Nystagmus Associated With the Absence of MYOD Expression Across the Lifespan in Extraocular and Limb Muscles.

Purpose: The extraocular muscles (EOMs) undergo significant levels of continuous myonuclear turnover and myofiber remodeling throughout life, in contrast to limb skeletal muscles. Activation of the myogenic pathway in muscle precursor cells is controlled by myogenic transcription factors, such as MYOD. Limb muscles from MyoD-/- mice develop normally but have a regeneration defect, and these mice develop nystagmus. We examined MyoD-/- mice to determine if they have an aging phenotype. Methods: Eye movements of aging MyoD-/- mice and littermate controls (wild type) were examined using optokinetic nystagmus (OKN). We assessed limb muscle function, changes to myofiber number, mean cross-sectional area, and abundance of the PAX7 and PITX2 populations of myogenic precursor cells. Results: Aging did not significantly affect limb muscle function despite decreased mean cross-sectional areas at 18+ months. Aging wild type mice had normal OKN responses; all aging MyoD-/- mice had nystagmus. With OKN stimulus present, the MyoD-/- mice at all ages had shorter slow phase durations compared to wild type age matched controls. In the dark, the MyoD-/- mice had a shorter slow phase duration with age. This correlated with significantly decreased fiber numbers and cross-sectional areas. The EOM in MyoD-/- mice had increased numbers of PAX7-positive satellite cells and significantly decreased PITX2-positive myonuclei. Conclusions: The absence of MYOD expression in aging mice causes a decrease in on-going myofiber remodeling, EOM fiber size, and number, and is associated with the development of spontaneous nystagmus. These results suggest that muscle-specific mutations can result in nystagmus, with increasing aging-related changes in the MyoD-/- EOM.

Real-time text message surveys reveal student perceptions of personnel resources throughout a course-based research experience.

Course-based research experiences (CREs) are designed to engage students in authentic scientific experiences that are embedded into a standard curriculum. CREs provide valuable research experiences to large numbers of undergraduate biology students, however, CRE implementation can require many personnel. Because limited personnel may be a barrier to widespread CRE implementation, our goal was to discover which personnel students valued throughout a CRE and the ways they were valuable. We investigated students' perceptions of personnel resources throughout a semester-long CRE using two survey approaches. Using a text message survey administered multiple times per week, real-time data was collected about which personnel resource students perceived to be the most helpful. Using a web-based survey administered five times throughout the semester, retrospective data was collected about how often students used each personnel resource and how helpful students perceived each personnel resource to be. Graduate teaching assistants (TAs) were consistently selected as the most helpful personnel resource by the majority of respondents throughout the semester, with most respondents describing graduate TAs providing project-specific feedback. Although less frequently, undergraduate TAs were also consistently selected as the most helpful personnel resource. Respondents described undergraduate TAs providing project-specific feedback, general feedback, and project-specific resources. Data from the retrospective, web-based survey largely mirrored the real-time, text message survey data. Throughout the semester, most respondents reported using graduate TAs "Often" or "Always" and that graduate TAs were "Very" or "Extremely" helpful. Throughout most of the semester, most respondents also reported using undergraduate TAs "Often" or "Always" and that undergraduate TAs were "Very" or "Extremely" helpful. The results of this descriptive study underscore the importance of graduate and undergraduate TAs in the development and implementation of CREs, emphasizing the need for departments and course coordinators to be intentional in planning TA training that prepares TAs to fulfill their critical role in CRE implementation.

Meta-analysis of Gender Performance Gaps in Undergraduate Natural Science Courses.

To investigate patterns of gender-based performance gaps, we conducted a meta-analysis of published studies and unpublished data collected across 169 undergraduate biology and chemistry courses. While we did not detect an overall gender gap in performance, heterogeneity analyses suggested further analysis was warranted, so we investigated whether attributes of the learning environment impacted performance disparities on the basis of gender. Several factors moderated performance differences, including class size, assessment type, and pedagogy. Specifically, we found evidence that larger classes, reliance on exams, and undisrupted, traditional lecture were associated with lower grades for women. We discuss our results in the context of natural science courses and conclude by making recommendations for instructional practices and future research to promote gender equity.

Open Inquiry versus Broadly Relevant Short-Term Research Experiences for Non-Biology Majors.

