bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Skeletal Muscle Protein Translation during Energetic Stress
Authors: Wolff Christopher A , Peelor Frederick F , Petrilli Erin B , Casey Richard M , Allison Joseph T , Hamilton Karyn L , Miller Benjamin F 
Published: November 2023

Protein translation is an energetically costly process that is globally repressed during energetic stress, as AMPK is activated and mTOR is inhibited. However, our laboratory has demonstrated that mitochondrial protein translation is maintained or increased during the energetic stress signaling associated with multiple models of slowed aging. The mechanisms allowing for the selective translation of mitochondrial proteins during energetic stress are not well characterized in mammalian systems. Thus, the purpose of this experiment was to examine the mechanisms regulating mitochondrial protein translation during energetic stress signaling. To induce energetic stress signaling, we treated C2C12 myotubes with 6 concentrations (10μM, 50μM, 100μM, 500μM, 1mM, or 2mM) of either buformin, phenformin, or metformin to find the lowest effective concentration for AMPK activation and mTOR inhibition. Western blot analyses demonstrated that 100μM buformin, 50μM phenformin, and 2mM metformin had the most potent AMPK activation (2.7, 2.6, and 2.5 fold increases, respectively) accompanied by inhibition of mTOR, as assessed by ribosomal protein S6 phosphorylation (0.8, 0.6, and 0.4 fold reductions, respectively). These concentrations were subsequently utilized for the mechanistic experiments. To assess the mechanisms regulating protein translation during energetic stress signaling C2C12 myotubes were treated for 24 hours with Buformin, Rapamycin, Metformin+Rapamycin, or a vehicle‐control. After treatment, translation was halted using cycloheximide and RNA was extracted from the C2C12 myotubes. RNA was then nuclease treated to create ribosome protected mRNA fragments, which were purified, utilized to prepare the cDNA library, and analyzed on an Illumina NextSEQ 500. Ribosome protected mRNA fragments (RPFs) were aligned against the current mouse genome, and further bioinformatics processing is underway. Through ribosomal profiling we were able to determine which proteins were selectively translated during energetic stress and whether these proteins differed by specific drug treatment. Further, this analysis provides proteins of interest that may contribute to slowing the aging process. It is expected that these data will provide mechanistic insight into why energetic stress signaling and the associated increase in mitochondrial protein synthesis contributes to the slowed aging phenotype.Support or Funding InformationThis project was supported by National Institute on Aging Grant R01‐AG‐042569
Regulation Of Actin Dynamics By WNT‐5A: Implications For Human Airway Smooth Muscle Contraction
Authors: Koopmans Tim , Kumawat Kuldeep , Menzen Mark , Halayko Andrew , Gosens Reinoud 
Published: November 2023

An important pathophysiological feature of asthma is airway hyperresponsiveness (AHR), characterized by exaggerated bronchoconstriction in which the airway smooth muscle (ASM) is fundamentally involved. How the ASM in asthmatics differs from that in non‐asthmatics is a current focus for research. We have shown previously that WNT‐5A is increasingly expressed in ASM of mild to moderate asthmatics. WNT proteins have been implicated in the regulation of cytoskeletal filaments and microtubules, thus, we tested the hypothesis that WNT‐5A is a determinant of the contractile response of ASM through modulation of the cytoskeleton.Using bovine tracheal smooth muscle strips, we found that following pre‐incubation with recombinant WNT‐5A (500 ng/mL, 48 h), maximum isometric tension induced by histamine was increased (Emax = 1,67 fold of control, p < 0.01). We used immortalized human ASM cells to study the underlying mechanisms. In myocytes loaded with the ratiometric calcium indicator, Fura‐2, we found that neither direct exposure to WNT‐5A nor pre‐incubation (48 h) had any effect on calcium flux handling. Moreover, WNT‐5A treatment for 24 h did not yield any detectable change in mRNA expression of the major calcium channels or receptors (IP3R, SERCA2 and RyR1/2/3). However, while direct exposure to WNT‐5A (10–200 ng/mL, 24 h) had no effect on total alpha smooth muscle actin (α‐SMA) protein, it did lead to increased abundance of filamentous actin (labelled with fluorochrome‐conjugated phalloidin). These changes, which were visible after 2 hours, could be completely prevented by treatment with the Rho kinase inhibitor Y27632 (1 uM). Interestingly, ASM cells serum‐deprived for 7 days to adopt a contractile phenotype also showed greater WNT‐5A protein expression (1,99 fold of control, p < 0.05) compared to the proliferative phenotype (serum‐fed cells). We next assessed whether TGF‐β1 played an upstream role in the WNT‐5A response. We found that WNT‐5A siRNA not only blocked TGF‐β1‐induced actin polymerization, but could also abrogate TGF‐β1‐induced accumulation of α‐SMA protein. In line with this contention, adding WNT‐5A on top of TGF‐β1 synergistically induced α‐SMA protein abundance (2,21 fold of TGF‐β, p < 0.01).In summary, WNT‐5A promotes increased agonist‐induced airway smooth muscle contraction, most likely by increasing actin polymerization and actin expression in concerted action with TGF‐β1. These results may be relevant in the understanding of AHR and warrant further investigation.Support or Funding InformationThis work was financially supported by a NWO Vidi grant, 016.126.307.
Sodium Nitrate Supplementation Does Not Alter Mitochondrial Bioenergetics In The Heart
Authors: Monaco Cynthia M.F. , Miotto Paula M. , Holloway Graham P. 
Published: November 2023

