bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Myogenesis by a Caveolin Binding Motif in Na/K‐ATPase
Authors: Cai Liquan , Wang Xiaoliang , Pierre Sandrine , Xie Zijian
Published: April 2020

The caveolin binding motif (CBM) in Na/K‐ATPase (NKA) is highly conserved in metazoan animals and plays essential roles in cell signaling. The genetic mutation of this CBM sequence (F97A;F100A) in mice gave rise to the diminished somite and open neural tube formation, and eventually mutant embryos died at around E12.5. Subsequent analyses with RNA‐seq and real‐time RT‐PCR demonstrated that this CBM mutation drastically inhibited gene expression of transcription factors Myogenin and NeuroD, as well as component members in signaling pathways essential for embryogenesis, such as Wnt, Notch and Sonic Hedgehog.
In order to further dissect the molecular mechanisms behind this genetic control, we used human induced pluripotent stem cells (iPSC) and exemplified the regulation of CBM‐NKA on stem cell differentiation using skeletal muscle cells (Skm). Through CRISPR/Cas9 genome editing, we created the mutant CBM iPSC and further differentiated them into skeletal muscle cells with sequential induction system. Compared to the parental human iPSC as the control, the CBM mutant stem cells lost their ability to differentiate into skeletal muscle cells. Moreover, the differentiated cells from CBM mutants did not contain the banding structures shown in the EM imaging and immunofluorescence staining with the antibody against Actinin. As expected, these differentiated mutant cells dramatically reduced mRNA expression of marker genes at both early differentiation stages (PAX3, MYOD and MYOGENIN) and late stages (myosin heavy chain 3, MYH3; myosin heavy chain 8, MYH8; Troponin T1, TNNT1; and Caveolin
3, CAV3). Exogenous expression of a mouse MyoD gene via the lentiviral gene delivery regained the ability of CBM mutant iPSC cells to differentiate into muscle cells, reflected at the level of both marker gene expression and cell structure imaging.
Finally, we assessed one of the essential signaling pathway in vertebrate myogenesis, beta‐catenin/Wnt. The TOPFlash assay consisting of 7XTCF binding sequences as the Wnt reporter demonstrated a significant reduction of luciferase activity in CBM mutant iPSC. This reduced Wnt activity was further proven to associate with the disrupted cell membrane localization of the beta‐catenin protein, which was demonstrated through an immunostaining and confocal imaging, as well as the cell fractionation assay. Altogether, our study has revealed a novel pathway by which NKA controls myogenesis through Wnt signaling.
The protease inhibition with Ritonavir impairs endothelial function and promotes vascular smooth cell proliferation via RANTES/C‐C chemokine receptor type 5 and Nox1‐derived reactive oxygen species pathway.
Authors: do Nascimento Thiago Bruder , Belin de Chantemele Eric J.
Published: April 2020

Thanks to the advent of highly active antiretroviral therapy (HAART), the life of people living with HIV has been extended to close to that of healthy people. However, patients with HIV are now presenting with accelerated development of cardiovascular disease (CVD) and CVD is currently the leading cause of death in patients with HIV on HAART. Herein, we tested the hypothesis that the protease inhibitor ritonavir contributes to HAART‐associated CVD by impairing vascular function via C‐C chemokine receptor type 5 (CCR5)‐ and reactive oxygen species‐mediated mechanisms. We treated 8–10‐week‐old male C57BL6/J with ritonavir for 4 consecutive weeks (5mg/kg/day, i.p). At the end of the treatment, blood was collected and aortas excised to measure H2O2 via Amplex Red and vascular function via wire myography. Aorta from ritonavir treated animals exhibited higher H2O2 levels, impaired relaxation to acetylcholine (Ach) and increased contractility to phenylephrine (Phe). Nox1 inhibition with GKT771, (10μM) fully restored endothelial function in rings from ritonavir treated animals. Ritonavir treatment also elevated RANTES (CCR5 ligand) plasma levels, as well as CCR5, NADPH oxidase 1 (Nox1) and NoxA1 transcript levels in the aorta. No difference was observed for others oxidases, such as Nox2 and Nox4. Repetition of the ritonavir treatment in CCR5 deficient mice revealed that CCR5 deficiency protects vessels from ritonavir induced‐endothelial dysfunction and upregulation of Nox1 and NoxA1 enzymes. To analyze the direct effects of RANTES on the vasculature, thoracic aorta, endothelial, and vascular smooth muscle cells were exposed to RANTES in different times (20ng/mL). RANTES elevated endothelial NOX1 expression and impaired ACh‐mediated relaxation. Concomitantly, Nox1 inhibition prevented RANTES‐mediated endothelial dysfunction. In addition, RANTES time‐dependently stimulated rat aortic smooth muscle cell proliferation, as measured by Ki67 expression, which was partially reverted by Nox1 inhibition. Our data suggest that the protease inhibitor ritonavir contributes to vascular dysfunction and cardiovascular disease by increasing RANTES‐mediated CCR5 activation ultimately leading to increases in Nox1 expression, ROS production, endothelial dysfunction and vascular smooth muscle cell proliferation. Taken together, these findings provide potential explanation for the increased risk of cardiovascular disease developed by patients living with HIV and present CCR5 as a potential target for treatment.

