Effect of high-fat diet on isometric, concentric and eccentric contractile performance of skeletal muscle isolated from female CD-1 mice.

Despite evidence inferring muscle and contractile mode-specific effects of high-fat diet (HFD), no study has yet considered the impact of HFD directly on eccentric muscle function. The present work uniquely examined the effect of 20-week HFD on the isometric, concentric and eccentric muscle function of isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles. CD-1 female mice were randomly split into a control (n = 16) or HFD (n = 17) group and for 20 weeks consumed standard lab chow or HFD. Following this period, SOL and EDL muscles were isolated and assessments of maximal isometric force and concentric work loop (WL) power were performed. Each muscle was then subjected to either multiple concentric or eccentric WL activations. Post-fatigue recovery, as an indicator of incurred damage, was measured via assessment of concentric WL power. In the EDL, absolute concentric power and concentric power normalised to muscle mass were reduced in the HFD group (P < 0.038). HFD resulted in faster concentric fatigue and reduced eccentric activity-induced muscle damage (P < 0.05). For the SOL, maximal isometric force was increased, and maximal eccentric power normalised to muscle mass and concentric fatigue were reduced in the HFD group (P < 0.05). HFD effects on eccentric muscle function are muscle-specific and have little relationship with changes in isometric or concentric function. HFD has the potential to negatively affect the intrinsic concentric and eccentric power-producing capacity of skeletal muscle, but a lack of a within-muscle uniform response indicates disparate mechanisms of action which require further investigation.

Evaluation of a remote hybrid staffing model for investigational drug service pharmacists.

PURPOSE: This project compared the time to complete investigational prescription drug order verification by investigational drug service (IDS) pharmacists in an onsite vs offsite (hybrid) staffing model, evaluating the impact of remote work on verification time. METHODS: Parenteral IDS drug orders from August 2019 through June 2022 were analyzed. Electronic medical records were timestamped for each order, first verification, second verification, and medication administration. The project was divided into four 6-month time periods to allow for 2 independent comparisons of onsite vs hybrid staffing models. A survey was given retrospectively to IDS pharmacists for the last hybrid timeframe to assess work satisfaction and burnout. RESULTS: A total of 4,398 orders were evaluated. The time from order entry to first verification was 14 minutes (n = 1,248) during the first onsite time period compared to 13 minutes (n = 1,042) during the first hybrid period (P = 0.003). The time for order verification was similar between the second onsite and hybrid periods (12 minutes [n = 1,041] vs 10 minutes [n = 1,067], respectively; P < 0.0001). The staff satisfaction survey for the hybrid model showed high levels of job satisfaction and no self-reported burnout. CONCLUSION: The remote hybrid staffing model did not result in a meaningful change in the time from order placement to medication administration within the IDS pharmacy. Hybrid staffing also resulted in greater job satisfaction among IDS pharmacists. This study highlights the benefits of implementing remote work practices in the pharmacy practice setting.

Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma.

Introduction: Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. Methods: We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. Results: Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. Discussion: We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

Pediatric Provider and Staff Burnout in Federally Qualified Community Health Centers.

This study examined changes in provider and staff burnout in 4 Boston-area federally qualified community health centers (FQHCs) participating in a pediatric behavioral health integration project. Utilizing the Maslach Burnout Inventory-Human Services Survey for Medical Personnel (MBI-HSSMP), the study found that emotional exhaustion among primary care providers ( M = 20.5; confidence interval [CI], 17.8-23.2) was higher ( P = .001) than that among behavioral health clinicians ( M = 13.6; CI, 10.4-16.8) and ( P = .00005) community health workers ( M = 10.8; CI, 7.3-14.2). Emotional exhaustion among staff increased ( P = .04) from baseline ( M = 16.8; CI, 15.0-18.6) to follow-up ( M = 20.8; CI, 17.5-24.2), but burnout at follow-up was lower than national averages. FQHCs are integral in caring for marginalized patients; therefore, supporting a stable workforce by minimizing burnout is essential.

T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control.

Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet the mechanisms of lymphocyte transit are not well defined. Here we show that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression by effector CD8+ T cells. Only high-affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells, therefore, exit the tumor, which limits the pool of CD8+ T cells available to exert tumor control. CXCR4 inhibition or loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. These data indicate that strategies to limit T cell egress might be an approach to boost the quantity and quality of intratumoral T cells and thereby response to immunotherapy.