Sodium nitrate has been shown to protect against the development of hypertension mediated heart failure and improve the functional outcomes following doxorubicin‐induced cardiotoxicity. While oral nitrate has several beneficial effects on cardiovascular performance, the mechanism(s) underlying these responses remain poorly defined. In skeletal muscle, the oral consumption of nitrate has been shown to have various biological effects that would be advantageous for optimal cardiac performance, including an increase in excitation‐contraction properties and mitochondrial coupling efficiency. Therefore, the objective of this study was to determine whether oral sodium nitrate consumption improved mitochondrial bioenergetics in the hearts of healthy rats. Male Sprague Dawley rats (~ 20 weeks of age) were randomly assigned to a control (n=8) or supplemented group (n=8), which consisted of consuming sodium nitrate in their drinking water (1 g/L) for 7 days. Mitochondrial respiration in permeabilized muscle fibres from the left ventricle showed no changes in leak respiration, maximal substrate‐supported respiration, or ADP sensitivity (apparent Km) with and without 20 mM creatine and 10 mM phosphocreatine/10 mM creatine. In addition, sodium nitrate supplementation did not alter the rates of mitochondrial H2O2 emission in permeabilized cardiac fibres. Therefore, compared to skeletal muscle, these results suggest that short‐term sodium nitrate does not affect mitochondrial bioenergetics in the heart and additional mechanisms must explain the beneficial effects of consumption on cardiac performance.Support or Funding Informationsupported by NSERC, Canada
Elucidating Interactions Between Human Mas‐related G‐protein Coupled Receptors
Authors: Van Remoortel Samuel , Arora Rohit , Labro Alain J. , Snyders Dirk J. , Buckinx Roeland , Timmermans Jean‐Pierre 
Published: November 2023

IntroductionG‐protein coupled receptors (GPCR's) are important targets in drug discovery research because of their extensive regulatory effects on cellular functioning and their profound molecular diversity. A decade ago a new GPCR family, referred to as Mas‐related G‐protein coupled receptors (MRGPR), was discovered and since then shown to be involved in nociception and mast cell physiology. MRGPR are thus interesting targets in pain treatment and neuro‐immune communication. Previously, we were able to show the involvement of MRGPR in gastro‐intestinal neuro‐immune communication and observed a marked co‐expression of MRGPRD, MRGPRE and MRGPRF in the murine enteric nervous system (Avula et al. 2011; Avula et al. 2013). Given the well‐known concept of GPCR heteromerization, as well as its importance in functional modulation, we investigated heteromeric properties of the human orthologues of MRGPRD, MRGPRE and MRGPRF.Material and methodsHeteromeric MRGPR interactions were efficiently screened using a novel luciferase complementation‐based platform. Further validation of interacting partners was performed with Fluorescence Resonance Energy Transfer (FRET). Cells were transiently transfected with eCFP‐ and/or eYFP‐coupled receptors, and FRET efficiency was determined with acceptor photobleaching. For MRGPRD plasmids, receptor functionality was tested according to the known MRGPRD‐mediated inhibition of KCNQ2‐potassium currents.ResultsTransient transfections with fluorescently tagged vector constructs confirmed membrane expression of the vector protein product. Ligand‐induced activation of tagged MRGPRD resulted in reversible inhibition of KCNQ2‐mediated potassium currents, indicating functionality of fluorescently tagged MRGPR. Interaction screening with luciferase complementation provided clear evidence for heteromeric interactions between MRGPRE and MRGPRF. FRET analysis on transiently co‐transfected cells confirmed this MRGPRE‐MRGPRF interaction.ConclusionThis study provides novel insights on the interactions between human MRGPR. The observed MRGPRE‐MRGPRF interaction contributes to the further elucidation of the function of these orphan receptors, and strengthens the relevance of heteromeric interactions in their receptor biology. Furthermore, we provide a proof‐of‐concept for the applicability of a new complementation‐based screening assay as a high‐throughput screening tool for protein interactions.Support or Funding InformationThis study was financially supported by FWO grant G019314N.
Variation in Exercise Capacity and Responses to Training in Inbred Mouse Strains
Authors: Massett Michael P. , Avila Joshua J. , Kim Seung Kyum 
Published: November 2023