Support or Funding Information

1R01HL130301‐01 to EBC; K99: 1K99HL140139‐01A1; R00: 4R00HL140139‐03 to TBN
Trimethylamine N‐Oxide (TMAO) Impairs Endothelium‐Dependent Relaxation in Mouse Mesenteric Resistance Artery and Aorta
Authors: Priviero Fernanda , Justina Vanessa Dela , Arishe Olufunke , Webb R. Clinton
Published: April 2020

Trimethylamine N‐oxide (TMAO) is a gut microbiota‐dependent metabolite produced from choline and phosphatidylcholine and it has been related to risk for cardiovascular diseases. Studies have shown that increased levels of TMAO are associated with heart attack, stroke and heart failure. This study tested the hypothesis that TMAO has a deleterious effect on the vascular beds leading to vascular dysfunction. Sixteen‐week old male lean mice were euthanized and the aorta and mesenteric resistance arteries (MRA) were mounted in a wire myograph. Concentration response curves to acetylcholine (ACh; 0.001 – 30 μM), A23187 (0.1 – 10 μM) and sodium nitroprusside (SNP; 0.001 – 1 μM) were performed in the absence or in the presence of TMAO (100 μM – incubated for 30 min) in order to evaluate endothelium dependent and independent‐relaxation of the aorta and MRA. In the aorta, TMAO decreased the potency of ACh (pEC50: 7.96 ± 0.22 versus 7.25 ± 0.16) and EMAX to ACh (89 ± 06% versus 63 ± 4%). Similarly, potency to ACh was decreased by TMAO in the MRA (pEC50: 7.52 ± 0.43 versus
6.01 ± 0.15, with vs. without TMAO, respectively). TMAO did not alter endothelium‐dependent relaxation induced by the calcium ionophore A23817 in the MRA, but completely abolished the relaxation in the aorta. Endothelium‐independent relaxation elicited by SNP was not affected by incubation with TMAO in aortic rings, whereas it caused a significant decrease in the maximal response to SNP in the MRA. In conclusion, TMAO leads to endothelial dysfunction in aorta and MRA and impaired smooth muscle relaxation of the MRA which might contribute to the development of cardiovascular diseases.

Support or Funding Information

NIH: NL13604
Small Changes of Active Site Residues of an Acinetobacter Aminoglycoside Acetyltransferase Affect Antibiotic Susceptibility
Authors: Pradhananga Anjani , Barol Irina , Stogios Peter , Pennings Pleuni , Kuhn Misty L.
Published: April 2020

Antibiotics are drugs used to treat bacterial infections by inhibiting growth and proliferation of microorganisms. Commonly prescribed broad‐spectrum antibiotics include aminoglycosides, but many bacteria have developed resistance to these drugs by acquiring enzymes that acetylate them. We previously reported the structural and kinetic characterization of two of these enzymes from Acinetobacter. However, several outstanding questions regarding the role of specific residues in the active site of these proteins remain. In particular, we observed an active site residue that was different between these two enzymes and appeared to alter the aminoglycoside binding pocket. It has been previously shown that active site mutations within aminoglycoside acetyltransferase enzymes can adjust their ability to modify aminoglycosides and therefore their susceptibility. Since we cannot predict which point mutations will ultimately enhance their activity and therefore resistance to aminoglycosides, we performed saturation mutagenesis of this residue in the Acinetobacter haemolyticus AAC(6′)‐Ig enzyme and tested the antibiotic susceptibility of Escherichia coli to four different aminoglycosides when these mutants were overexpressed. We found the identity of the amino acid at this position was critical for altering the susceptibility of E. coli to these antibiotics. Additionally, these mutants exhibited different susceptibility patterns depending upon the identity of aminoglycoside tested. Our results show how small changes of active site residues of aminoglycoside acetyltransferase can drastically affect bacterial susceptibility to aminoglycosides, and may provide insight to which antibiotics could be effective for treating Acinetobacter infections.

