The autophagy inhibitor NSC185058 suppresses mTORC1-mediated protein anabolism in cultured skeletal muscle.

The mammalian target of rapamycin (mTOR), and specifically the mTOR complex 1 (mTORC1) is the central regulator of anabolism in skeletal muscle. Among the many functions of this kinase complex is the inhibition of the catabolic process of autophagy; however, less work has been done in investigating the role of autophagy in regulating mTORC1 signaling. Using an in vitro model to better understand the pathways involved, we activated mTORC1 by several different means (growth factors, leucine supplementation, or muscle contraction), alone or with the autophagy inhibitor NSC185058. We found that inhibiting autophagy with NSC185058 suppresses mTORC1 activity, preventing any increase in cellular protein anabolism. These decrements were the direct result of action on the mTORC1 kinase, which we demonstrate, for the first time, cannot function when autophagy is inhibited by NSC185058. Our results indicate that, far from being a matter of unidirectional action, the relationship between mTORC1 and the autophagic cascade is more nuanced, with autophagy serving as an mTORC1 input, and mTORC1 inhibition of autophagy as a form of homeostatic feedback to regulate anabolic signaling. Future studies of cellular metabolism will have to consider this fundamental intertwining of protein anabolism and catabolism, and how it ultimately serves to regulate muscle proteostasis.

St. Jude Survivorship Portal: sharing and analyzing large clinical and genomic datasets from pediatric cancer survivors.

Childhood cancer survivorship studies generate comprehensive datasets comprising demographic, diagnosis, treatment, outcome, and genomic data from survivors. To broadly share this data, we created the St. Jude Survivorship Portal (https://survivorship.stjude.cloud), the first data portal for sharing, analyzing, and visualizing pediatric cancer survivorship data. Over 1,600 phenotypic variables and 400 million genetic variants from over 7,700 childhood cancer survivors can be explored on this free, open-access portal. Summary statistics of variables are computed on-the-fly and visualized through interactive and customizable charts. Survivor cohorts can be customized and/or divided into groups for comparative analysis. Users can also seamlessly perform cumulative incidence and regression analyses on the stored survivorship data. Using the portal, we explored the ototoxic effects of platinum-based chemotherapy, uncovered a novel association between mental health, age, and limb amputation, and discovered a novel haplotype in MAGI3 strongly associated with cardiomyopathy specifically in survivors of African ancestry.

Metformin treatment results in distinctive skeletal muscle mitochondrial remodeling in rats with different intrinsic aerobic capacities.

The rationale for the use of metformin as a treatment to slow aging was largely based on data collected from metabolically unhealthy individuals. For healthspan extension metformin will also be used in periods of good health. To understand potential context specificity of metformin treatment on skeletal muscle, we used a rat model (HCR/LCR) with a divide in intrinsic aerobic capacity. Outcomes of metformin treatment differed based on baseline intrinsic mitochondrial function, oxidative capacity of the muscle (gastroc vs soleus), and the mitochondrial population (IMF vs SS). Metformin caused lower ADP-stimulated respiration in LCRs, with less of a change in HCRs. However, a washout of metformin resulted in an unexpected doubling of respiratory capacity in HCRs. These improvements in respiratory capacity were accompanied by mitochondrial remodeling that included increases in protein synthesis and changes in morphology. Our findings raise questions about whether the positive findings of metformin treatment are broadly applicable.

A spatial cell atlas of neuroblastoma reveals developmental, epigenetic and spatial axis of tumor heterogeneity.

Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage with complex inter- and intra-tumoral heterogeneity. To chart this complexity, we generated a comprehensive cell atlas of 55 neuroblastoma patient tumors, collected from two pediatric cancer institutions, spanning a range of clinical, genetic, and histologic features. Our atlas combines single-cell/nucleus RNA-seq (sc/scRNA-seq), bulk RNA-seq, whole exome sequencing, DNA methylation profiling, spatial transcriptomics, and two spatial proteomic methods. Sc/snRNA-seq revealed three malignant cell states with features of sympathoadrenal lineage development. All of the neuroblastomas had malignant cells that resembled sympathoblasts and the more differentiated adrenergic cells. A subset of tumors had malignant cells in a mesenchymal cell state with molecular features of Schwann cell precursors. DNA methylation profiles defined four groupings of patients, which differ in the degree of malignant cell heterogeneity and clinical outcomes. Using spatial proteomics, we found that neuroblastomas are spatially compartmentalized, with malignant tumor cells sequestered away from immune cells. Finally, we identify spatially restricted signaling patterns in immune cells from spatial transcriptomics. To facilitate the visualization and analysis of our atlas as a resource for further research in neuroblastoma, single cell, and spatial-omics, all data are shared through the Human Tumor Atlas Network Data Commons at www.humantumoratlas.org.

ppBAM: ProteinPaint BAM track for read alignment visualization and variant genotyping.

SUMMARY: ProteinPaint BAM track (ppBAM) is designed to assist variant review for cancer research and clinical genomics. With performant server-side computing and rendering, ppBAM supports on-the-fly variant genotyping of thousands of reads using Smith-Waterman alignment. To better visualize support for complex variants, reads are realigned against the mutated reference sequence using ClustalO. ppBAM also supports the BAM slicing API of the NCI Genomic Data Commons (GDC) portal, letting researchers conveniently examine genomic details of vast amounts of cancer sequencing data and reinterpret variant calls. AVAILABILITY AND IMPLEMENTATION: BAM track examples, tutorial, and GDC file access links are available at https://proteinpaint.stjude.org/bam/. Source code is available at https://github.com/stjude/proteinpaint.

Combined effects of heavy ion exposure and simulated Lunar gravity on skeletal muscle.

BACKGROUND: The limitations to prolonged spaceflight include unloading-induced atrophy of the musculoskeletal system which may be enhanced by exposure to the space radiation environment. Previous results have concluded that partial gravity, comparable to the Lunar surface, may have detrimental effects on skeletal muscle. However, little is known if these outcomes are exacerbated by exposure to low-dose rate, high-energy radiation common to the space environment. Therefore, the present study sought to determine the impact of highly charge, high-energy (HZE) radiation on skeletal muscle when combined with partial weightbearing to simulate Lunar gravity. We hypothesized that partial unloading would compromise skeletal muscle and these effects would be exacerbated by radiation exposure. METHODS: For month old female BALB/cByJ mice were -assigned to one of 2 groups; either full weight bearing (Cage Controls, CC) or partial weight bearing equal to 1/6th bodyweight (G/6). Both groups were then divided to receive either a single whole body absorbed dose of 0.5 Gy of 300 MeV 28Si ions (RAD) or a sham treatment (SHAM). Radiation exposure experiments were performed at the NASA Space Radiation Laboratory (NSRL) located at Brookhaven National Laboratory on Day 0, followed by 21 d of CC or G/6 loading. Muscles of the hind limb were used to measure protein synthesis and other histological measures. RESULTS: Twenty-one days of Lunar gravity (G/6) resulted in lower soleus, plantaris, and gastrocnemius muscle mass. Radiation exposure did not further impact muscle mass. 28Si exposure in normal ambulatory animals (RAD+CC) did not impact gastrocnemius muscle mass when compared to SHAM+CC (p>0.05), but did affect the soleus, where mass was higher following radiation compared to SHAM (p<0.05). Mixed gastrocnemius muscle protein synthesis was lower in both unloading groups. Fiber type composition transitioned towards a faster isoform with partial unloading and was not further impacted by radiation. The combined effects of partial loading and radiation partially mitigated fiber cross-sectional area when compared to partial loading alone. Radiation and G/6 reduced the total number of myonuclei per fiber while leading to elevated BrdU content of skeletal muscle. Similarly, unloading and radiation resulted in higher collagen content of muscle when compared to controls, but the effects of combined exposure were not additive. CONCLUSIONS: The results of this study confirm that partial weightbearing causes muscle atrophy, in part due to reductions of muscle protein synthesis in the soleus and gastrocnemius as well as reduced peripheral nuclei per fiber. Additionally, we present novel data illustrating 28Si exposure reduced nuclei in muscle fibers despite higher satellite cell fusion, but did not exacerbate muscle atrophy, CSA changes, or collagen content. In conclusion, both partial loading and HZE radiation can negatively impact muscle morphology.