Improvements in cardiorespiratory fitness can significantly reduce the risk of all‐cause mortality. However, 7 – 45% of individuals show minimal or no improvements in exercise capacity. The aim of this study was to characterize differences in exercise capacity and training responses across mouse strains and estimate the genetic contribution to exercise capacity. Male mice from 25 inbred strains (n = 5–6/strain) performed a graded exercise test before and after 4 weeks of exercise training. Genome wide association mapping was performed using an efficient mixed model association (EMMA) algorithm to identify quantitative trait loci (QTL) for pre‐ and post‐training exercise capacity and the response to training. Exercise capacity was significantly different among strains when expressed as time (range = 21 – 42 min) and work performed (range = 0.4 – 3.9 kg·m). The responses to training were also different among strains. For the change in time, there was an approximately 4.5‐fold difference between the highest (+7.5 min) and lowest (−1.6 min) responding strains. This difference reached 7 fold when defined as change in work (−0.3 vs. 2.1 kg·m). Significant associations (P < 0.05) for exercise time were identified on 16 different chromosomes. Similarly, significant associations were identified on nearly all chromosomes for work performed. For training responses, significant associations were identified on 8 different chromosomes. Significant QTL on Chromosomes 3, 6, and 14 were common to most exercise phenotypes. These results indicate genetic background significantly influences responses to exercise training. Furthermore, the novel QTL identified in the current study provide new targets for investigating the mechanisms underlying variation in exercise capacity and training responses.Support or Funding InformationSupported by NIH grant HL085918 to MPM
Effects of Staphylococcus aureus Biofilm and Planktonic Secreted Products on Myofibroblast Differentiation
Authors: Hall Preston M , Brennan Robert E , Vaughan Melville B 
Published: November 2023

Wounds often become chronic by staying in the inflammatory stage of the healing process for too long. The ability of fibroblasts to differentiate into myofibroblasts is key for the normal progression of wound healing. Recent experimental evidence showed products secreted by planktonic S. aureus and S. aureus biofilms differentially affect viability and inflammatory cytokine production by human fibroblasts, along with human keratinocytes. Our goal was to determine whether media conditioned by S. aureus affected myofibroblast differentiation, using immunostaining techniques. We grew normal human fibroblasts on coverslips in media containing 1ng/ml TGF‐β plus or minus 100 μL/ml of the planktonic S. aureus secretions or the S. aureus biofilm secretions; these were compared to media‐only control. We found that products secreted by S. aureus, especially planktonic S. aureus, significantly decreased myofibroblast differentiation in three separate experiments across two different cell types which suggests that wound healing won't progress causing the wound to become chronic. Bacterial biofilms are difficult to treat, so promoting myofibroblasts may aid the healing of chronic wounds. These findings should spark future studies related to wound healing.Support or Funding Information3M Corporation and UCO Office of Research and Grants
Japanese herbal medicine, Juzentaihoto, suppress the lymphocyte infiltration in the aging mouse salivary glands
Authors: Nonaka Naoko , Nakamura Masanori 
Published: November 2023

Xerostomia or dry mouth is a systemic disease resulting from the change in the composition of saliva or reduced saliva flow. The symptom is caused by various factors such as the dysfunction of salivary glands and nervous system, the side effect of medicines and so on. The amount of salivation also decreases with aging. This symptom is very common today. Artificial saliva is usually prescribed for the treatment of xerostomia. We previously indicated that pituitary adenylate cyclase activating polypeptide (PACAP) stimulated the increase of salivation and the concentration of epidermal growth factor in secreted saliva. Furthermore, we found the severe lymphocyte infiltration in salivary glands of old C57B/6 mice. Juzentaihoto (JTT) is one of the Kampo Japanese herbal medicines and caused the induction of IL‐12 and subsequent activation of natural killer cells. In this study, we have examined the effect of JTT on lymphocyte infiltration in the major salivary glands (parotid, submandibular and sublingual glands). Male C57BL/6 mice were bred for 2 years under SPF condition. To examine the effect of the JTT, feed with/without JTT was given to mice during the breeding period. At the indicated time, salivary glands were dissected and processed for histological examination. Histological examination clearly indicated the severe lymphocyte infiltration in parotid glands and submandibular glands, but not in sublingual glands of control mice. Histomorphometric analysis indicated that the area of the lymphocyte infiltration in control mice was 4.35 ± 1.67% in parotid glands and 8.58 ± 4.43 % in submandibular glands respectively. On the other hand, the ratio of the infiltration was reduced to 1.94 ± 1.10 % in parotid glands and 1.74 ± 0.59 % in submandibular glands of JTT‐treated group. These results expect the old C57BL/6 (male) mice as a model of autoimmune disease such as Sjogren's syndrome and suggest JTT might be the effective therapeutic drug for the salivary gland disease with aging or inflammation.Support or Funding InformationGrant sponsors: Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant numbers: 20592148, 21592342 and 15K11022.
The Facial Nerve: The Path of Its Branches to the Muscles of the Infraorbital Region that Lift the Upper Lip in Relation to the Deep Fascia of the Midface
Authors: Amodeo Chiara Andretto , Delmas Vincent , Casasco Andrea , Keller Gregory S 
Published: November 2023