Support or Funding Information

Research reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).
Diagnostic Conundrum of an Osteocartilaginous Exostosis of the Patella: An “Ancient” Osteochondroma?
Authors: Cook Ryan L. , Ohan Hovsep , Adelanwa Ayodele , Herrold Christina B. , Bacaj Patrick , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

The patella is an uncommon site for a tumor. This report details the incidental finding of an excrescence on the right patella identified in a cadaver during a gross anatomy course. Histological assessment revealed no periosteal surface, a very thin cartilage cap, minimal endochondral ossification, bone intermediate to more cartilage, and degenerative changes. The assessment remains unclear. What is clear, however, is that this incidental finding has served as an excellent interdisciplinary exercise for anatomists and pathologists and has benefited the education of undergraduate and graduate medical students. Accordingly, in addition to the gross pathology and histopathology, a workflow is presented.

Support or Funding Information

T. Walley Williams Summer Anatomy Research Fellowship; WV Research Challenge Fund [HEPC.dsr.17.06]
Fiber‐Type Effects of KATP Channel Inhibition via Glibenclamide on the Recovery of Interstitial PO2 Following Muscle Contractions in Rats
Authors: Schulze Kiana M. , Colburn Trenton D. , Weber Ramona E. , Hageman K. Sue , Behnke Bradley J. , Poole David C. , Musch Timothy I.
Published: April 2020

KATP channels allow influx of potassium (K+) which hyperpolarizes cell membranes. Glibenclamide (GLI) is a second‐order sulphonylurea often prescribed to Type II diabetic patients to increase insulin release via the inhibition of pancreatic KATP channels. Unfortunately, in vivo studies demonstrate that GLI impairs vasodilation and O2 delivery‐utilization matching (assessed via interstitial PO2, PO2is) in contracting skeletal muscle. Determination of GLI’s impact on PO2is kinetics (T63; time taken to reach 63% of the final response) across muscles comprised of different fiber types during post‐exercise recovery may provide novel insights into mechanisms of fatigue in patients prescribed this medication, particularly in repeated bouts of activity.

Purpose

To test the hypothesis that KATP channel inhibition via GLI will slow PO2is kinetics, thus lengthening recovery time following contractions in rat fast‐(mixed gastrocnemius, MG) and slow‐(soleus, SOL) twitch muscles.

Methods

In twenty Sprague‐Dawley rats (male (n=5), female (n=8) and ovariectomized female (F‐OVX; n=7)), PO2is was determined, before and after GLI superfusion (5 mg kg−1), via phosphorescence quenching (G4). Measurements were taken for 3 min throughout the duration of, and 3 min following, the cessation of electrically‐induced contractions (180 s, 1 Hz, 7V). Blood flow was measured via fluorescent‐labeled microspheres in the last 30 seconds of contractions (15 μm).

Results

In both muscles, PO2is recovered to its precontracting level in control and GLI conditions within the 3 min measurement window. However, GLI significantly slowed PO2is recovery following contractions in the MG (T63: 87.7 ± 4.6 vs 117.4 ± 7.9 s; p < 0.05), but not in the SOL (73.7 ± 8.8 vs 84.8 ± 11.2 s; p > 0.05). The PO2is value at which T63 occurred was not significantly different between the control and GLI conditions for MG or SOL. During contractions, blood flow was attenuated in the MG following GLI (53 ± 4 vs 39 ± 3 ml/100 g/min; p < 0.05), but not in the SOL (37 ± 4 vs 35 ± 5; p > 0.05).

Conclusion

These data highlight the role of KATP channel function in supporting PO2is recovery of fast‐twitch oxidative fiber types due in part to elevated blood flow. These effects of sulphonylurea medication (i.e. GLI) may explain, in part, the early onset of exhaustion in patients performing high‐intensity exercise and/or activities of daily living that recruit fast‐twitch muscles.

Support or Funding Information

Supported by NIH Grants: HL108328 (T.I.M and D.C.P) and F31HL145981 (T.D.C)
ERG is a Potent Regulator of Permeability in Human Lung Microvascular Endothelial Cells
Authors: Hunter Felicia , Randolph Sonya , Byrd Crystal , Tharakan Binu , Childs Ed
Published: April 2020