Exercise and mitochondrial remodeling to prevent age-related neurodegeneration.

Healthy brain activity requires precise ion and energy management creating a strong reliance on mitochondrial function. Age-related neurodegeneration leads to a decline in mitochondrial function and increased oxidative stress, with associated declines in mitochondrial mass, respiration capacity, and respiration efficiency. The interdependent processes of mitochondrial protein turnover and mitochondrial dynamics, known together as mitochondrial remodeling, play essential roles in mitochondrial health and therefore brain function. This mini-review describes the role of mitochondria in neurodegeneration and brain health, current practices for assessing both aspects of mitochondrial remodeling, and how exercise mitigates the adverse effects of aging in the brain. Exercise training elicits functional adaptations to improve brain health, and current literature strongly suggests that mitochondrial remodeling plays a vital role in these positive adaptations. Despite substantial implications that the two aspects of mitochondrial remodeling are interdependent, very few investigations have simultaneously measured mitochondrial dynamics and protein synthesis. An improved understanding of the partnership between mitochondrial protein turnover and mitochondrial dynamics will provide a better understanding of their role in both brain health and disease, as well as how they induce protection following exercise.

Single-cell multiomics identifies clinically relevant mesenchymal stem-like cells and key regulators for MPNST malignancy.

Malignant peripheral nerve sheath tumor (MPNST), a highly aggressive Schwann cell (SC)-derived soft tissue sarcoma, arises from benign neurofibroma (NF); however, the identity, heterogeneity and origins of tumor populations remain elusive. Nestin+ cells have been implicated as tumor stem cells in MPNST; unexpectedly, single-cell profiling of human NF and MPNST and their animal models reveal a broad range of nestin-expressing SC lineage cells and dynamic acquisition of discrete cancer states during malignant transformation. We uncover a nestin-negative mesenchymal neural crest-like subpopulation as a previously unknown malignant stem-like state common to murine and human MPNSTs, which correlates with clinical severity. Integrative multiomics profiling further identifies unique regulatory networks and druggable targets against the malignant subpopulations in MPNST. Targeting key epithelial-mesenchymal transition and stemness regulators including ZEB1 and ALDH1A1 impedes MPNST growth. Together, our studies reveal the underlying principles of tumor cell-state evolution and their regulatory circuitries during NF-to-MPNST transformation, highlighting a hitherto unrecognized mesenchymal stem-like subpopulation in MPNST disease progression.

The genomic landscape of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Ablation of Ghrelin Receptor Mitigates the Metabolic Decline of Aging Skeletal Muscle.

The orexigenic hormone ghrelin has multifaceted roles in health and disease. We have reported that ablation of the ghrelin receptor, growth hormone secretagogue receptor (GHS-R), protects against metabolic dysfunction of adipose tissues in aging. Our further observation interestingly revealed that GHS-R deficiency phenocopies the effects of myokine irisin. In this study, we aim to determine whether GHS-R affects the metabolic functions of aging skeletal muscle and whether GHS-R regulates the muscular functions via irisin. We first studied the expression of metabolic signature genes in gastrocnemius muscle of young, middle-aged and old mice. Then, old GHS-R knockout (Ghsr-/-) mice and their wild type counterparts were used to assess the impact of GHS-R ablation on the metabolic characteristics of gastrocnemius and soleus muscle. There was an increase of GHS-R expression in skeletal muscle during aging, inversely correlated with the decline of metabolic functions. Remarkedly the muscle of old GHS-R knockout (Ghsr-/-) mice exhibited a youthful metabolic profile and better maintenance of oxidative type 2 muscle fibers. Furthermore, old Ghsr-/- mice showed improved treadmill performance, supporting better functionality. Also intriguing to note was the fact that old GHS-R-ablated mice showed increased expression of the irisin precursor FNDC5 in the muscle and elevated plasma irisin levels in circulation, which supports a potential interrelationship between GHS-R and irisin. Overall, our work suggests that GHS-R has deleterious effects on the metabolism of aging muscle, which may be at least partially mediated by myokine irisin.

