Biology subjects

Moore, D. R.

Publications and source records attributed to Moore, D. R..

6 recordsLinked to original sources

bioRxiv2025

Stable isotope tracer captures the anabolic response of human skeletal muscle microtissues undetected by puromycin labeling

Skeletal muscle microtissues are valuable in vitro models for studying the stimuli regulating muscle protein synthesis (MPS), the key determinant of changes in muscle mass. Differentiated between opposing posts, microtissues contain aligned, contractile myotubes, providing a controlled system for investigating the responses of skeletal muscle to nutrient and contractile stimulation. However, microtissue MPS responses to these stimuli remain under-characterized. Stable isotope-labeled amino acid tracers deliver sarcoplasmic and myofibrillar fractional synthetic rates (FSR) for MPS in human studies, but have not been implemented in engineered skeletal muscle. We close these gaps by characterizing stimulation-induced MPS, in microtissues and 2D myotubes derived from the same primary myoblast line, using stable isotope tracers and puromycin incorporation. In microtissues, sarcoplasmic FSR increased significantly during the two-hour period following amino acid treatment (p < 0.0001), whereas myofibrillar FSR remained unchanged (p = 0.159). However, both fractions were unresponsive to ketone stimulation and contraction (all p [≥] 0.703). 2D myotubes showed significant increases in sarcoplasmic and myofibrillar FSR in response to amino acid treatment (both p = 0.002). Notably, microtissues demonstrated a more stable myofibrillar protein fraction, with a sarcoplasmic-to-myofibrillar FSR ratio of [~]2:1 which closely resembled that of native human muscle. The puromycin-based approach failed to detect MPS responses to any stimulus (all p [≥] 0.677), highlighting the superior sensitivity of tracer-based measurements, particularly where longer timescales are needed to capture an effect. These findings support the use of engineered muscle and isotope-derived measurements of MPS in future studies of stimuli regulating skeletal muscle mass. New FindingsO_ST_ABSWhat is the central question of this study?C_ST_ABSStable isotope tracers are emerging as a powerful approach to measure fraction-specific protein synthesis. However, their efficacy relative to conventional puromycin labeling remains unreported, and they have not been applied to engineered skeletal muscle. What is the main finding and its importance?By implementing stable isotope tracers in engineered muscle, we showcase the ability to capture anabolic responses that are undetected by puromycin-based methods. We found that the myofibrillar protein fraction of microtissues is more stable than the sarcoplasmic fraction, a property of native muscle, absent in 2D myotubes. These findings demonstrate the physiological relevance of engineered muscle and support the adoption of isotope-derived measurements in future studies.

bioengineering↗

bioRxiv2025

Exploring the multifactorial causes and therapeutic strategies for anabolic resistance in sarcopenia: A systems modeling study

BackgroundSarcopenia is the progressive loss of skeletal muscle mass, strength, and function with age, driven by dysregulation in the rates of muscle protein synthesis (MPS) and breakdown (MPB). Although MPB contributes to net protein balance (NB), a primary contributor of sarcopenia is anabolic resistance, defined as the blunted MPS response to anabolic stimuli such as feeding. While candidate mechanisms of anabolic resistance have been identified, none singularly accounts for the observed reduction in MPS. Instead, multiple mechanisms likely act simultaneously and interactively to suppress MPS. Studying these interactions experimentally is challenging. Mathematical modeling is well suited to analyzing complex biological phenomena such as anabolic resistance. MethodsWe analyzed a previously developed kinetic model of leucine-mediated signaling and protein metabolism in human skeletal muscle to systematically investigate potential mechanisms contributing to anabolic resistance. Using global sensitivity analysis, we identified key controllers of MPS, MPB, and NB. We then simulated amino acid feeding in older adults, classified the responses as either anabolic sensitive or resistant, and compared the resulting parameter distributions of the two groups. We next performed targeted analysis to evaluate the effects of individual and combined putative mechanisms of anabolic resistance on muscle metabolism. Finally, we simulated therapeutic interventions aimed at restoring muscle metabolism. ResultsThe sensitivity analysis revealed that MPS and MPB are primarily controlled by their proximal signaling processes, while NB is largely driven by MPS dynamics. Exploratory simulations showed that several parameters and signaling protein concentrations, particularly those controlling MPS, differed significantly between anabolic sensitive and resistant groups. The targeted simulations indicated that multiple dysregulated mechanisms were required to account for the experimentally observed reductions in MPS in older adults. Therapy simulations showed that single-target interventions could largely restore MPS when isolated mechanisms were perturbed (e.g., increasing mTORC1 sensitivity, enhancing p70S6K levels), but a multifactorial approach was required to recover muscle metabolism when all anabolic resistance mechanisms were present. ConclusionThis study highlights the multifactorial nature of anabolic resistance and the implications for therapy. Specifically, the results motivate new hypotheses regarding the mechanisms most likely to be impaired and argue for multi-target therapeutic strategies to help restore muscle protein metabolism in aging.