Proteome Analysis of Whole Blood Collected by Volumetric Absorptive Microsampling.

Proteomic biomarker discovery and analysis from human biofluids using liquid chromatography-mass spectrometry (LC-MS) is an area of intense biomedical research. There is a growing interest to analyze microsampled patient blood specimens as this is potentially more patient-friendly enabling at-home and bedside self-collection of small blood volumes. However, there are limited studies applying LC-MS proteomic analysis of whole blood as it is dominated by red blood cell proteins such as hemoglobin which suppresses the detection of other less abundant proteins. Volumetric absorptive microsampling (VAMS) devices overcome this issue in part by providing a trapping matrix which allows depletion of abundant blood cell proteins through washing, prior to proteolysis and LC-MS. This approach allows the analysis of proteins from erythrocytes, leukocytes, and plasma and leads to deeper proteomic coverage compared to conventional plasma proteomics, increasing the prospects to discover novel biomarker proteins.

Piloting a new model of personalised care for people with fibromyalgia in primary care with secondary care multidisciplinary support.

OBJECTIVE: An estimated 5.5 million people in England have high-impact chronic pain, which is severe pain associated with significant disability. Current models of healthcare often fail to address their broad range of symptoms and address their complex non-medical needs. METHODS: A pilot project was designed with the aim of improving the quality of care and addressing unmet needs of patients high-impact musculoskeletal (MSK) pain through providing a personalised approach to their pain and wider psychosocial needs. The model comprised a longer initial appointment with a general practitioner, a later follow-appointment, and support of the multidisciplinary team (MDT) (informally and through a formal MDT meeting) with both primary care clinicians and specialists based in secondary care. RESULTS: Forty six patients were seen using this model, with prominent themes of consultations including self-management, social needs, mental health and understanding their diagnosis. Evaluation of the pilot demonstrated improvements in MSK and non-MSK symptoms, together with improved patient confidence in self-management and knowledge and understanding of their condition. Multidisciplinary working proved to be invaluable in addressing patients' wider needs but also upskilling and supporting primary care clinicians. Primary care staff also found it to be a satisfying way to care for patients, and developed increasing skills and confidence in supporting patients with chronic pain. CONCLUSION: This model of care appears to be an effective way to help primary care teams to provide more holistic personalised care to a group of patients who are highly complex and so often forgotten.

Activation of the myosin motors in fast-twitch muscle of the mouse is controlled by mechano-sensing in the myosin filaments.

Myosin motors in resting muscle are inactivated by folding against the backbone of the myosin filament in an ordered helical array and must be released from that conformation to engage in force generation. Time-resolved X-ray diffraction from single fibres of amphibian muscle showed that myosin filament activation could be inhibited by imposing unloaded shortening at the start of stimulation, suggesting that filaments were activated by mechanical stress. Here we improved the signal-to-noise ratio of that approach using whole extensor digitorum longus muscles of the mouse contracting tetanically at 28°C. Changes in X-ray signals associated with myosin filament activation, including the decrease in the first-order myosin layer line associated with the helical motor array, increase in the spacing of a myosin-based reflection associated with packing of myosin tails in the filament backbone, and increase in the ratio of the 1,1 and 1,0 equatorial reflections associated with movement of motors away from the backbone, were delayed by imposing 10-ms unloaded shortening at the start of stimulation. These results show that myosin filaments are predominantly activated by filament stress, as in amphibian muscle. However, a small component of filament activation at zero load was detected, implying an independent mechanism of partial filament activation. X-ray interference measurements indicated a switch-like change in myosin motor conformation at the start of force development, accompanied by transient disordering of motors in the regions of the myosin filament near its midpoint, suggesting that filament zonal dynamics also play a role in its activation. KEY POINTS: Activation of myosin filaments in extensor digitorum longus muscles of the mouse is delayed by imposing rapid shortening from the start of stimulation. Stress is the major mechanism of myosin filament activation in these muscles, but there is a small component of filament activation during electrical stimulation at zero stress. Myosin motors switch rapidly from the folded inhibited conformation to the actin-attached force-generating conformation early in force development.

Gonadal sex patterns p21-induced cellular senescence in mouse and human glioblastoma.