The myogenesis program drives clonal selection and drug resistance in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric cancer with features of skeletal muscle; patients with unresectable or metastatic RMS fare poorly due to high rates of disease recurrence. Here, we use single-cell and single-nucleus RNA sequencing to show that RMS tumors recapitulate the spectrum of embryonal myogenesis. Using matched patient samples from a clinical trial and orthotopic patient-derived xenografts (O-PDXs), we show that chemotherapy eliminates the most proliferative component with features of myoblasts within embryonal RMS; after treatment, the immature population with features of paraxial mesoderm expands to reconstitute the developmental hierarchy of the original tumor. We discovered that this paraxial mesoderm population is dependent on EGFR signaling and is sensitive to EGFR inhibitors. Taken together, these data serve as a proof of concept that targeting each developmental state in embryonal RMS is an effective strategy for improving outcomes by preventing disease recurrence.

Maximizing Mobilization of Resources for Unplanned Certified Registered Nurse Anesthetist Staffing Deficiencies in a Large Midwest Multi-Hospital Trauma Center.

Acute staffing deficiencies pose a challenge for hospitals because shortages can negatively impact patient safety. The purpose of this study was to examine the most effective staff recall system (SRS) for Certified Registered Nurse Anesthetists (CRNAs) during times of acute staffing deficiencies, whether from unforeseen emergencies or mass call outs. A preposttest design was used to explore the efficacy of the hospital's current CRNA SRS compared to a newly developed short message system (SMS) Acute Staffing Deficiency Recall Plan. Two 60-minute simulation drills were conducted to test the two systems with a convenience sample of 317 CRNAs. Data were gathered related to CRNAs: contacted, availability, and length of time to contact. Surveys were utilized pre/ post to evaluate knowledge, confidence, and selfefficacy regarding the recall systems. During the first simulation drill, 26% of CRNAs were contacted and 19% confirmed availability within 60 minutes using the standard CRNA SRS. During the second simulation, 86% of the CRNAs were contacted and 38% confirmed availability using the SMS messaging system within 60 minutes (P < .00001 and P < .0001 respectively). The SMS messaging improved CRNA contact by 60% and availability improved by 19% over the standard SRS.

Culprits or consequences: Understanding the metabolic dysregulation of muscle in diabetes.

The prevalence of type 2 diabetes (T2D) continues to rise despite the amount of research dedicated to finding the culprits of this debilitating disease. Skeletal muscle is arguably the most important contributor to glucose disposal making it a clear target in insulin resistance and T2D research. Within skeletal muscle there is a clear link to metabolic dysregulation during the progression of T2D but the determination of culprits vs consequences of the disease has been elusive. Emerging evidence in skeletal muscle implicates influential cross talk between a key anabolic regulatory protein, the mammalian target of rapamycin (mTOR) and its associated complexes (mTORC1 and mTORC2), and the well-described canonical signaling for insulin-stimulated glucose uptake. This new understanding of cellular signaling crosstalk has blurred the lines of what is a culprit and what is a consequence with regard to insulin resistance. Here, we briefly review the most recent understanding of insulin signaling in skeletal muscle, and how anabolic responses favoring anabolism directly impact cellular glucose disposal. This review highlights key cross-over interactions between protein and glucose regulatory pathways and the implications this may have for the design of new therapeutic targets for the control of glucoregulatory function in skeletal muscle.

Use of deuterium oxide (2H2O) to assess muscle protein synthesis in juvenile red drum (Sciaenops ocellatus) fed complete, and valine-deficient diets.