The mimetic muscles are innervated by the facial nerve. The function of these muscles is very important because they partecipate to the eye protection, the speach and the nutrition. Furthermore from an evolutionary point of view the facial expressions are a major way of communication between human beings. Classic anatomy describes the path of the branches of the facial nerve in the midface, anterior to the parotid fascia, as running in the subcutaneous tissue, a recent study related to the fascial layers of this region has shown that a deep fascia exists in the infraorbital region, it continues from the temporal area and it covers the mimetic muscles that lift the upper lip. The aim of this study is to describe the path of the branches of the facial nerve in this facial area in relation to the deep fascia recently described.Materials and Methods24 hemifaces have been dissected at the Laboratoire d'Anatomie de Paris Descartes to study the relationship between the branches of the facial nerve and the deep fascia of the infraorbital region.ResultsThis study demonstrates that the branches of the facial nerve that go to the muscles of the infraorbital region are always covered and protected by the deep fascia of this region. Some branches run deep to the muscles, on the contrary some run superficial to the zygomaticus major, minor, levaetor labii superioris etc and anastomize with other branches to reach the glabellar region, all these branches run deep to the deep fascia.ConclusionsThe existance of a deep fascia in the infraorbital region has been shown. This fascia covers and protects the branches of the facial nerve that run to the muscles that lift the upper lip, this finding changes the classic anatomical concept of the facial nerve running without protection in the subcutaneous tissue of the midface and it has important implications in both descriptive and applied anatomy.
Quantification of Telomerase Activity in Colonial Ascidians
Authors: Durham Megan , Beck Chelsey , Keeling Elena 
Published: November 2023

Colonial ascidians are marine invertebrate chordates that reproduce sexually and asexually, and are also capable of whole body regeneration in response to damage. Telomerase is required to maintain chromosome length in all eukaryotic cells that divide indefinitely; it is therefore expressed in stem cells and is hypothesized to be necessary for both continuous asexual budding and for regeneration. Here, we investigate telomerase activity in the colonial ascidian Botrylloides. Telomerase activity was determined using a qPCR‐based assay and normalized by total protein. Results so far show that adult ascidian tissue has high levels of telomerase activity; this may be related to stem cell abundance and regeneration abilities of Botrylloides. Work in progress is examining telomerase activity in different tissues, including probable stem cell niches, as well as in different developmental and regeneration stages.Support or Funding InformationCSU Council on Ocean Affairs, Science & Technology (COAST); Biological Sciences Department, California Polytechnic State University
Expression and Assembly of Active Human Telomerase in Escherichia coli
Authors: Hansen Jeffrey Lewis , Thiyagarajan Thirumagal , Larson Amy Kraft , Rideout April , Hansen Debra Tumbula 
Published: November 2023

The assembly of human telomerase is a complicated process in which over a dozen different human proteins are implicated. These proteins include the Hsp90 heat shock complex, the box H/ACA snoRNP complex, and other proteins. The specific roles of these proteins in telomerase assembly are unclear. Telomerase expression and assembly in reported recombinant systems occurs either in eukaryotic cells or in eukaryotic cell extracts. We are studying human telomerase expression and assembly in a non‐eukaryotic system, Escherichia coli. In order to study the assembly of active human telomerase in E. coli cells, we developed a modular system of plasmids compatible for co‐transformation and co‐expression of various components of telomerase assembly. This approach differs from previous recombinant approaches because co‐expression allows for co‐translational and co‐transcriptional assembly. In fact, we detected telomerase activity by the trap assay in cell extracts from a small subset of our library of co‐expression strains. Surprisingly, we found that co‐expression of hTERT with hTR (the protein component and RNA component of telomerase) alone in E. coli is sufficient for the assembly of active telomerase, with the certainty that no other eukaryotic factors are present. We are now relying on the modular nature of our system to efficiently produce additional E. coli strains containing new combinations of plasmids in an effort to increase the yield of active telomerase.Support or Funding InformationThis research was originally supported by startup funding at the Medical University in South Carolina.Currently support is through internal funding at A.T. Still University.