ERG (ETS‐related gene) is a member of the ETS (Erythroblast‐transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. In order to study the role of ERG in regulating microvascular permeability, we grew primary human lung microvascular endothelial cells (HLMEC) on Transwell inserts as a monolayer and transfected them with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid or their respective control plasmids. Recombinant VEGF was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Monolayer permeability was evaluated based on FITC‐albumin extravasation and measurement of its fluorescence intensity. The viability of cells and apoptosis/necrosis were studied in cells grown on chamber slides or 96 well‐plate using EZviable Calcein AM fluorometric assay and an apoptosis/necrosis visualization assay, respectively. CRISPR/cas9‐based ERG knockdown resulted in monolayer hyperpermeability comparted to ERG knockdown control group. VEGF treatment induced monolayer hyperpermeability significantly. ERG activation attenuated VEGF‐induced hyperpermeability. ERG knockdown or VEGF treatment had no significant effect on cell viability and did not induce apoptosis/necrosis. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability and ERG enhancement has potential benefits against barrier dysfunction and hyperpermeability. Furthermore, our results show that ERG knockdown‐induced hyperpermeability is not due to changes in cell viability or apoptosis/necrosis.
Methods and Sensors For Functional Genomic Studies of Cell‐Cycle Transitions in Single Cells
Authors: Zambon Alex , Hsu Tom , Klinck Miranda , Patam Leomar , Hickerson Anna
Published: April 2020

Our understanding of the genetic control mechanisms governing the cell cycle has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our fundamental understanding of much of what we know about how the cell cycle is regulated, these approaches also mask the signaling and genetic signatures of rare subpopulations. Quiescent (G0) or very slow dividing (SD) cells are two such related populations. Our data and that of others show that the majority of single clonal MCF7 breast cancer cells cultured at subconfluency can complete a full round of cell division in as little as 24h, however subpopulations within that culture (1–5%) can require over 100h to complete the cell cycle. Moreover, it is not clear how these phenotypes are transferred to daughter cells. Understanding the proteins and networks that control G0 or SD cells may lead to the identification of new drug targets for aging or tissue regeneration. This study establishes an integrated computational, imaging and molecular pipeline that enables the phenotyping of G0 or SD cultured cells within a population. We define imaging parameters and open source R scripts to generate and analyze time‐lapse images to track and quantify cell cycle transition in individual cells expressing a genetically encoded cell cycle reporter system (Ki67p‐FUCCI). We report how these cells can be used to identify transcriptomic signatures of G0, G1 and G2 phase cells within a population. Additionally, we present single‐cell microinjection parameters to optimize single cell transfection by direct injection of mRNA in MCF7 cells with detectable reporter gene expression (GFP) within 5h of microinjection. These findings coupled with the possibility of targeting several hundreds of single cells within a field of view while simultaneously conducting 4 channel time‐lapse microscopy dramatically increase throughput and sensitivity over conventional methods for functional genomics studies to identify new genes that control cell cycle transitions.
Probiotic Mitigates High Fat Diet‐Induced Mammary Gland Inflammation and Matrix Remodeling
Authors: Molnar John Christian , Homme Rubens Petit , Smolenkova Irina , Mallonee Carissa Jo , Stanisic Dragana , George Akash , Singh Mahavir , Tyagi Suresh C.
Published: April 2020

Hidradenitis Suppurativa (HS) is a chronic inflammatory condition about which little is known. HS results from occlusion of the apocrine sweat glands of the skin. The blocked follicles will become inflamed, swell, and burst, leaving scar tissue after lesional skin heals. As the disease progresses, tracts develop beneath the skin, which connect lesional areas to new regions. MMP activity is elevated in lesional, perilesional, and tract biopsies. Obesity is known to exacerbate the condition, while the mechanism is unknown. A High Fat Diet (HFD) causes gut dysbiosis and we hypothesize that this shift in the microbiome leads to an increase in circulating inflammatory cytokines and inflammation in the mammary gland (MG), a modified apocrine gland. Mice fed a HFD showed elevated inflammatory cytokines in serum and MG tissue. MMP activity was increased in mice fed a HFD compared to normal diet (ND), but was mitigated when given probiotic (PB). MMP inhibitors such as TIMP‐3 were significantly over expressed in the HFD+PB group. Administration of probiotic and restoration of the gut microflora has a protective effect against inflammation caused by a HFD.

Support or Funding Information

Research supported by NIH grants HL74185, HL139047, and AR‐71789.
Adrenodoxin Modulates Substrate Binding in the Vitamin‐D Metabolizing Human Cytochrome P450 24A1
Authors: Jay Natalie , Estrada Fernando
Published: April 2020

