are present with at least one protective genotype per gene, showing an average 65% reduction in progression risk with high dietary ITC exposure. CONCLUSION: Despite that genetic variations in ITC-metabolizing/functioning genes may modify the effect of dietary ITCs on NMIBC prognosis, dietary recommendation of CV consumption may help improve NMIBC survivorship.

Dicyanorhodanine-Pyrrole Conjugates for Visible Light-Driven Quantitative Photoswitching in Solution and the Solid State.

Small molecule photoswitches capable of toggling between two distinct molecular states in response to light are versatile tools to monitor biological processes, control photochemistry, and design smart materials. In this work, six novel dicyanorhodanine-based pyrrole-containing photoswitches are reported. The molecular design avails both the Z and E isomers from synthesis, where each can be isolated using chromatographic techniques. Inter- and intramolecular hydrogen bonding (H-bonding) interactions available to the E and Z isomers, respectively, uniquely impart thermal stability to each isomer over long time periods. Photoisomerization could be assessed by solution NMR and UV-vis spectroscopic techniques along with complementary ground- and excited-state computational studies, which show good agreement. Quantitative E → Z isomerization occurs upon 523 nm irradiation of the parent compound (where R = H) in solution, whereas Z → E isomerization using 404 nm irradiation offers a photostationary state (PSS) ratio of 84/16 (E/Z). Extending the π-conjugation of the pyrrole unit (where R = p-C6H4-OMe) pushes the maximum absorption to the yellow-orange region of the visible spectrum and allows bidirectional quantitative isomerization with 404 and 595 nm excitation. Comparator molecules have been prepared to report how the presence or absence of H-bonding affects the photoswitching behavior. Finally, studies of the photoswitches in neat films and photoinactive polymer matrices reveal distinctive structural and optical properties of the Z and E isomers and ultimately afford reversible photoswitching to spectrally unique PSSs using visible light sources including the Sun.