Undergraduate student participation in course-based research experiences results in many positive outcomes, but there is a lack of evidence demonstrating which elements of a research experience are necessary, especially for non-biology majors. Broad relevance is one element that can be logistically challenging to incorporate into research experiences in large-enrollment courses. We investigated the impacts of broad relevance in a short-term research experience in an introductory biology course for non-majors. Students either participated in an open-inquiry research experience (OI-RE), where they developed their own research question, or a broadly relevant research experience (BR-RE), where they investigated a question assigned to them that was relevant to an ongoing research project. We found a significant association between the type of research project experienced and students' preference for an experience, with half of the students in the OI-RE group and nearly all students in the BR-RE group preferring a broadly relevant research experience. However, since science confidence increased over the course for both groups, these findings indicate that while students who participated in a BR-RE valued it, broadly relevant research experiences may not be necessary for positive outcomes for non-majors.

A Call for Data-Driven Networks to Address Equity in the Context of Undergraduate Biology.

National efforts to improve equitable teaching practices in biology education have led to an increase in research on the barriers to student participation and performance, as well as solutions for overcoming these barriers. Fewer studies have examined the extent to which the resulting data trends and effective strategies are generalizable across multiple contexts or are specific to individual classrooms, institutions, or geographic regions. To address gaps in our understanding, as well as to establish baseline information about students across contexts, a working group associated with a research coordination network (Equity and Diversity in Undergraduate STEM, EDU-STEM) convened in Las Vegas, Nevada, in November of 2019. We addressed the following objectives: 1) characterize the present state of equity and diversity in undergraduate biology education research; 2) address the value of a network of educators focused on science, technology, engineering, and mathematics equity; 3) summarize the status of data collection and results; 4) identify and prioritize questions and interventions for future collaboration; and 5) construct a recruitment plan that will further the efforts of the EDU-STEM research coordination network. The report that follows is a summary of the conclusions and future directions from our discussion.

Effects of retinoic acid signaling on extraocular muscle myogenic precursor cells in vitro.

One major difference between limb and extraocular muscles (EOM) is the presence of an enriched population of Pitx2-positive myogenic precursor cells in EOM compared to limb muscle. We hypothesize that retinoic acid regulates Pitx2 expression in EOM myogenic precursor cells and that its effects would differ in leg muscle. The two muscle groups expressed differential retinoic acid receptor (RAR) and retinoid X receptor (RXR) levels. RXR co-localized with the Pitx2-positive cells but not with those expressing Pax7. EOM-derived and LEG-derived EECD34 cells were treated with vehicle, retinoic acid, the RXR agonist bexarotene, the RAR inverse agonist BMS493, or the RXR antagonist UVI 3003. In vitro, fewer EOM-derived EECD34 cells expressed desmin and fused, while more LEG-derived cells expressed desmin and fused when treated with retinoic acid compared to vehicle. Both EOM and LEG-derived EECD34 cells exposed to retinoic acid showed a higher percentage of cells expressing Pitx2 compared to vehicle, supporting the hypothesis that retinoic acid plays a role in maintaining Pitx2 expression. We hypothesize that retinoic acid signaling aids in the maintenance of large numbers of undifferentiated myogenic precursor cells in the EOM, which would be required to maintain EOM normalcy throughout a lifetime of myonuclear turnover.

A Call to Develop Course-Based Undergraduate Research Experiences (CUREs) for Nonmajors Courses.

Course-based undergraduate research experiences (CUREs) for non-science majors (nonmajors) are potentially distinct from CUREs for developing scientists in their goals, learning objectives, and assessment strategies. While national calls to improve science, technology, engineering, and mathematics education have led to an increase in research revealing the positive effects of CUREs for science majors, less work has specifically examined whether nonmajors are impacted in the same way. To address this gap in our understanding, a working group focused on nonmajors CUREs was convened to discuss the following questions: 1) What are our laboratory-learning goals for nonmajors? 2) What are our research priorities to determine best practices for nonmajors CUREs? 3) How can we collaborate to define and disseminate best practices for nonmajors in CUREs? We defined three broad student outcomes of prime importance to the nonmajors CURE: improvement of scientific literacy skills, proscience attitudes, and evidence-based decision making. We evaluated the state of knowledge of best practices for nonmajors, and identified research priorities for the future. The report that follows is a summary of the conclusions and future directions from our discussion.

Sparing of the extraocular muscles in mdx mice with absent or reduced utrophin expression: A life span analysis.

