bluuebunny/crossref_metadata_2025 huggingface dataset:
Systematic characterization of enzyme activity on ENPP1 deficiency disease phenotype
Authors: Ansh Anenya J. , Stabach Paul R. , Carpenter Thomas O. , Ferreira Carlos R. , Braddock Demetrios
Published: May 2022

ENPP1 codes for a type 2 transmembrane glycoprotein in the ectonucleotide pyrophosphatase/phosphodiesterase protein family. The enzyme’s catalytic activity hydrolyzes extracellular ATP to generate pyrophosphate and adenosine monophosphate (AMP), thereby contributing to downstream purinergic signaling pathways. Pathogenic variants in ENPP1 are associated with diseases such as Type 2 Diabetes, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2), Cole Disease, and early onset osteoporosis. The human phenotypes associated with these diseases are varied, and include life threatening large arterial calcifications, phosphate wasting rickets due to FGF23 elevation, cutaneous hypopigmentation and punctate keratosis, ankylosis and ossification of spinal ligaments, premature osteoporosis, as well as insulin resistance and cardiovascular disease.

To determine whether the above disease phenotypes correlate with ENPP1 enzyme activity we quantitated enzyme velocities of ENPP1 variants identified in 42 ENPP1‐deficient patients followed at the NIH, together with an additional 22 variants reported in the literature. We simultaneously evaluated catalytic velocity of all variants in triplicate, enabling a direct comparison of enzyme activity across disease phenotypes. Cole disease is induced by variants in the SMB domains of ENPP1, and variants associated with the Autosomal Dominant form reduced enzyme velocity to a greater extent than the Autosomal Recessive form – between 32% to 8% of wild type (WT) vs. 33% of WT levels, respectively. Further supporting the presence of a gene dose effect, monoallelic variants in ENPP1 associated with elevation of FGF23, phosphate wasting, and early onset osteoporosis, occurred in patients with variants that reduced enzyme velocity below 50% of WT, while the polymorphisms associated with insulin resistance and cardiovascular disease reduced enzyme velocity to 73% of WT levels. Additionally, variants associated with life‐threatening vascular calcifications in GACI, which only occur in autosomal recessive biallelic ENPP1 deficient patients, reduced ENPP1 enzyme activity over a surprisingly wide range, from 25% to 100% of WT levels. Finally, disease severity in GACI correlated with the more severely damaging ENPP1 variant rather than the average of both variant allele products, suggesting that the effects of biallelic ENPP1 variants on in vivo enzyme activity are dominated by the more damaging ENPP1 allele. In summary, comparison of enzyme velocities among various ENPP1 deficient disease phenotypes supports the notion that specific variants determine these variable phenotypes, and are sometimes, but not always, related to enzyme activity. ENPP1 gene dose and dominance of the severe isoform in biallelic disease are notable mechanisms for determination of phenotype in some forms of ENPP1 deficiency.
Increased Levels of Stress Induces a More Metastatic Phenotype in Human Melanoma Cells
Authors: Fajardo Abigail , Barkoudeh Laila , Contreras Abigail , Gutierrez Francisca , Diaz Heidi , Phan Kati H. , Lam Daniel , Rodriguez Guadalupe J. , Rosero Rebecca A. , Klettke Elizabeth , Amomoy Jerry L. , Nguyen Cecilia , Phung Vanessa , Tran Jenny M. , Zarghooni Melody , Armaiz‐Peña Guillermo N. , Villares Gabriel J.
Published: May 2022

Growing evidence suggests that stress plays a vital role in metastasis and tumor development by activating the sympathetic nervous system. The catecholamines released into the tumor microenvironment (TME), specifically norepinephrine (NE), results in a cascade effect leading to a variety of pro‐metastatic activities that sustain tumor growth and increase melanoma aggressiveness. Most notable are the secretion of cytokines and the stimulation of tumor‐associated macrophages (TAMs). Using a co‐culture system of several human melanoma cancer cells and macrophages, we found that sustained exposure of NE in the TME induced the release of tumor‐growth and progression‐associated cytokines from TAMs. Several of the upregulated cytokines found, including IL‐11, IL‐24, GRO‐a, MIF, DKK‐1, and angiopoietin‐2, are known to induce growth, migration, invasion, and tumor development in human melanoma. An ELISA assay of human‐MIF was used to validate the cytokine array in which upregulation of MIF was noted in co‐culture with NE present. At a molecular level, these findings suggest how physiological stress in cancer patients can further lead to cancer progression. These preliminary findings may lead to supportive therapies that emphasize mental health and the treatment of the psychological state of the patient. Our next steps include continuing the validation of the cytokine arrays using different ELISA assays to quantify protein levels of stress‐secreted cytokines in the TME that augment the metastatic phenotype of human melanoma cells.
Modulation of miR‐21 can reprogram the TGF‐β signaling pathway to alter HSC phenotype in vitro
Authors: Srivastava Ankita , Parrish Austin , Hoek Jan B. , Vadigepalli Rajanikanth
Published: May 2022