Vitamin‐D insufficiency is associated with rickets, osteomalacia, cardiovascular disease, and chronic kidney disease and affects nearly 50% of the population. In humans, vitamin D is metabolized by a series of cytochrome P450 enzymes, including inactivation by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 in humans hydroxylates either the carbon‐23 or carbon‐24 position of 1,25‐(OH)2D3 followed by a series of reactions to produce additional metabolites. The human isoform of CYP24A1 is not easily expressed and purified recombinantly, likely due to the labile nature of the enzyme. Therefore, most current structural studies are based on the rat isoform, which does not display carbon‐23 regioselective hydroxylation. As a way to study the human isoform, we have initiated use of a fusion construct of human CYP24A1 with its reduction‐oxidation protein partner adrenodoxin (Adx). The fusion product contains a short linker sequence connected to a cleavable tobacco etch virus protease recognition site, thus allowing for production and then separation of human CYP24A1 from Adx. This construct provides a platform for interrogating the structural effects of an Adx‐CYP24A1 complex on substrate, analog, and inhibitor binding in CYP24A1. Notably, we’ve observed that when Adx is present, titration with 1,25(OH)2D3 primarily populates a low‐spin state. This effect is removed upon cleavage and separation of Adx. Our interpretation is that the presence of adrenodoxin stabilizes a closed form of CYP24A1 that prevents water from departing the active site as the sixth ligand. This finding points toward close structural coupling between substrate binding and redox partner recognition in mitochondrial P450 enzymes.

Support or Funding Information

R00GM112862
Investigating the Structure and Function of the Arf GEFs Gea1 and Gea2.
Authors: Muccini Arnold , Fromme J. Chris
Published: April 2020

In eukaryotes, vesicle formation at the Golgi complex is initiated by the conserved small GTPase Arf1. In budding yeast, Arf1 localization and activity is controlled by three separate guanine nucleotide exchange factors (GEFs) Gea1, Gea2, and Sec7. Sec7 activates Arf1 at the trans‐Golgi network, and mechanisms regulating the activity and recruitment of Sec7 to the Golgi have been well characterized. For example, Sec7 is recruited to the TGN by four small GTPases including its substrate Arf1. However, Gea1 and Gea2, which activate Arf1 at earlier Golgi, do not share the same regulatory mechanisms as Sec7. While initially thought to be redundant paralogs, we have found that Gea1 and Gea2 localize to separate regions of the Golgi despite sharing common regulatory elements. As such, the purpose and mechanism for their differential localization remains unclear. I will present my work in trying to understand the role of Gea1 and Gea2 in the formation of retrograde vesicles and my advances in using Cryo‐EM to obtain a structure of these proteins.
Intestinal TMEM16A function as a luminal chloride channel
Authors: Kazi Mirajul Hoque , Saha Tultul , Aoun Joydeep , Hayashi Mikio , Sheikh Irshad Ali , Leblanc Normand , Sarkar Paramita , Ameen Nadia , Woodward Owen M.
Published: April 2020

There has been a high level of uncertainty whether TMEM16A channels are truly expressed on the apical membranes of enterocytes and contribute to luminal Cl− secretion. Here, we describe a series of independent experimental approaches including qPCR, western blot, immuno‐staining and electrophysiology using Tmem 16a+/− mouse intestinal tissue, human colonic tissue and T84 cells to characterize the expression, localization and physiological function of TMEM16A and its regulation by the multi‐PDZ domain‐containing protein NHERF1.

qPCR analysis revealed that Tmem16a transcripts are differentially expressed across the intestinal epithelial tissue with highest expression in the colon. Tmem16a mRNA expression did not correlate with TMEM16A protein expression. Western blot analysis of TMEM16A in mouse intestinal tissue revealed that one (Boster Bio PA2290) out of five commercially available polyclonal antibodies appreciably recognized a band at the expected size of below 100 kDa monomeric protein in jejunum, ileum, proximal and distal colon, which was absent in Tmem16a+/− mice. Ussing chamber experiments confirmed that TMEM16A protein abundance correlated with Cl− secretion in different regions of native intestine activated by the Ca2+‐dependent agonist carbachol (CCH). In Tmem16a+/− colon, basal and CCH stimulated Isc was largely reduced. Immunostaining of intestinal tissue with PA2290 antibody confirmed luminal expression of TMEM16A proteins compared to Tmem16a+/− mice. Luminal but not serosal application of MONNA, a TMEM16A specific inhibitor, resulted in significant reduction of CCH stimulated Isc, indicating that TMEM16A is primarily expressed and functions as a luminal Cl− channel. Luminal expression of TMEM16A in human colon was confirmed by confocal microscopy. Localization of TMEM16A to the luminal membrane may be facilitated through interactions with the scaffold protein NHERF1 which we found immunoprecipitated TMEM16A. Our results indicate that TMEM16A is prominently expressed in different segments of native mouse intestine as a luminal membrane protein and in human colonic tissue. TMEM16A also interacts with NHERF1. These data are consistent with TMEM16A localizing to the luminal membrane via the actin cytoskeleton and NHERF1 to influence CCH stimulated luminal Clsecretion.