Sparing of the extraocular muscles in muscular dystrophy is controversial. To address the potential role of utrophin in this sparing, mdx:utrophin(+/-) and mdx:utrophin(-/-) mice were examined for changes in myofiber size, central nucleation, and Pax7-positive and MyoD-positive cell density at intervals over their life span. Known to be spared in the mdx mouse, and contrary to previous reports, the extraocular muscles from both the mdx:utrophin(+/-) and mdx:utrophin(-/-) mice were also morphologically spared. In the mdx:utrophin(+/)(-) mice, which have a normal life span compared to the mdx:utrophin(-/-) mice, the myofibers were larger at 3 and 12 months than the wild type age-matched eye muscles. While there was a significant increase in central nucleation in the extraocular muscles from all mdx:utrophin(+/)(-) mice, the levels were still very low compared to age-matched limb skeletal muscles. Pax7- and MyoD-positive myogenic precursor cell populations were retained and were similar to age-matched wild type controls. These results support the hypothesis that utrophin is not involved in extraocular muscle sparing in these genotypes. In addition, it appears that these muscles retain the myogenic precursors that would allow them to maintain their regenerative capacity and normal morphology over a lifetime even in these more severe models of muscular dystrophy.

Mitochondrial Aging and Physical Decline: Insights From Three Generations of Women.

Decline in mitochondrial DNA (mtDNA) copy number, function, and accumulation of mutations and deletions have been proposed to contribute to age-related physical decline, based on cross sectional studies in genetically unrelated individuals. There is wide variability of mtDNA and functional measurements in many population studies and therefore we assessed mitochondrial function and physical function in 18 families of grandmothers, mothers, and daughters who share the same maternally inherited mtDNA sequence. A significant age-related decline in mtDNA copy number, mitochondrial protein expression, citrate synthase activity, cytochrome c oxidase content, and VO2 peak were observed. Also, a lower abundance of SIRT3, accompanied by an increase in acetylated skeletal muscle proteins, was observed in grandmothers. Muscle tissue-based full sequencing of mtDNA showed greater than 5% change in minor allele frequency over a lifetime in two locations, position 189 and 408 in the noncoding D-loop region but no changes were noted in blood cells mtDNA. The decline in oxidative capacity and muscle function with age in three generations of women who share the same mtDNA sequence are associated with a decline in muscle mtDNA copy number and reduced protein deacetylase activity of SIRT3.

Disease course in mdx:utrophin+/- mice: comparison of three mouse models of Duchenne muscular dystrophy.

The mdx mouse model of Duchenne muscular dystrophy (DMD) is used to study disease mechanisms and potential treatments, but its pathology is less severe than DMD patients. Other mouse models were developed to more closely mimic the human disease based on knowledge that upregulation of utrophin has a protective effect in mdx muscle. An mdx:utrophin(-/-) (dko) mouse was created, which had a severe disease phenotype and a shortened life span. An mdx:utrophin(+/-) mouse was also created, which had an intermediate disease phenotype compared to the mdx and dko mice. To determine the usefulness of mdx:utrophin(+/-) mice for long-term DMD studies, limb muscle pathology and function were assessed across the life span of wild-type, mdx, mdx:utrophin(+/-), and dko mice. Muscle function assessment, specifically grip duration and rotarod performance, demonstrated that mdx:utrophin(+/-) mice were weaker for a longer time than mdx mice. Mean myofiber area was smaller in mdx:utrophin(+/-) mice compared to mdx mice at 12 months. Mdx:utrophin(+/-) mice had a higher percentage of centrally nucleated myofibers compared to mdx mice at 6 and 12 months. Collagen I and IV density was significantly higher in mdx:utrophin(+/-) muscle compared to mdx at most ages examined. Generally, mdx:utrophin(+/-) mice showed an intermediate disease phenotype over a longer time course compared to the mdx and dko mice. While they do not genetically mirror human DMD, mdx:utrophin(+/-) mice may be a more useful animal model than mdx or dko mice for investigating long-term efficacy of potential treatments when fibrosis or muscle function is the focus.

The role of Pitx2 in maintaining the phenotype of myogenic precursor cells in the extraocular muscles.

Many differences exist between extraocular muscles (EOM) and non-cranial skeletal muscles. One striking difference is the sparing of EOM in various muscular dystrophies compared to non-cranial skeletal muscles. EOM undergo continuous myonuclear remodeling in normal, uninjured adults, and distinct transcription factors are required for the early determination, development, and maintenance of EOM compared to limb skeletal muscle. Pitx2, a bicoid-like homeobox transcription factor, is required for the development of EOM and the maintenance of characteristic properties of the adult EOM phenotype, but is not required for the development of limb muscle. We hypothesize that these unique properties of EOM contribute to the constitutive differences between EOM and non-craniofacial skeletal muscles. Using flow cytometry, CD34(+)/Sca1(-/)CD45(-/)CD31(-) cells (EECD34 cells) were isolated from extraocular and limb skeletal muscle and in vitro, EOM EECD34 cells proliferated faster than limb muscle EECD34 cells. To further define these myogenic precursor cells from EOM and limb skeletal muscle, they were analyzed for their expression of Pitx2. Western blotting and immunohistochemical data demonstrated that EOM express higher levels of Pitx2 than limb muscle, and 80% of the EECD34 cells expressed Pitx2. siRNA knockdown of Pitx2 expression in EECD34 cells in vitro decreased proliferation rates and impaired the ability of EECD34 cells to fuse into multinucleated myotubes. High levels of Pitx2 were retained in dystrophic and aging mouse EOM and the EOM EECD34 cells compared to limb muscle. The differential expression of Pitx2 between EOM and limb skeletal muscle along with the functional changes in response to lower levels of Pitx2 expression in the myogenic precursor cells suggest a role for Pitx2 in the maintenance of constitutive differences between EOM and limb skeletal muscle that may contribute to the sparing of EOM in muscular dystrophies.