microRNAs are small non‐coding RNAs capable of modulating gene expression through post‐transcriptional modification affecting fundamental regulatory mechanisms to control cell function and play a significant role in the transition between cell states. Previous studies have demonstrated the differential expression of specific microRNAs in the context of disease states including alcoholic liver disease. Research from our group and others have identified several miRNAs as potential regulators of liver regeneration, such as miR‐21, whose role in liver regeneration and liver disease is well‐characterized, and several other miRNAs that show differential expression in response to altered miR‐21 expression. Dysregulation of miR‐21 expression has been implicated in altered hepatic stellate cell (HSC) activation. A potential mechanism for ethanol‐mediated impairment of liver regeneration is due to TGF‐β/SMAD/SMURF signaling in activated HSCs. In this study, we have characterized the HSCs response to miR‐21 dysregulation followed by TGF‐β stimulation using human hepatic stellate cell line LX2. We manipulated miR‐21 activity using locked nucleic acid oligos to either inhibit or mimic miR‐21. Our findings show that inhibition of miR‐21 activity followed by TGF‐β stimulation leads to the altered expression of components of the TGF‐β signaling pathway that in effect attenuated the downstream response to TGF‐β. By contrast, enhancing miR‐21 activity using a mimic amplified response to TGF‐β signal. This reprogramming of the TGF‐β pathway by exogenous alterations in miR‐21 activity was reflected in the modulation of HSC activation signature and migration phenotype. Taken together, our results show that changes in miR‐21 activity rewires the TGF‐β signaling network with consequences on HSC phenotype in vitro.
Identity, Power, and Legitimacy: Faculty Conceptions of Diversity in Higher Education
Authors: Lo Stanley M. , Suarez Nicole A. , Wang Song , Brydges Stacey
Published: May 2022

Institutions have increasingly made the commitment to diversify higher education, and faculty play integral roles in creating an inclusive culture and learning environments. Our study asks: How do faculty conceptualize diversity in higher education, and how do these conceptions influence curriculum and instruction? Using phenomenography as the theoretical and methodological framework, we examined the qualitatively different ways in which faculty experience and understand diversity. These ways of understanding are organized into a phenomenographic outcome space with specific aspects that describe the phenomenon of diversity and variations within each aspect that distinguish the individual experiences. Data were collected through semi‐structured interviews with 30 faculty from two‐year and four‐year minority‐serving institutions. Transcripts were analyzed using grounded theory, and data were coded by two independent researchers to ensure reliability and analyzed by four researchers with diverse positionalities to support validity. Qualitative codes were constructed to capture how each participant described diversity. Five aspects were identified from our data: student features, legitimized membership, intelligence mindset, faculty role, and learning environment. Variations among experiences were organized into an outcome space with three distinct conceptions of diversity. In Conception I (which we termed essentialist), faculty attend to demographic features of students and view students with a fixed mindset of intelligence and as outsiders to higher education. This is aligned with equal treatments of all students and a curriculum approach that considers diversity as an impediment to learning. In Conception II (functionalist), faculty attend to different student viewpoints and consider students with a deficit mindset and as guests who transiently pass through higher education institutions. This is aligned with accommodations for student needs and a curriculum approach that supports struggling students. Conception III (existentialist) includes and expands on Conception II by attending to how lived experiences intersect with demographic features and viewpoints to shape the kinds of learners that individual students become in the classroom. Implicit power dynamics are considered, and students are viewed as rightfully present in higher education regardless of their backgrounds. Specific curriculum approaches are intentionally implemented to foster productive conversations around different student characteristics and to center social justice issues, and diversity enriches learning in the classroom. Overall, our results indicate that faculty acknowledge different student features and have varying understanding for what diversity means and why it is important in higher education, and some conceptions of diversity do not necessarily suggest an inclusive culture.
Sleep Deficits, Repetitive Behaviors, and Increased Slow Gamma Power in an Ank3 Mouse Model of Epilepsy‐Mood Disorder Comorbidity
Authors: Riveira Nicholas J. , Villacres Perez Juan E. , Kim Sohmee , Colgin Laura L. , Lopez Angel Y.
Published: May 2022

The ANK3 gene is a leading bipolar disorder candidate gene in humans. Previous studies showed that a bipolar disorder (BD)‐associated variant of ANK3 (ANK3‐1b) leads to increased firing threshold and diminished action potential dynamic range of parvalbumin (PV) interneurons and absence epilepsy in mice, thus providing a potential mechanistic link between epilepsy and BD. To better understand the impact of PV interneurons on network activity and behavior in these mice, we examined spectral correlates of behaviors seen in Ank3‐1b knockout (KO) mice during overnight home‐cage activity using paired video‐EEG recordings. PV interneuron dysfunction and aberrant gamma rhythms have been implicated in BD. Also, the fast‐spiking properties of PV interneurons make them important for generating and modulating high frequency gamma oscillations. Thus, we anticipated changes in the power of EEG signals in the gamma frequency range (> 25 Hz) during behaviors related to BD symptoms seen in human patients such as changes in sleep and activity levels. Ank3‐1b KO mice exhibited an overall increase in slow gamma (~25‐45 Hz) power compared to controls, and slow gamma power correlated with seizure phenotype severity across behaviors. Notably, during sleep, increased slow gamma power co‐occurred with decreases in time spent in the rapid eye movement (REM) phase. We also identified a repetitive behaviors phenotype in Ank3‐1b KO mice that co‐occurred with increased slow gamma power. These results further support the association of Ank3‐1b with BD and suggest modulation of gamma oscillations as a potential therapeutic target.
Abscisic Acid Controlled Redox Proteome of Arabidopsis and its Regulation by Heterotrimeric G‐proteins
Authors: Smythers Amanda L. , McConnell Evan , Pandey Sona , Hicks Leslie M.
Published: May 2022