Males exhibit higher incidence and worse prognosis for the majority of cancers, including glioblastoma (GBM). Disparate survival may be related to sex-biased responses to treatment, including radiation. Using a mouse model of GBM, we show that female cells are more sensitive to radiation, and that senescence represents a major component of the radiation therapeutic response in both sexes. Correlation analyses revealed that the CDK inhibitor p21 and irradiation induced senescence were differentially regulated between male and female cells. Indeed, female cellular senescence was more sensitive to changes in p21 levels, a finding that was observed in wildtype and transformed murine astrocytes, as well as patient-derived GBM cell lines. Using a novel Four Core Genotypes model of GBM, we further show that sex differences in p21-induced senescence are patterned during early development by gonadal sex. These data provide a rationale for the further study of sex differences in radiation response and how senescence might be enhanced for radiation sensitization. The determination that p21 and gonadal sex are required for sex differences in radiation response will serve as a foundation for these future mechanistic studies.

Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.

Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.

Age-Related Skeletal Muscle Dysfunction Is Aggravated by Obesity: An Investigation of Contractile Function, Implications and Treatment.

Obesity is a global epidemic and coupled with the unprecedented growth of the world's older adult population, a growing number of individuals are both old and obese. Whilst both ageing and obesity are associated with an increased prevalence of chronic health conditions and a substantial economic burden, evidence suggests that the coincident effects exacerbate negative health outcomes. A significant contributor to such detrimental effects may be the reduction in the contractile performance of skeletal muscle, given that poor muscle function is related to chronic disease, poor quality of life and all-cause mortality. Whilst the effects of ageing and obesity independently on skeletal muscle function have been investigated, the combined effects are yet to be thoroughly explored. Given the importance of skeletal muscle to whole-body health and physical function, the present study sought to provide a review of the literature to: (1) summarise the effect of obesity on the age-induced reduction in skeletal muscle contractile function; (2) understand whether obesity effects on skeletal muscle are similar in young and old muscle; (3) consider the consequences of these changes to whole-body functional performance; (4) outline important future work along with the potential for targeted intervention strategies to mitigate potential detrimental effects.

Age-related changes in isolated mouse skeletal muscle function are dependent on sex, muscle, and contractility mode.

The present study aimed to simultaneously examine the age-related, muscle-specific, sex-specific, and contractile mode-specific changes in isolated mouse skeletal muscle function and morphology across multiple ages. Measurements of mammalian muscle morphology, isometric force and stress (force/cross-sectional area), absolute and normalized (power/muscle mass) work-loop power across a range of contractile velocities, fatigue resistance, and myosin heavy chain (MHC) isoform concentration were measured in 232 isolated mouse (CD-1) soleus, extensor digitorum longus (EDL), and diaphragm from male and female animals aged 3, 10, 30, 52, and 78 wk. Aging resulted in increased body mass and increased soleus and EDL muscle mass, with atrophy only present for female EDL by 78 wk despite no change in MHC isoform concentration. Absolute force and power output increased up to 52 wk and to a higher level for males. A 23-36% loss of isometric stress exceeded the 14-27% loss of power normalized to muscle mass between 10 wk and 52 wk, although the loss of normalized power between 52 and 78 wk continued without further changes in stress (P > 0.23). Males had lower power normalized to muscle mass than females by 78 wk, with the greatest decline observed for male soleus. Aging did not cause a shift toward slower contractile characteristics, with reduced fatigue resistance observed in male EDL and female diaphragm. Our findings show that the loss of muscle quality precedes the loss of absolute performance as CD-1 mice age, with the greatest effect seen in male soleus, and in most instances without muscle atrophy or an alteration in MHC isoforms.

The impact of a high-fat diet in mice is dependent on duration and age, and differs between muscles.