The use of 2H2O in tank water to assess protein synthesis rates in fish is a relatively novel methodology that could allow for a better understanding of the effects of particular nutritional and environmental variables on rates of protein accretion. As such, this study involved an assessment and comparison of protein synthesis rates in the muscle of juvenile red drum fed a control diet (nutritionally complete) versus a valine (Val)-deficient diet. Six groups of 12 juvenile red drum, initially weighing ~ 4.5 g/fish, were stocked in six separate 38-L aquaria operating as a recirculating system. Fish were acclimatized to experimental conditions for 2 weeks while being fed the control diet. Just prior to initiating the protein synthesis assay, one aquarium of fish was fed the control diet while a second aquarium of fish was fed the Val-deficient diet. Immediately after consuming the experimental diets, each group of fish was moved to an independent aquarium containing 2H2O, and the fractional synthetic rate (FSR) of protein synthesis was obtained at 12, 24, 36 and 48 h after feeding by collecting two fish per treatment at each time point. This protein synthesis assay procedure was performed in three separate sessions, and considered as replicates over time (n = 3) for fish fed the control or Val-deficient diets immediately before initiating the session. Results indicated that a one-time feeding of a diet deficient in Val significantly reduced protein synthesis rates in the muscle of red drum. In addition, a significant effect of time after feeding was found, where observed FSR values peaked at 12 h after feeding and decreased as time progressed. In conclusion, deuterium methodologies were applicable to red drum, and this approach had the sensitivity to assess differences in protein synthesis rates when dietary perturbations were introduced.

Retinoblastoma from human stem cell-derived retinal organoids.

Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Children with germline mutations in RB1 have a high likelihood of developing retinoblastoma and other malignancies later in life. Genetically engineered mouse models of retinoblastoma share some similarities with human retinoblastoma but there are differences in their cellular differentiation. To develop a laboratory model of human retinoblastoma formation, we make induced pluripotent stem cells (iPSCs) from 15 participants with germline RB1 mutations. Each of the stem cell lines is validated, characterized and then differentiated into retina using a 3-dimensional organoid culture system. After 45 days in culture, the retinal organoids are dissociated and injected into the vitreous of eyes of immunocompromised mice to support retinoblastoma tumor growth. Retinoblastomas formed from retinal organoids made from patient-derived iPSCs have molecular, cellular and genomic features indistinguishable from human retinoblastomas. This model of human cancer based on patient-derived iPSCs with germline cancer predisposing mutations provides valuable insights into the cellular origins of this debilitating childhood disease as well as the mechanism of tumorigenesis following RB1 gene inactivation.

Rider Energy Expenditure During High Intensity Horse Activity.

Despite the fact that horseback riding is a popular sport, there is little information available on horseback riding as a physical activity. The objective of this experiment was to quantify energy expenditure of participants (n=20) during three riding tests: a 45min walk-trot-canter ride (WTC), a reining pattern ride and a cutting simulation ride while wearing a telemetric gas analyzer. Total energy expenditure (tEE), mean and peak metabolic equivalents of task (MET), heart rate (HR), respiratory frequency (RF), relative oxygen consumption (relVO2), and respiratory exchange ratio (RER) were assessed. Mean MET and HR responses were greater (P < .05) for riders during the long trot portion of the WTC (6.19 ± 0.21 MET, 152.14 ± 4.4 bpm) and cutting (4.53±0.21 MET, 146.88 ± 4.4 bpm) vs the overall WTC (3.81 ± 0.16 MET, 131.5 ± 4.2 bpm). When WTC was evaluated by gait, mean MET increased as gait speed increased. As expected, METs were greater (P < .05) for riders during long trot (6.19 ± 0.21 MET) and canter (5.95 ± 0.21 MET) than during the walk (2.01 ± 0.21 MET) or trot (3.2 ± 0.21 MET). Previous horseback riding studies have not reported METs, but the peaks of all three activities in the present study were similar to METs measured during activities like jogging, playing soccer and rugby. Riders engaged in cutting and reining experienced more intense exercise in short durations, while, as expected on the basis of the duration of the activity, WTC provided a greater overall total energy expenditure. These results suggest that it is possible for health benefits to be achieved through accumulated horseback riding exercise, particularly if riding at the more intense gaits.