Reactive oxygen species (ROS) are highly efficient, membrane permeable cell signals that are essential for cellular regulation. These signals can mark macromolecules for degradation, act as a retrograde signal for transcriptional regulation, as well as directly modulate enzymatic activity via the reversible oxidation of cysteine thiols. Although this reversible cysteine oxidation is an essential regulatory mechanism, the resulting modifications are labile and infrequent compared to the global proteome, making them challenging to maintain ex vivo and detect reproducibly. In our lab, we have optimized an approach for oxidized resin assisted capture (OxRAC) that enables thorough, reproducible analysis of the full reversible cysteine redoxome. By employing iodoacetamide as a blocking reagent during cell lysis, we ensure that cysteines reduced in vivoare excluded before non‐specifically reducing reversibly oxidized cysteines for enrichment and analysis via label free quantitative LC‐MS/MS.

In this work, our optimized OxRAC platform was implemented to probe the dependence of heterotrimeric G‐proteins on reversible cysteine oxidation for the mediation of abscisic acid (ABA) signaling pathways in Arabidopsis thaliana. By cross comparing the wildtype Col0plants with the Gβ protein (AGB1) null mutant, agb1, we were able to quantify 6,891 unique oxidized cysteine‐containing peptides and reveal 923 significant changes in oxidation following ABA treatment. Divergent pathways, including primary metabolism, ROS response, translation, and photosynthesis, exhibited both ABA and G‐protein dependent redox changes, many of which occurred on proteins not previously linked to either ABA or G‐proteins. Together, these data uncover a complex network of reversible oxidations that allow ABA and G‐proteins to rapidly adjust cellular signaling to adapt to changing environments and suggest that a functional G‐protein complex is required to maintain intracellular redox homeostasis and fully execute plant stress responses.
Novel Dopamine D4 Receptor‐Selective Antagonists For Neuropsychiatric Disorders
Authors: Keck Thomas M. , Kelshikar Shreya , Shah Nisha , McBride Connor , Ximello Sanchez Moises , Panasis Diandra , Maslonka Brianna , Muccilli Abigail , Ladik Maria , Akca Ebrar , Ginet Sophia , Nilson Ashley , Boldizsar Noelia , Free R. Benjamin , Sibley David R. , Boateng Comfort A.
Published: May 2022

The neurotransmitter dopamine signals through G protein‐coupled receptors that regulate a variety of neurophysiological functions, including movement, emotional regulation, motivation, and cognition. Dopamine D4 receptors (D4Rs) are enriched in the prefrontal cortical regions critical to attention, cognition, decision‐making, and executive function. The physiological relevance of D4R signaling in the brain is not well understood and but a variety of evidence indicates its importance in neuropsychiatric disorders related to cognition and behavioral control, including Alzheimer’s disease, ADHD, and substance use disorders (SUDs). Preclinical studies indicate that D4R‐selective ligands can improve outcomes in animal models of these disorders, but the field is limited by the availability of highly selective ligands with suitable pharmacological characterization. Herein we report the development and structure‐activity relationships of a novel class of highly D4R‐selective ligands, primarily full antagonists, based on a phenylpiperazine (PP) scaffold and benzothiazole secondary pharmacophore. Following comprehensive in vitro binding and functional analyses, selected ligands were evaluated for pharmacokinetics in rat and human liver microsomes, followed by preliminary in vivo behavioral analysis. Overall, we identified several novel compounds with high binding affinity and D4R selectivity (Ki ≤ 100 nM and >100‐fold vs. other D2‐like receptors). Lead compound CAB‐01‐019, one of the most selective and high‐affinity D4R antagonists in this set, has favorable in vivo pharmacokinetics in rats, and inhibited cocaine self‐administration in rats. This new class of compounds represents a new set of tools to enhance medications development at D4R and to better understand the role of D4R signaling in complex behaviors.
Manganese Homeostasis in Bacteria: Interaction of the Small Protein MntS and Manganese Exporter MntP in E. coli
Authors: Seymour Mackenzie , Wright Zachary , Waters Lauren
Published: May 2022

Manganese (Mn) serves as a vital cofactor for bacterial survival, protecting bacteria from reactive oxygen species, radiation, and acidic environments. Bacterial metal ion regulation is associated with pathogenicity, as many virulent strains of bacteria upregulate their ability to obtain metal ions from host species. However, excess metal ions quickly become toxic for most bacteria, resulting in tightly regulated cytoplasmic metal ion concentrations. To better understand regulation of metal levels, our research has focused on the interactions of proteins responsible for Mn homeostasis in Escherichia coli (E. coli), specifically small protein interactions. Small proteins of less than 50 amino acids in length are an emerging group of understudied proteins, yet those that have been studied appear to have important regulatory roles. This research has focused on the small protein called MntS, just 42 amino acids long, which is regulated by the manganese activated transcription factor MntR. In E. coli, MntR represses the production of MntS as well as MntH, a manganese importer, and activates the synthesis of MntP, a manganese exporter. Interestingly, when MntS and MntP are expressed at the same time in the presence of high Mn concentrations, E. coli becomes sensitive to high levels of Mn, suggesting that MntS could interfere with the ability of MntP to export Mn. Indeed, using two‐hybrid assays and co‐purifications we have demonstrated an interaction between MntS and MntP. Additionally, while searching for MntS homologs, we discovered similarity between MntS and the signal peptide (SP) of certain SitA proteins. SitA is a periplasmic protein responsible for cation import, typically Mn. While E. colilacks a SitA protein completely, in the species with the SPs most similar to MntS, the sitAgene is encoded next to mntR. This suggested that the SPs might have similar activity as MntS. We have demonstrated that they produce similar phenotypes in both Mn sensitivity and two‐hybrid assays, suggesting both an evolutionary relationship and a necessary regulatory function. These results provide deeper insight into E. coli’s metal ion regulation, which in the long‐run could allow better treatment of bacterial infections by targeting metal ion regulators.
Response of enthesopathy in ENPP1 deficiency to enzyme replacement therapy in murine models and enthesopathy comorbidities and quality of life in ENPP1‐deficient adults
Authors: Ansh Anenya J. , Nester Catherine , O'Brien Christine , Stabach Paul R. , Murtada Sae‐Il , Lester Ethan R. , Khursigara Gus , Molloy Liz , Carpenter Thomas O. , Ferreira Carlos R. , Braddock Demetrios T.
Published: May 2022

Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency leads to cardiovascular calcification in infancy, FGF23‐mediated hypophosphatemic rickets in childhood, and osteomalacia in adulthood. Excessive enthesis mineralization and cervical spine fusion have been previously reported in patients with biallelic ENPP1 deficiency, but their effect on quality of life is unknown. We describe additional musculoskeletal complications in patients with ENPP1 deficiency, namely osteoarthritis and interosseous membrane ossification, and for the first time evaluate health‐related quality of life (HRQoL) in patients with this disease, both subjectively via narrative report, and objectively via the Brief Pain Inventory – Short Form, and a Patient Reported Outcome Measurement Information System Physical Function (PROMIS PF) short form. Residual pain, similar in magnitude to that identified in adult patients with X‐linked hypophosphatemia, was experienced by the majority of patients despite use of analgesic medications. Impairment in physical function varied from mild to severe. To assess murine ENPP1 deficiency for the presence of enthesopathy, and for the potential response to enzyme replacement therapy, we maintained Enpp1asj/asj mice on regular chow for 23 weeks and treated cohorts with either vehicle or a long‐acting form of recombinant ENPP1. Enpp1asj/asj mice treated with vehicle exhibited robust calcification throughout their Achilles tendons, whereas two‐thirds of those treated with ENPP1 enzyme replacement exhibited complete or partial suppression of the Achilles tendon calcification. Our combined results document that musculoskeletal complications are a significant source of morbidity in homozygous ENPP1 Deficiency, a phenotype which is closely recapitulated in Enpp1asj/asjmice. Finally, we show that a long‐acting form of recombinant ENPP1 prevents the development of enthesis calcification at the relatively modest dose of 0.3 mg/kg per week, suggesting that suppression of enthesopathy may be attainable upon dose escalation.
Topographical Organization and Morphology of Substance‐P Axons in the Flat‐Mounts of the Mouse Whole Stomach
Authors: Mistareehi Anas , Ma Jichao , Madas Jazune , Kwiat Andrew , Chen Jin , Bendowski Kohlton , Nguyen Duyen , Powley Terry , Cheng Zixi J.
Published: May 2022

More than 50 million Americans suffer from chronic pain. However, the anatomical and physiological mechanisms of peripheral nociceptive processes have not been well elucidated which has seriously impeded the progress of designing novel bioengineering manipulations/treatments for chronic pain. In this study, we performed a comprehensive topographical mapping of pain‐related neural circuitry in the flat‐mount of whole mouse stomach (male, n=8, 3‐5 months). We used Substance P (SP) as a marker for nociceptive axons and applied a combination of state‐of‐the‐art techniques, including flat‐mount tissue processing and immunohistochemistry of the whole stomach, confocal microscopy, Zeiss Imager microscopy to determine the distribution and morphology of SP‐IR axons and terminals in the whole stomach. We found that 1) SP‐IR axons formed extensive terminal networks in both the ventral and dorsal stomachs. 2) SP‐IR axons were much denser in the antrum and corpus regions than in the fundus and cardia. 3) SP‐IR varicose axons ran in parallel with the circular and longitudinal muscle layers. 4) SP‐IR axons innervated blood vessels. 5) SP‐IR varicose axons formed terminals wrapping around individual myenteric neurons. 6) There were no confirmed SP‐IR myenteric neurons. 7) SP‐IR afferent innervation of the stomach showed a similar pattern of SP‐IR axons and terminals between the ventral and dorsal stomachs. In control mice (n=8), we injected tracer DiI into the ventral and dorsal stomach muscular layers or Fluorogold injection (i.p) and found that the main extrinsic source of SP‐IR afferent axons in the stomach was from the T7‐T11 DRG and to a lesser extent the VNG, but not from the celiac ganglia and dorsal motor nucleus of vagus. Our data provide for the first time a comprehensive topographical map for SP‐IR axons and terminals in the whole stomach with single cell/axon/synapse resolution. This work will contribute to a 3D digital representation of a brain‐stomach nociceptive atlas in a stomach scaffold, thus providing a novel anatomical foundation for functional mapping of nociceptive afferent axons and their pathological remodeling in the stomach. The first two authors contributed equally to this work.
Blood Pressure and Arterial Stiffness after a Maximal Aerobic Exercise in Young Adult Binge Drinkers and Moderate Drinkers
Authors: Jackson Ronald , Tawade Samiksha , Drab Yuliya , Baynard Tracy , Clifford Philip , Piano Mariann , Phillips Shane , Hwang Chueh‐Lung
Published: May 2022