Loss of synaptic vesicles from neuromuscular junctions in aged MRF4-null mice.

MRF4 belongs to the basic helix-loop-helix class of transcription factors and this and other members of its family profoundly influence skeletal muscle development. Less is known about the role of these factors in aging. As MRF4 is preferentially expressed in subsynaptic nuclei, we postulated it might play a role in maintenance of the neuromuscular junction. To test this hypothesis, we examined the junctional regions of 19-20-month-old mice and found decreased levels of SV2B, a marker of synaptic vesicles, in MRF4-null mice relative to controls. There was a corresponding decrease in grip strength in MRF4-null mice. Taken together, these data suggest that the intrinsic muscle factor, MRF4 plays an important role in maintenance of neuromuscular junctions.

Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis.

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin(-/-) (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of mpcs, the EOMCD34 cells, that are retained in significantly higher percentages in normal, mdx and DKO mice EOM, appear to be resistant to elevated levels of oxidative stress and toxins, and actively proliferate throughout life. Current studies are focused on further defining the EOMCD34 cell subtype molecularly, with the hopes that this may shed light on a cell type with potential therapeutic use in patients with sarcopenia, cachexia, or muscular dystrophy.

Mitochondrial DNA alterations and reduced mitochondrial function in aging.

Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed.

Protein and energy metabolism in type 1 diabetes.

Profound metabolic changes occur in people with type 1 diabetes mellitus during insulin deprivation. These include an increase in basal energy expenditure and reduced mitochondrial function. In addition, protein metabolism is significantly affected during insulin deprivation. A greater increase in whole-body protein breakdown than protein synthesis occurs resulting in a net protein loss. During insulin deprivation the splanchnic bed has a net protein accretion which accounts for the total increase in whole-body protein synthesis while muscle is in a net catabolic state.

Basic helix-loop-helix factors recruit nuclear factor I to enhance expression of the NaV 1.4 Na+ channel gene.

We have previously shown that the basic helix-loop-helix (bHLH) transcription factors coordinate Na(V) 1.4 Na(+) channel gene expression in skeletal muscle, but the identity of the co-factors they direct is unknown. Using C2C12 muscle cells as a model system, we test the hypothesis that the bHLH factors counteract negative regulation exerted through a repressor E box (-90/-85) by recruiting positive-acting transcription factors to the nucleotides (-135/-57) surrounding the repressor E box. We used electrophoretic mobility shift assays to identify candidate factors that bound the repressor E box or these adjacent regions. Repressor E box-binding factors included the known transcription factor, ZEB/AREB6, and a novel repressor E box-binding factor designated REB. Mutations of the repressor E box that interfere with the binding of these factors prevented repression. The transcription factor, nuclear factor I (NFI), bound immediately upstream and downstream of the repressor E box. Mutation of the NFI-binding sites diminished the ability of myogenin and MRF4 to counteract repression. Based on these observations we suggest that bHLH factors recruit NFI to enhance skeletal muscle Na(+) channel expression.

The Ca(V) 1.2 Ca(2+) channel is expressed in sarcolemma of type I and IIa myofibers of adult skeletal muscle.

Although Ca(2+)-dependent signaling pathways are important for skeletal muscle plasticity, the sources of Ca(2+) that activate these signaling pathways are not completely understood. Influx of Ca(2+) through surface membrane Ca(2+) channels may activate these pathways. We examined expression of two L-type Ca(2+) channels in adult skeletal muscle, the Ca(V) 1.1 and Ca(V) 1.2, with isoform-specific antibodies in Western blots and immunocytochemistry assays. Consistent with a large body of work, expression of the Ca(V) 1.1 was restricted to skeletal muscle where it was expressed in T-tubules. Ca(V) 1.2 was also expressed in skeletal muscle, in the sarcolemma of type I and IIa myofibers. Exercise-induced alterations in muscle fiber types cause a concomitant increase in the number of both Ca(V) 1.2 and type IIa-positive fibers. Taken together, these data suggest that the Ca(V) 1.2 Ca(2+) channel is expressed in adult skeletal muscle in a fiber type-specific manner, which may help to maintain oxidative muscle phenotype.