systems biology↗

bioRxiv2021

Mechanistic target of rapamycin complex 1 (mTORC1) activity occurs predominantly in the periphery of human skeletal muscle fibers, in close proximity to focal adhesion complexes, following anabolic stimuli

Following anabolic stimuli (e.g. mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1 activity occurs prior to or following this translocation. We therefore aimed to determine the cellular location of mTORC1 activity in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7, 23{+/-}5yrs, 76.8{+/-}3.6kg, 13.6{+/-}3.8%BF, FED) or following a whole-body resistance exercise bout (n=7, 22{+/-}2yrs, 78.1{+/-}3.6kg, 12.2{+/-}4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. The spatial regulation of mTORC1 activity was assessed through immunofluorescent staining of p-RPS6Ser240/244, an mTORC1-specific phosphorylation event. p-RPS6Ser240/244 measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining was displayed a similar pattern, suggesting mTORC1 activity occurs predominantly in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that mTORC1 activity occurs in the region of human muscle fibers to which mTORC1 translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 activation in vivo.

cell biology↗

bioRxiv2019

Listening difficulties in children: Behavior and brain activation produced by dichotic listening of CV syllables

Listening difficulties (LiD), also known as auditory processing disorders, are common in children with and without hearing loss. Impaired interactions between the two ears have been proposed as an important component of LiD. Previous studies have focused on testing using multiple sequential dichotic digits that carry a substantial memory load and executive control demands. We examined the ability of 6-13 year old children with normal audiometric thresholds to identify and selectively attend to dichotically presented CV syllables using the Bergen Dichotic Listening Test (BDLT; www.dichoticlistening.com). Children were recruited as typically developing (TD; n=39) or having LiD (n=35) based primarily on composite score of the ECLiPS caregiver report. Different single syllables (ba, da, ga, pa, ta, ka) were presented simultaneously to each ear (6x36 trials). Children reported the syllable heard most clearly (non-forced, NF) or the syllable presented to the right (forced, FR) or left (FL) ear. Interaural level differences (ILDs) manipulated bottom-up perceptual salience. Dichotic listening data (correct responses, Laterality Index) were analyzed initially by group (LiD, TD), age, report method (NF, FR, FL) and ILD (0, {+/-} 15 dB) and compared with speech-in-noise thresholds (LiSN-S) and cognitive performance (NIH Toolbox). fMRI measured brain activation produced by a receptive speech task that segregated speech, phonetic and intelligibility components. Some activated areas (planum temporale, inferior frontal gyrus and orbitofrontal cortex) were correlated with dichotic results in TD children only.\n\nNeither group, age nor report method affected the Laterality Index of right/left recall. However, a significant interaction was found between ear, group and ILD. Children with LiD were more influenced by large ILDs, especially favoring the left ear, than were TD children. Neural activity associated with Speech, Phonetic and Intelligibility sentence cues did not significantly differ between groups. Significant correlations between brain activity level and BDLT were found in several frontal and temporal locations for the TD but not for the LiD group.\n\nChildren with LiD were more influenced by large ILDs, especially favoring the left ear, than were TD children and were thus less able to modulate performance through attention, and more driven by the physical properties of the acoustic stimuli.