Exaggerated systolic blood pressure (SBP) response to exercise predicts the development of hypertension. In normotensive young adults, alcohol consumption is associated with increased arterial stiffness, which potentially influence exercise SBP response. Young adults who have alcohol drinking habits usually exercise regularly, but their exercise SBP response has received little attention. Whether alcoholic drinking pattern influences exercise SBP response remains to be determined. The purpose of this study is two‐fold: 1) to investigate SBP and arterial stiffness in response to maximal aerobic exercise; and 2) to examine if alcoholic drinking pattern influences the response. Twenty healthy young adults (9 women; age: 27±3 years; BMI: 24.1±2.1 kg/m2; mean±SD) with a history of alcohol consumption over the past year were included in this study. Based on the self‐reported alcoholic drinking pattern, participants were categorized as binge drinkers (n=10; BD) or moderate drinkers (n=10; MOD). Brachial SBP and central SBP and arterial stiffness (carotid to femoral pulse wave velocity; cfPWV) were measured before and after a maximal graded exercise test on a cycle ergometer. At the end of maximal exercise, brachial SBP increased (BD: 110±13 vs. 172±15 mmHg; pre‐ vs. post‐ex; P<0.0005 and MOD: 110±9 vs. 158±22 mmHg; P<0.0005), regardless of drinking pattern (P=0.1 for BD vs. MOD). Central systolic BP (BD: 101±10 vs. 113±7 mmHg and MOD: 102±8 vs. 112±8 mmHg; P≤0.01 for both groups) increased in all participants after exercise (obtained ~ 4min post‐ex), regardless of drinking pattern (P=0.5 for BD vs. MOD). After exercise (~15 min), cfPWV did not increase in MOD (5.6±0.8 vs. 5.7±1.1 m/s; P=0.8), while cfPWV increased in BD (5.5±0.5 vs. 6.3±0.9 m/s; P=0.02). In conclusion, our study found that SBP response to maximal aerobic exercise was similar between BD and MOD. However, arterial stiffness was higher following maximal aerobic exercise in BD, suggesting a negative adaptation in vasculature associated with binge drinking. Future studies with larger sample sizes and a group of alcohol abstainers are needed to confirm our findings.
Inhibition of YAP signaling improves recovery in injured skeletal muscle
Authors: Iyer Shama R. , Shah Sameer B. , Ward Christopher W. , Stains Joseph P. , Folker Eric S. , Lovering Richard M.
Published: May 2022

Skeletal muscle experiences mechanical forces that regulate its development, hypertrophy, and homeostasis. YAP (Yes‐associated protein) not only serves as an indicator of nuclear mechanical signals, but can also induce a wide range of downstream signaling cascades. We previously reported increased YAP signaling in healthy (wild type, WT) muscle following isometric contractile loading, and constitutively active YAP signaling in the muscles of mdxmice (dystrophin‐null, murine model of Duchenne muscular dystrophy, or DMD) and aged mice. We tested the hypothesis that increased YAP signaling contributes to the loss of muscle function following eccentric contraction‐induced injury in WT muscle. We also tested the hypothesis that pharmacological inhibition of YAP signaling (verteporfin, 100 mg/kg delivered via intraperitoneal injection every other day immediately following injury for 10 days) improves muscle function following injury. We found increased YAP signaling following eccentric contraction‐induced muscle injury in healthy muscles, indicated by increased gene expression of YAP and its downstream targets. Moreover, pharmacological inhibition of YAP activity resulted in hastened recovery following eccentric contraction injury. Following 50 eccentric contractions to the quadriceps muscles, vehicle (DMSO) treated muscles had a loss of force of 44.8 ± 14.1% and 42.9 ± 17.1% at days 2 and 4 following injury, while verteporfin treated muscles had a loss of force of 13.7 ± 24.7% and 9.05 ± 24.5% at days 2 and 4 following injury. Both groups were fully recovered at 6 days following injury. We conclude that reducing YAP signaling improves muscle function following injury to WT muscle, and could play a significant role in ameliorating muscle pathology in dystrophic and aged muscle.
Repeated Treadmill Running Attenuates Indomethacin‐caused Gastric Injury in Rats with Streptozotocin‐induced Diabetes
Authors: Filaretova Ludmila , Podvigina Tatiana , Komkova Olga , Yarushkina Natalia
Published: May 2022