Support or Funding Information

University Grant Commission, and Indian Council of Medical Research, Govt. of India
Resting Muscle Sympathetic Nerve Activity Is Not Different in Symptomatic Concussed College Athletes versus Healthy Controls
Authors: Worley Morgan L. , Sackett James R. , Schlader Zachary J. , Leddy John J. , Johnson Blair D.
Published: April 2020

Heart rate variability, primarily an indicator of cardiac parasympathetic function, has been shown to be reduced at rest and during physiological stressors (e.g., aerobic exercise, handgrip exercise, and face cooling) following a concussion, which suggests altered autonomic function. Previous work in our lab has demonstrated attenuated increases in sympathetically‐mediated increases in blood pressure due to unaltered total peripheral resistance during face cooling and the cold pressor test in symptomatic concussed college athletes (CA) compared to healthy controls (HC). These responses suggest that CA have blunted sympathetic activation. It is unknown if a direct measure of sympathetic nerve activity (e.g., muscle sympathetic nerve activity (MSNA)) is altered at rest in CA compared to HC.

PURPOSE

We tested the hypothesis that MSNA at rest would be lower in CA compared to HC.

METHODS

Seven CA (age: 19±2 y, 4 females, 4±2 days post‐injury) and seven HC (age: 21±3 y, 3 females) completed one study visit. Heart rate (3‐lead electrocardiogram), mean arterial pressure (photoplethosmography), and MSNA (microneurography) from the left radial nerve were continuously recorded at 1 kHz for 30 s following at least 20 minutes of supine rest. MSNA burst frequency, burst incidence, and total activity were assessed using commercially available software. Cardiac output was calculated as the product of heart rate and stroke volume. Total peripheral resistance was calculated as mean arterial pressure divided by cardiac output. We used unpaired t‐tests to determine if the main outcome variables were different between groups. Values are reported as mean ± SD.

RESULTS

Resting heart rate (CA: 58±10 vs. HC: 61±12 bpm; P = 0.31), mean arterial pressure (CA: 95±9 vs. HC: 88±10 mmHg; P = 0.11), cardiac output (CA: 6±1 vs. HC: 7±3 L/min; P = 0.31), and total peripheral resistance (CA: 16±3 vs. HC: 16±11 mmHg/L/min; P = 0.45) were not different between CA and HC. Resting MSNA was not different between CA and HC for burst frequency (CA: 27±6 vs. HC: 29±9 beats/min; P = 0.33), burst incidence (CA: 47±10 vs. HC: 46±11 bursts/100 heartbeats; P = 0.44), or total activity (CA: 4883±1520 vs. HC: 6321±1689 AU/min; P = 0.07).

CONCLUSION

These preliminary data indicate that muscle sympathetic nerve burst frequency, burst incidence, and cardiovascular variables during resting conditions are not different in symptomatic concussed college athletes compared to healthy controls. Research is warranted to determine if muscle sympathetic nerve activity is different between symptomatic concussed colleges athletes and healthy controls during a sympathoexcitatory maneuver.

Support or Funding Information

Supported by the National Center for Advancing Translational Sciences of the National Institutes of Health Pilot Grants Award Program under award Number UL1TR001412.
Variations of the Premaxillary Suture in Fetal and Infant Development: a Study of Position, Completeness, and Branching
Authors: Cusick Abbey Marie , Zdilla Matthew J.
Published: April 2020

The premaxillary suture joins the pre‐maxillary (i.e., incisive) and proper maxillary regions of the maxilla. This suture is important in maxillary development at early stages in life, including intrauterine and infantile growth. By adolescence, the suture becomes fused and is usually unapparent. This study aims to classify the premaxillary suture in maxillae from individuals aged five‐months intrauterine to five‐months post‐natal. Sutures were categorized based on completeness, positioning with regard to the dental arch, and branching pattern. The development of the premaxillary‐maxillary junction likely plays an important role in the pathogenesis of cleft lip. Therefore, a thorough understanding of the variations in the premaxillary sutures may elucidate the pathogenesis of cleft lip.

Support or Funding Information

Funding: WV Research Challenge Fund [HEPC.dsr.17.06]
Standard and High Fat Food Intake is Suppressed by PF5190457, the Ghrelin Growth Hormone Secretagogue 1α Receptor Inverse Agonist/Antagonist
Authors: Brehm Victoria Dobbie , Anastasio Noelle C. , Merritt Christina R. , Moeller F. Gerard , Cunningham Kathryn A.
Published: April 2020

Ghrelin is a 28‐amino acid peptide produced by epithelial cells in the stomach mucosa and is well‐characterized to modulate gastrointestinal function, growth hormone secretion, glucose regulation, and appetite, and is increasingly implicated in substance use disorder (SUD) phenotypes. Ghrelin actions are transduced through the growth hormone secretagogue receptor 1α (GHS1αR) which is noted to exhibit constitutive activity in in vitro recombinant cell lines, and in dispersed pancreas islet cells, studies which support the concept of endogenous ghrelin tone. The small molecule PF5190457 exhibits relatively equipotent actions as a neutral GHS1αR antagonist and inverse agonist without apparent partial agonist actions (Kong et al. Br J Pharmacol 173:1452, 2016). To expand our knowledge of the physiological impact of GHS1αR function in vivo and determine an effective dose of PF5190457 for future studies of SUD phenotypes, we tested the hypotheses that PF5190457 would antagonize GHS1αR agonist‐induced intake of standard chow (SD) or high fat food (HFF) and act as an inverse agonist to inhibit basal HFF intake and weight gain.