neuroscience↗

bioRxiv2019

Improving Sensitivity of the Digits-in-Noise Test using Antiphasic Stimuli

ObjectiveThe digits-in-noise test (DIN) has become increasingly popular as a consumer-based method to screen for hearing loss. Current versions of all DINs either test ears monaurally or present identical stimuli binaurally (i.e., diotic noise and speech, NoSo). Unfortunately, presentation of identical stimuli to each ear inhibits detection of unilateral sensorineural hearing loss (SNHL), and neither diotic nor monaural presentation sensitively detects conductive hearing loss (CHL). Following an earlier finding of enhanced sensitivity in normally hearing listeners, this study tested the hypothesis that interaural antiphasic digit presentation (NoS{pi}) would improve sensitivity to hearing loss caused by unilateral or asymmetric SNHL, symmetric SNHL, or CHL.\n\nDesignThis cross-sectional study, recruited adults (18-84 years) with various levels of hearing, based on a four-frequency pure tone average (PTA) at 0.5, 1, 2 and 4kHz. The study sample was comprised of listeners with normal hearing (n=41; PTA [≤] 25 dB HL in both ears), symmetric SNHL (n=57; PTA > 25 dB HL), unilateral or asymmetric SNHL (n=24; PTA > 25 dB HL in the poorer ear) and CHL (n=23; PTA > 25 dB HL and PTA air-bone gap [≥] 20 dB HL in the poorer ear). Antiphasic and diotic speech reception thresholds (SRTs) were compared using a repeated-measures design.\n\nResultsAntiphasic DIN was significantly more sensitive to all three forms of hearing loss than the diotic DIN. SRT test-retest reliability was high for all tests (ICC r > 0.89). Area under the receiver operating characteristics (ROC) curve for detection of hearing loss (> 25 dB HL) was higher for antiphasic DIN (0.94) than for diotic DIN (0.77) presentation. After correcting for age, PTA of listeners with normal hearing or symmetric SNHL was more strongly correlated with antiphasic (rpartial[96]=0.69) than diotic (rpartial=0.54) SRTs. Slope of fitted regression lines predicting SRT from PTA was significantly steeper for antiphasic than diotic DIN. For listeners with normal hearing or CHL, antiphasic SRTs were more strongly correlated with PTA (rpartial[62]=0.92) than diotic SRTs (rpartial[62]=0.64). Slope of regression line with PTA was also significantly steeper for antiphasic than diotic DIN. Severity of asymmetric hearing loss (poorer ear PTA) was unrelated to SRT. No effect of self-reported English competence on either antiphasic or diotic DIN among the mixed first-language participants was observed\n\nConclusionsAntiphasic digit presentation markedly improved the sensitivity of the DIN test to detect SNHL, either symmetric or asymmetric, while keeping test duration to a minimum by testing binaurally. In addition, the antiphasic DIN was able to detect CHL, a shortcoming of previous monaural or binaurally diotic DIN versions. The antiphasic DIN is thus a powerful tool for population-based screening. This enhanced functionality combined with smartphone delivery could make the antiphasic DIN suitable as a primary screen that is accessible to a large global audience.

pathology↗

bioRxiv2019

Genome-wide association study identifies 44 independent genomic loci for self-reported adult hearing difficulty in the UK Biobank cohort

Age-related hearing impairment (ARHI) is the most common sensory impairment in the aging population; a third of individuals are affected by disabling hearing loss by the age of 651. ARHI is a multifactorial condition caused by both genetic and environmental factors, with estimates of heritability between 35% and 55%2-4. The genetic risk factors and underlying biological pathology of ARHI are largely unknown, meaning that targets for new therapies remain elusive. We performed genome-wide association studies (GWAS) for two self-reported hearing phenotypes, hearing difficulty (HDiff) and hearing aid use (HAid), using over 250,000 UK Biobank5 volunteers aged between 40-69 years. We identified 44 independent genome-wide significant loci (P<5E-08), 33 of which have not previously been associated with any form of hearing loss. Gene sets from these loci are enriched in auditory processes such as synaptic activities, nervous system processes, inner ear morphology and cognition. Immunohistochemistry for protein localisation in adult mouse cochlea indicate metabolic, sensory and neuronal functions for NID2, CLRN2 and ARHGEF28 identified in the GWAS. These results provide new insight into the genetic landscape underlying susceptibility to ARHI.

genetics↗