Diabetes is one of the most common chronic diseases leading to disability and death. Physical activity has a beneficial effect on all vital functions of the body under the norm as well as pathology including diabetes. Previously we have shown that treadmill running attenuates the vulnerability of gastric mucosa to ulcerogenic action of indomethacin (IM). The aim of the study was to investigate the effect of treadmill running on the vulnerability of the gastric mucosa to ulcerogenic action of IM in rats with streptozotocin‐induced diabetes. Diabetes was induced by a single injection of streptozotocin (STZ, 70 mg/kg, ip). Two regimens of treadmill running, repeated (Experiment 1) and single (Experiment 2), were used. There were four groups of rats in each experiment: two groups (running and sedentary) treated with STZ and two groups (running and sedentary) treated with vehicle of STZ (control). In Experiment 1 daily repeated treadmill running was initiated 7 days before STZ (or its vehicle) administration and continued daily 7 days after STZ administration. The intensity and duration of treadmill running gradually increased from 10 to 15 cm/s and from 10 to 20 min/day, respectively. In Experiment 2 only single treadmill session (15 cm/s, 15 min) was initiated 7 days after STZ (or its vehicle) administration. In both experiments, IM (35 mg/kg, sc) was administered immediately after completion of treadmill session in preliminary (24 h) fasted rats 7 days after STZ injection. Gastric erosion was examined 4 h after IM administration. Glucose level, body weight, water consumption and plasma corticosterone levels were also measured. Signs of diabetes (an increase of glucose level, loss of body weight, an increase of water consumption and elevation of plasma corticosterone levels) were developed 1 week after STZ administration. IM administration caused the gastric erosion 4 h after the injection. STZ‐induced diabetes aggravated ulcerogenic action of IM: the mean area of gastric erosion was significantly greater in rats with STZ injection compared to that in control animals. Repeated as well as single treadmill running did not significantly affect the signs of diabetes. However, repeated daily treadmill running resulted in a significant decrease of the area of gastric erosion in rats with STZ‐induced diabetes as well as control (non‐diabetic) rats compared to corresponding sedentary groups. A single running, before IM administration, reduced the area of gastric erosion in non‐diabetic rats, but it did not affect the gastric injury in rats with STZ‐induced diabetes. The data suggest that regular physical activity started before STZ‐induced diabetes can protect the gastric mucosa against the ulcerogenic action of IM.
A novel and important role of UFM1‐binding protein 1 (UFBP1) in the regulation of ER and cardiac homeostasis
Authors: Tandra Varsha , Ma Wenxia , Zhang Aizhen , Ayala Juan , Li Honglin , Su Huabo , Li Jie
Published: May 2022

Protein modifications by ubiquitin and ubiquitin‐like proteins are emerging as an important mechanism regulating heart function. UFM1 (ubiquitin‐fold modifier 1) is a novel ubiquitin‐like protein that modifies protein targets via UFM1‐specific conjugation enzymes. Dysregulation of UFM1 modification, termed ufmylation, has been linked to multiple human diseases but its importance in the heart remains unclear. We have previously reported that ufmylation is upregulated in compensated, hypertrophic mouse hearts but decreased in failing human hearts. Inhibition of ufmylation by targeted ablation of the E3 UFM1 ligase 1 (UFL1) in mouse hearts resulted in dilated cardiomyopathy during ageing and increased the propensity to heart failure in response to hemodynamic stress. However, how ufmylation exerts its cardioprotective effect remains unclear. Here, we report that UFBP1 (UFM1 binding protein 1), an ER‐resident ufmylation target, is an important downstream effector of ufmylation in the heart. While deletion of UFBP1 in mouse hearts has no impact on ufmylation, the knockout (KO) mice developed dilated cardiomyopathy at resting condition, as indicated by left ventricular wall thinning, chamber dilatation and significantly decreased cardiac contractility at 6 months of age. Moreover, loss of UFBP1 exacerbated pressure overload‐induced cardiac remodeling and dysfunction, recapitulating multiple aspects of the phenotypes of UFL1‐deficient hearts. Mechanistically, UFL1 controls the expression of UFBP1. Depletion of both UFL1 and UFBP1 in cultured cardiomyocytes aggravated ER stress‐induced cell injury. Furthermore, excess ER stress is implicated in UFBP1KO hearts and is aggravated with the progression of cardiomyopathy. Interestingly, ER stress inducers upregulated the expression of ufmylation pathway components at both transcript and protein levels in cultured cardiomyocytes and mouse hearts, indicating an intimate cross‐talk between ufmylation and ER stress. Collectively, our data identify a novel UFM1‐UFL1‐UFBP1 axis in constraining pathological cardiac remodeling and maintaining ER homeostasis.
Long‐Term Cardioprotective Potential of Exogenous Ubiquitin in Myocardial Ischemia/Reperfusion Injury
Authors: Shook Paige , Dalal Suman , Singh Mahipal , Singh Krishna
Published: May 2022

Background

Coronary heart disease (CHD) is a leading cause of death worldwide. Myocardial ischemia/reperfusion (I/R) injury is a clinical manifestation of CHD. Timely reperfusion of coronary vessels in an ischemic injury is essential to limit the infarct size. However, reperfusion itself can induce cardiac cell death, and lead to increased adverse remodeling after an I/R injury. Previously, our lab has shown that pretreatment with ubiquitin (UB; a highly evolutionarily conserved protein) decreases inflammatory response and preserves heart function 3 days post‐I/R. The objective of this study is to investigate the long‐term cardioprotective potential of UB post‐I/R. To enhance the clinical relevance, UB treatment was started at the time of reperfusion. It is hypothesized that UB treatment has long‐term cardioprotective potential, improves cardiac function, and attenuates cardiac remodeling post injury.

Methods

C57BL/6 male mice underwent ligation of the left anterior descending coronary artery (LCA) for 45 min. Mice were then treated with UB (1 µg/g body weight, i.p. injection) or saline at time of reperfusion of LCA. Sham animals underwent the same surgery without the ligation of LCA. Mice were treated with UB or saline twice/day (i.p. injections) for 3 days followed by treatment with UB (1 μg/g/h)or saline using micro‐osmotic pumps for 28 days. Heart function was measured at 3, 7, 14 and 28 days post‐I/R using echocardiography. Mice were sacrificed at 28 days post‐I/R. Left ventricular (LV) tissue was used to measure remodeling parameters and serum samples were used to measure a panel of 23 cytokines/chemokines using multiplex assay.