Methods

Male Sprague‐Dawley rats (n=16) maintained on ad libitum SD were pretreated with intraperitoneal injection of PF5190457 (20 mg/kg) or vehicle 60 min prior to subcutaneous (SC) injection of the GHS1αR agonist hexarelin (80 μg/kg) and assay of SD intake over a 6‐hr period. In a second study, rats (n=32) were administered acute or repeated PF5190457 (20 mg/kg) prior to maintenance on ad libitum HFF for three days. On day 3, all rats were treated with PF5190457 or vehicle 60 min prior to hexarelin (80 μg/kg; SC). Food intake and body weight were recorded throughout the study.

Results

PF5190457 significantly suppressed hexarelin‐induced SD intake vs. vehicle (p < 0.05). Both acute and repeated PF5190457 administration suppressed hexarelin‐induced HFF intake vs. control (p < 0.05). Repeated PF5190457 suppressed basal HFF intake and body weight gain (p < 0.05).

Conclusions

The novel GHS1αR inverse agonist/antagonist PF5190457 suppressed measures of both hexarelin‐induced SD and HFF intake, demonstrating antagonist activity in vivo. Similar efficacy of acute and repeated PF5190457 to suppress hexarelin‐induced HFF intake suggests that tolerance did not develop. Further, PF5190457 suppressed basal HFF intake and HFF‐associated weight gain, indicating inverse agonist activity in vivo. Together, these data suggest that PF5190457 will be a useful pharmacological tool to study the involvement of GHS1αR in SUD phenotypes.

Support or Funding Information

Supported by NIDA UG3 DA050317 and F30 DA049501
The Inflammatory Lipid, 2‐Chlorohexadecanal: Cell Toxicity and Potential Scavenger Protection
Authors: Shakya Shubha , Ford David A.
Published: April 2020

Hypochlorous acid is produced by either leukocyte myeloperoxidase activity or from chlorine gas reactions with water. 2‐Chlorofatty aldehydes (2‐ClFALD) are: 1) formed when hypochlorous acid attacks the plasma membrane phospholipid plasmalogen molecular subclass and 2) produced following leukocyte activation as well in the lung of mice exposed to chlorine gas. The biological role of 2‐ClFALD is largely unknown. Recently, our lab showed the electrophilic α‐chlorinated carbon of the 2‐ClFALD molecular species, 2‐chlorohexadecanal (2‐ClHDA) is targeted by nucleophilic attack of the cysteinyl‐sulfhydryl group of glutathione. Here, we synthesized an alkyne analog of 2‐ClHDA (2‐ClHDyA) to demonstrate 2‐ClHDyA protein modification in RAW 264.7 cells using click chemistry strategies. Furthermore, confocal microscopy showed that 2‐ClHDyA co‐localizes to the endoplasmic reticulum, Golgi, and mitochondria. Additionally, diffuse distribution of 2‐ClHDyA suggested cytoplasmic protein modification. The intensity of the fluorescence signal of 2‐ClHDyA increased in a dose‐dependent manner suggesting increased protein modification. Cell viability/metabolic activity assessed by 3‐(4,5‐Dimethyl‐2‐Thiazolyl)‐2,5‐Diphenyl‐2H‐Tetrazolium Bromide (MTT) test revealed that 2‐ClHDA reduced RAW 264.7 cell viability in a dose‐dependent manner. Cell viability was further reduced in RAW 264.7 cells treated with 2‐ClHDA in DMEM with Fetal Bovine Serum (FBS) supplementation. Click chemistry showed 2‐ClHDyA modified FBS proteins. These observations suggested 2‐ClFALD modified proteins might have accounted for cell death via cell signaling outside or inside the cell. Next, we investigated traditional aldehyde sequesterers including N‐acetyl cysteine (NAC), carnosine, and aminoguanidine to identify potential protectors that would prevent cellular 2‐ClFALD‐protein interaction resulting in reduced toxicity caused by 2‐ClFALD. Among tested sequesterers, NAC proved to be an effective sequesterer of 2‐ClHDA. NAC prevented 2‐ClHDyA interaction with FBS proteins and rescued cell death significantly. Next, we characterized the conjugate of 2‐ClHDA and NAC using direct infusion electrospray mass spectrometry. The conjugate appeared to be a result of a nucleophilic substitution reaction where nucleophilic sulfur of NAC attacked the alpha carbon of 2‐ClHDA leading to the release of chloride ion. These studies demonstrate that the interaction of 2‐ClFALD with cellular proteins with free sulfhydryl groups cause toxicity and the interaction of 2‐ClFALD with N‐acetyl cysteine may protect against toxicity caused by 2‐ClFALD.