Ral signaling pathway in health and cancer.

The Ral (Ras-Like) signaling pathway plays an important role in the biology of cells. A plethora of effects is regulated by this signaling pathway and its prooncogenic effectors. Our team has demonstrated the overactivation of the RalA signaling pathway in a number of human malignancies including cancers of the liver, ovary, lung, brain, and malignant peripheral nerve sheath tumors. Additionally, we have shown that the activation of RalA in cancer stem cells is higher in comparison with differentiated cancer cells. In this article, we review the role of Ral signaling in health and disease with a focus on the role of this multifunctional protein in the generation of therapies for cancer. An improved understanding of this pathway can lead to development of a novel class of anticancer therapies that functions on the basis of intervention with RalA or its downstream effectors.

Long-term influence of chemotherapy on steatosis-associated advanced hepatic fibrosis.

To determine whether chemotherapy treatment at least 6 months prior to the detection of hepatic steatosis is associated with advanced hepatic fibrosis. Demographics, comorbid conditions, and laboratory data for cancer patients with hepatic steatosis were reviewed. The primary end point of this study was a low probability of fibrosis as calculated by the AST-to-platelet ratio index (APRI)-a surrogate for the absence of histologic bridging fibrosis and/or cirrhosis. Of 279 patients, 117 (41.9 %) were treated with chemotherapy and 197 (66.3 %) had a low probability of fibrosis by APRI. A smaller proportion of patients treated with chemotherapy had a low probability of hepatic fibrosis compared with untreated patients (64.1 vs. 75.3 %, p = 0.04). On multivariable analysis, chemotherapy treatment was a negative predictive factor for a low probability of fibrosis (OR 0.366 [95 % CI 0.184-0.708], p < 0.01). Among chemotherapy-treated patients, 75 (64.1 %) had a low probability of fibrosis. There were no differences in chemotherapy duration (mean 7.8 vs. 7.5 cycles) and interval from last dose to steatosis diagnosis (24.3 vs. 21.4 months) between patients with and without a low probability of fibrosis. A smaller proportion of patients treated with irinotecan or 5-fluorouracil had a low probability of fibrosis (37.3 vs. 66.7 %, p = 0.04). On multivariable analysis, irinotecan or 5-fluorouracil treatment was a negative predictive factor for low probability of fibrosis (OR 0.277 [95 % CI 0.091-0.779], p = 0.02). Prior chemotherapy treatment, especially with 5-fluorouracil or irinotecan, is a negative predictor for the absence of advanced hepatic fibrosis among patients with steatosis.

Breast cancer patients' perspectives on and use of complementary and alternative medicine: a study by the Susan G. Komen Breast Cancer Foundation.

The purpose of this study was to examine patterns and predictors of complementary and alternative medicine (CAM) among breast cancer patients. A review of the existing survey literature on CAM use for breast cancer was conducted with a series of eight focus groups (N = 67) to further examine the perspectives of breast cancer patients on CAM. The rates of CAM use varied from 17 to 75%, with a mean of 45%. Vitamins and minerals and herbs were the most frequently cited categories. Users tended to be younger, more educated, and more likely to have used CAM prior to their diagnosis. Focus group data indicate that breast cancer patients use a wide array of CAM for a variety of reasons, including symptom management, improving quality of life, and enhancing immune function. Although women rely on a variety of resources for information, they frequently experience frustration owing to the absence or conflicting nature of such information. Communication with conventional providers about CAM is frequently experienced as either unsupportive or not helpful by many patients. The results point to the value of developing better evidence-based informational resources related to CAM and cancer and the need for physicians to become better educated about CAM and how to communicate more effectively with their breast cancer patients about it.