Results

I/R+saline group exhibited a significant reduction in heart function as observed by decreased percent fractional shortening (%FS) and percent ejection fraction (%EF) vs sham (p<0.05) at 3, 7, 14 and 28 days post‐I/R. However, I/R+UB group showed significantly higher %FS and %EF vs I/R+saline (p<0.05) throughout the observation period. Increases in fibrosis (measured by trichrome staining), myocyte apoptosis (measured using TUNEL assay), and hypertrophy (measured using WGA staining) were significantly lower in I/R+UB vs I/R+saline (p<0.05). Expression of MMP‐2 and TIMP‐4 was lower, while expression of MMP‐9 (active) was significantly higher in I/R+UB vs I/R+saline (p<0.05). Serum levels of IL‐6, IL‐2 and G‐CSF were significantly lower in I/R+UB vs I/R+saline (p<0.05).

Conclusion

Post‐ischemic UB treatment improves heart function, and associates with decreased myocardial remodeling parameters (fibrosis, apoptosis, and hypertrophy) as well as decreased serum cytokines/chemokines levels.
Compromised membrane repair in muscle cells from GNE myopathy patients
Authors: Hallak Diana , Lopez Miguel , Bulgart Hannah , Koczwara Kathrine , Lek Monkil , Weisleder Noah
Published: May 2022

GNE myopathy (GNEM) is an adult onset autosomal recessive neuromuscular disease that initially presents as distal muscle weakness and slowly progresses to atrophy of skeletal muscles throughout the body. GNEM results from mutations in the GNE gene that is located on chromosome 9 and encodes for the N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) which is the rate‐limiting enzyme for the biosynthesis of sialic acid. Sialic acid plays an important role in cellular interactions through modification of the terminal carbohydrate chains on glycoproteins in the plasma membrane. While minimal GNE activity is required for viability, hyposialylation has been observed in GNEM patients. Even though the mechanism of the disease requires further investigations beyond the impaired sialic acid pathway, hyposialylation is still thought to be responsible, at least partially, for the disease pathology. This study aims to elucidate the molecular mechanism of GNEM that links hyposialylation to muscle atrophy and weakness. The association of sialic acid with membrane proteins suggests possible implications for the plasma membrane integrity and repair capacity. We hypothesized that GNEM involves reduced membrane repair capacity in skeletal muscle. To test this hypothesis, we performed laser injury assays on myoblasts transdifferentiated from human skin fibroblasts of GNE patients. Isolated fibroblasts were transdifferentiated into myoblasts using overexpression of MyoD. A multiphoton laser was used to injure the myoblasts in the presence of FM4‐64 lipophilic dye. The resealing capacity is measured by observing the change in fluorescent dye uptake following injury. Our results indicate reduced sarcolemmal membrane repair in GNE myoblasts when compared to healthy controls. To test if sialic acid is required for effective membrane repair, we assessed the level of plasma membrane damage using a glass bead wounding assay on C2C12 myoblasts treated with neuraminidase to remove sialic acid from these cells. The results indicate that sialic acid depleted myoblasts have a more fragile plasma membrane. To further elucidate the mechanism of GNEM we measured the expression levels of key membrane repair proteins in myoblasts and myotubes derived from GNE patients and healthy volunteers. Overall, our results indicate that hyposialylation is correlated with reduced sarcolemmal membrane integrity and repair capacity. To confirm this as a mechanism of GNE myopathy, future studies will use shRNA to knock down GNE in electroporated wild‐type mouse muscle. Muscles from the electroporated mice will be tested using laser injury and western blots to confirm the results we observed in vitro. Future studies also include analyzing expression levels of sarcolemma repair proteins in GNEM patients. Preliminary data from our studies show that sialic acid deficiency is associated with impaired sarcolemma repair leading to muscle atrophy in GNEM.
Developing a Pipeline for Personalized Diagnoses of Glut1 Deficiency Syndrome
Authors: Griffith Laiken , Donohue Katherine , Abul‐Khoudoud Maya , Gentry Matthew
Published: May 2022

Glut1 deficiency syndrome (Glut1DS) is a brain metabolic disorder, caused by the impairment of glucose transport in the brain. Glut1DS occurs when there is a mutation in the SLC2A1 gene, which encodes for the protein glucose transporter protein type 1. Around 500 people worldwide suffer from this disease, and most deal with symptoms such as seizures, movement disorders, and in more severe cases, microcephaly. A common treatment for Glut1DS is the ketogenic diet, which provides the brain with an alternate energy source. Patients suffering from Glut1DS have been shown to respond more positively to this treatment when they are able to start it early in life.

Unfortunately, due to the variety of phenotypes associated with Glut1DS, there is often a large gap between onset of symptoms and diagnosis. This is problematic because a delay in diagnosis means a delay in treatment, leading to more severe disease progression. In order to bridge the gap, our lab applied a suite of bioinformatics techniques to characterize novel mutations and help patients achieve faster diagnoses. Because Glut1DS patients with different mutations experience varying loss of function of the Glut1 transporter, linking mutation type to phenotype could help patients receive a faster diagnosis, allowing them to start treatment earlier.