Prolonged high-fat diets (HFDs) can cause intramyocellular lipid (IMCL) accumulation that may negatively affect muscle function. We investigated the duration of a HFD required to instigate these changes, and whether the effects are muscle specific and aggravated in older age. Muscle morphology was determined in the soleus, extensor digitorum longus (EDL) and diaphragm muscles of female CD-1 mice from 5 groups: young fed a HFD for 8 weeks (YS-HFD, n=16), young fed a HFD for 16 weeks (YL-HFD, n=28) and young control (Y-Con, n=28). The young animals were 20 weeks old at the end of the experiment. Old (70 weeks) female CD-1 mice received either a normal diet (O-Con, n=30) or a HFD for 9 weeks (OS-HFD, n=30). Body mass, body mass index and intramyocellular lipid (IMCL) content increased in OS-HFD (P≤0.003). In the young mice, this increase was seen in YL-HFD and not YS-HFD (P≤0.006). The soleus and diaphragm fibre cross-sectional area (FCSA) in YL-HFD was larger than that in Y-Con (P≤0.004) while OS-HFD had a larger soleus FCSA compared with that of O-Con after only 9 weeks on a HFD (P<0.001). The FCSA of the EDL muscle did not differ significantly between groups. The oxidative capacity of fibres increased in young mice only, irrespective of HFD duration (P<0.001). High-fat diet-induced morphological changes occurred earlier in the old animals than in the young, and adaptations to HFD were muscle specific, with the EDL being least responsive.

An Acetylenic Lipid from the New Zealand Ascidian Pseudodistoma cereum: Exemplification of an Improved Workflow for Determination of Absolute Configuration of Long-Chain 2-Amino-3-alkanols.

An acetylenic 2-amino-3-alcohol, distaminolyne B (2), isolated from the New Zealand ascidian Pseudodistoma cereum, is reported. The isolation and structure elucidation of 2 and assignment of 2S,3S absolute configuration (AC) using the exciton coupled circular dichroism technique are described. Using a methodologically facile workflow, the same AC was also established by analysis of specific rotation, terminal methyl C-1 δC chemical shift, and NH δH and J values of the N,O-diacetate derivative.

The impact of pelvic lateral rotation on hindlimb kinematics and stride length in the red-legged running frog, Kassina maculata.

Some frog species, such as Kassina maculata (red-legged running frog), use an asynchronous walking/running gait as their primary locomotor mode. Prior comparative anatomy work has suggested that lateral rotation of the pelvis improves walking performance by increasing hindlimb stride length; however, this hypothesis has never been tested. Using non-invasive methods, experimental high-speed video data collected from eight animals were used to create two three-dimensional kinematic models. These models, each fixed to alternative local anatomical reference frames, were used to investigate the hypothesis that lateral rotation of the mobile ilio-sacral joint in the anuran pelvis plays a propulsive role in walking locomotion by increasing hindlimb stride length. All frogs used a walking gait (duty factor greater than 0.5) despite travelling over a range of speeds (0.04-0.23 m s-1). The hindlimb joint motions throughout a single stride were temporally synchronized with lateral rotation of the pelvis. The pelvis itself, on average, underwent an angular excursion of 12.71° (±4.39°) with respect to the body midline during lateral rotation. However, comparison between our two kinematic models demonstrated that lateral rotation of the pelvis only increases the cranio-caudal excursion of the hindlimb modestly. Thus, we propose that pelvic lateral rotation is not a stride length augmenting mechanism in K. maculata.

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

Does Dietary-Induced Obesity in Old Age Impair the Contractile Performance of Isolated Mouse Soleus, Extensor Digitorum Longus and Diaphragm Skeletal Muscles?

Ageing and obesity independently have been shown to significantly impair isolated muscle contractile properties, though their synergistic effects are poorly understood. We uniquely examined the effects of 9 weeks of a high-fat diet (HFD) on isometric force, work loop power output (PO) across a range of contractile velocities, and fatigability of 79-week-old soleus, extensor digitorum longus (EDL) and diaphragm compared with age-matched lean controls. The dietary intervention resulted in a significant increase in body mass and gonadal fat pad mass compared to the control group. Despite increased muscle mass for HFD soleus and EDL, absolute isometric force, isometric stress (force/CSA), PO normalised to muscle mass and fatigability was unchanged, although absolute PO was significantly greater. Obesity did not cause an alteration in the contractile velocity that elicited maximal PO. In the obese group, normalised diaphragm PO was significantly reduced, with a tendency for reduced isometric stress and fatigability was unchanged. HFD soleus isolated from larger animals produced lower maximal PO which may relate to impaired balance in older, larger adults. The increase in absolute PO is smaller than the magnitude of weight gain, meaning in vivo locomotor function is likely to be impaired in old obese adults, with an association between greater body mass and poorer normalised power output for the soleus. An obesity-induced reduction in diaphragm contractility will likely impair in vivo respiratory function and consequently contribute further to the negative cycle of obesity.