Immunocytochemical localization of TNF type 1 and type 2 receptors in the rat spinal cord.

Tumor necrosis factor-alpha (TNF-alpha) is secreted in numerous pathophysiological situations by a variety of cell types. Tactile hypersensitivity (allodynia) is one component of a constellation of "illness behaviors" triggered by TNF-alpha. TNF-alpha is also implicated in neuropathic pain after peripheral nerve injury and apoptosis after spinal cord injury (SCI). It is possible that SCI, illness- and peripheral injury-induced hypersensitivity may share a similar spinal mediated etiology. These studies identify the locus of type-1 TNF (TNFR1 or p55) and type-2 TNF (TNFR2 or p75) receptors within the spinal cord. At all spinal levels, TNFR1 receptor immunoreactivity (TNFR1-ir) was constitutively expressed on cells and afferent fibers within the dorsal root ganglia, afferent fibers of the dorsal root, dorsal root entry zone (REZ) and within lamina I and II of the dorsal horn. Unilateral dorsal rhizotomy eliminated the characteristic pattern of TNFR1-ir at the rhizotomized REZ. In contrast, TNFR2-ir was consistently absent from dorsal root fibers and the region of the root entry zone. Consistent with our previous report, medullary afferent fibers in the solitary tract and spinal trigeminal tract labelled for TNF1-ir, but did not express TNFR2-ir. The presence TNFR1-ir on dorsal horn afferents, suggests that TNF-alpha may be a mechanism responsible for tactile hypersensitivity during illness. The presence of TNFR1 receptors, and perhaps their long-term activation or plasticity, may also play a critical role in the chronic allodynia and hyperreflexia observed after SCI or peripheral nerve damage.

TNF alpha-p55 receptors: medullary brainstem immunocytochemical localization in normal and vagus nerve-transected rats.

Tumor necrosis factor alpha (TNF(alpha)) is a potent modulator of autonomic reflex mechanisms that control the stomach. Evidence suggests that TNF(alpha) action directly on vago-vagal reflex control circuits causes the autonomic misregulation of digestion manifested as gastrointestinal stasis, nausea, and emesis associated with illness. Neurophysiological studies indicated that TNF(alpha) may have effects on vagal afferents in the solitary nucleus, as well as neurons of the solitary nucleus (NST) and dorsal motor nucleus (DMN) of the vagus. The aim of this study was to determine the location of the TNFR1 receptor (p55) in the medulla using immunocytochemical methods. We devised a technique for localizing the p55 receptor using heat-induced antigen recovery in fixed tissue sections. This protocol allowed us to demonstrate that dense p55-immunoreactivity (p55-ir) is constitutively present on central (but not peripheral) vagal afferents in the solitary tract (ST) and nucleus; p55-ir is also present on afferents entering the spinal trigeminal nucleus. Unilateral supra-nodose vagotomy eliminated p55-ir from ipsilateral central vagal afferents. Virtually all neurons in the brainstem appeared to express p55-ir at a low level, i.e., just above background. However, vagotomy caused a dramatic up-regulation of p55-ir in vagal motor neurons. This increase in p55-ir in axotomized neurons may play a pivotal role in the connection between the occurrence of the injury and the initiation of apoptotic processes resulting in elimination of damaged neurons.

Leptin-induced changes in body composition in high fat-fed mice.

Female C57BL/6J mice were adapted to 10% or 45% kcal fat diets for 8 weeks. Continuous intraperitoneal infusion of 10 micro g of leptin/day from a miniosmotic pump transiently inhibited food intake in low fat-fed but not high fat-fed mice. In contrast, both low and high fat-fed leptin-infused mice were less fat than their phosphate-buffered saline (PBS) controls after 13 days. Leptin infusion inhibited insulin release but did not change glucose clearance in low fat-fed mice during a glucose tolerance test. A single intraperitoneal injection of 30 micro g of leptin inhibited 24-hr energy intake and inhibited weight gain in both low and high fat-fed mice. Insulin responsiveness was improved in high fat-fed mice during an insulin sensitivity test due to an exaggerated elevation of circulating insulin concentrations. Thus, leptin infusion reduced adiposity independently of energy intake in high fat-fed mice and improved insulin sensitivity in low fat-fed mice, whereas leptin injections, which produced much greater, but transient, increases in serum leptin concentration, inhibited energy intake in both low and high fat-fed mice.