In order to do this, we analyzed and classified different mutations in the SLC2A1 gene based on predicted pathogenicity. By comparing these data to known Glut1DS patient phenotypes, we were able to able to define five mutation classes based on severity. This type of analysis can be used to provide a faster diagnosis and personalized treatment plans for patients based on their mutation class. As personalized medicine for rare genetic diseases progresses, a database of mutations and severity class could help patients start life‐changing treatment much earlier. Additionally, this model for pipeline development could be expanded to assist in the diagnosis of other rare genetic diseases.
Fiber Type Differences in SDHmax and Mitochondrial Volume Density between Diaphragm and Tibialis Anterior Muscles
Authors: Hernandez‐Vizcarrondo Genesis A. , Brown Alyssa D. , Davis Leah A. , Fogarty Matthew J. , Mantilla Carlos B. , Sieck Gary C.
Published: May 2022

The diaphragm (DIAm) and tibialis anterior (TA) muscles comprise both fatigue resistant (type I and IIa) and fatigable (type IIx/IIb) muscle fibers. Type I and IIa fibers in the DIAm are very frequently recruited for breathing, whereas in the TA, type I and IIa fibers are recruited for balance and locomotion. More fatigable type IIx/IIb fibers are only infrequently recruited for higher force behaviors in both the DIAm and TA. The repetitive activation of the DIAm during breathing is associated with a duty cycle of 40‐50%, making the DIAm one of, if not the most active muscle in the body ‐ much more active than the TA. These differences in activation history lead to differences in energetic demands between the DIAm and TA. We hypothesized that type I and IIa fibers in the DIAm exhibit a greater mitochondrial volume density and respiratory capacity (SDHmax) compared to type I and IIa fibers in the TA. Muscle segments from both DIAm and TA were dissected from adult female and male Sprague‐Dawley rats (~300 g), pinned to cork at optimal sarcomere length and rapidly frozen in melting isopentane. Subsequently, transverse serial sections of muscle fibers were cut at 10 µm thickness. In a series of transverse sections, muscle fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. In an alternate series of sections of the same muscle fibers, mitochondria were labeled using MitoTracker Green. In a third series of alternate sections, the maximum velocity of the succinate dehydrogenase reaction (SDHmax) was measured using a quantitative histochemical assay in which accumulation of the reaction product, nitroblue tetrazolium diformazan (NBTdfz), was measured as a change in optical density (OD) in muscle fibers every 15 s for a 10‐min period. Muscle fiber sections were imaged in 3D using an Olympus Fluoview confocal microscope (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. In the mitoTracker labeled sections, each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI= SA3/16µ2V2) using ImageJ and NIS elements. Mitochondrial volume density and morphometry were analyzed using ImageJ. The mitochondrial volume density and SDHmax were greater in type I and IIa fibers of the DIAm compared to the TA. On the other hand, mitochondrial volume density and SDHmax of type IIx/IIb fibers were comparable between the DIAm and TA. These results support mitochondrial structural and functional adaptations in type I and IIa DIAm fibers based on activation history and greater energetic demand.
Neddylation is critical for cardiomyocyte maturation by promoting perinatal metabolic transition
Authors: Zou Jianqiu , Ayala Juan , Wang Wenjuan , Wang Jinxi , Zhou Jiliang , Song Long‐Sheng , Li Jie , Su Huabo
Published: May 2022

The heart undergoes significant structural, metabolic, gene expression and functional alterations during the perinatal to postnatal transition. Although recent studies have delineated several novel epigenetic and transcriptional regulators of cardiac maturation, post‐transcriptional mechanisms regulating cardiomyocyte (CM) maturation remain poorly defined. We hypothesized that neddylation, a protein modification that conjugates a small ubiquitin‐like protein NEDD8 to protein substrates via an E1‐E2‐E3 cascade, plays a critical role in driving cardiac maturation program. Taking advantage of an AAV9‐cTnT‐Cre system and the mTmG reporter, we achieved CM‐specific, mosaic knockout of NAE1, a critical neddylation E1 enzyme, in postnatal hearts. Mice with high levels (>90%) of NAE1 deletion in CMs exhibited a cardiomyopathy as evidenced by significant chamber dilatation and greatly compromised cardiac contractility. While mice with modest levels (30%) of NAE1 deletion in CMs had preserved cardiac function, inhibition of neddylation caused severe disruption of transverse tubules (T‐tubules) network and a disturbance in binucleation and cell enlargement in NAE1‐null CMs, as well as upregulation of fetal genes and downregulation of their adult forms, indicating a key, cell‐autonomous role for neddylation in CM maturation. Transcriptome analysis revealed an upregulation of glycolytic genes and a downregulation of oxidative metabolic genes in NAE1‐deficient hearts. Biochemical analyses confirmed defects in fatty acid oxidative metabolism and mitochondrial dysfunction in NAE1‐deficient hearts and neonatal CMs treated with a neddylation inhibitor. Mechanistically, we found that HIF1α, a transcription factor known to promote glycolysis and suppress oxidative metabolism, is a putative NEDD8 target. Inhibition of neddylation stabilized HIF1α and led to abnormal accumulation of HIF1α in NAE1‐deficient hearts and neonatal CMs treated with neddylation inhibitor, which may contribute to the observed metabolic dysfunction. Together, our findings suggest that neddylation serves as a novel post‐translational mechanism regulating CM maturation through sustaining the glycolytic to oxidative metabolic switch in postnatal hearts.