Support or Funding Information

NIH grants: R01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697 and U01 ES028182
RhoA induces mitophagy through PINK1 stabilization to confer cardioprotection
Authors: Tu Michelle , Yu Justin D. , Kono Maiko , Tan Valerie P. , Smith Jeffrey M. , Miyamoto Shigeki
Published: April 2020

An established feature of cardiac disease involves an increasing presence of dysfunctional mitochondria in the heart. Mitophagy is a key player in maintaining mitochondrial quality and thereby regulates cellular homeostasis. For instance, inhibition of mitophagy has been shown to exacerbate cardiac injury induced by oxidative stress. RhoA is a small G‐protein that serves as a proximal downstream effector of numerous GPCRs and is also responsive to oxidative stress. Activation of RhoA has been previously shown to protect the heart against stress; however, its specific role in mitophagy has not been determined. Here, we explored the possibility that RhoA signaling regulates mitophagy and in turn, provides cardioprotection. Adenoviral overexpression of RhoA in neonatal rat ventricular myocytes increased both endogenous and exogenous PINK1 levels as well as mitochondrial Parkin levels, leading to mitophagy assessed by EM analysis. Although RhoA expression did not affect PINK1 mRNA levels, it did inhibit PINK1 protein degradation, thus stabilizing PINK1 protein. Depolarization of the mitochondrial membrane potential is well established to stabilize PINK1 at the mitochondria. Interestingly, however, RhoA expression did not change the mitochondrial membrane potential. Thus the basis for PINK1 accumulation at mitochondria in response to RhoA appears to be unique. siRNA‐mediated knockdown and pharmacological inhibition of PKD, an established downstream effector of RhoA, reversed RhoA‐induced stabilization of PINK1. However, overexpression of PKD alone was not sufficient to stabilize PINK1. Active RhoA accumulates at mitochondria and this response is completely inhibited by PKD inhibition, suggesting a potential role of PKD dependent mitochondrial RhoA distribution in regulating PINK1 stability. While extrapolating the role of RhoA signaling in cardiac disease, myocardial infarction induced by ligation of the left anterior descending artery was reduced in adult mice expressing constitutively active RhoA through cardiac‐specific MLC2v promoter driven adeno‐associated virus serotype 9 when compared to that of wild‐type (WT) mice. Furthermore, although both WT and cardiac specific RhoA knockout (KO) mice around 18 weeks old revealed no overt signs of cardiac dysfunction, at around 45 weeks old, KO mice not only showed hypertrophy and fibrosis, but also revealed contractile dysfunction. Currently, we are investigating the specific contribution of RhoA‐induced mitophagy in these cardiac disease models. Taken together, our results suggest that RhoA stabilizes PINK1 through its PKD‐mediated translocation to the mitochondria, subsequently inducing mitophagy in a manner uniquely independent of the conventional pathway and thus, could confer cardioprotection against stresses.

Support or Funding Information

This work was supported by American Heart Association grant 19TPA34910011 to S. Miyamoto.
Isolation and Characterization of an Antibiotic‐producing Advenella Species
Authors: Meza Vedejenne , Peterson Stacey N.
Published: April 2020

Antibiotic resistance mechanisms in pathogenic bacteria paired with the general overuse of antibiotics has caused an exponential decrease in the efficiency of many antibiotics. To search for producers of novel antibiotics, this scientific research has turned to the most naturally abundant microbial source, soil. Specifically, the isolation and identification of antibiotic‐producing bacteria from a soil sample was the primary objective of this research. A soil sample collected in late January of 2019 from a mountain edge located at the Chalon campus of Mount Saint Mary’s University‐Los Angeles underwent serial dilutions and plating to isolate bacteria. Once isolated, the bacteria were screened against various ESKAPE relatives, and a viable producer against Bacillus subtilis was identified. The producer’s 16S rRNA gene was amplified through colony PCR, purified through gel electrophoresis, and sequenced. The gene sequence of the producer identified it as a lesser known soil microbe, Advenella kashmirensis (98.41%). Prior research has described Advenella kashmirensis as a sulfur‐oxidizing, chemolithotrophic, gram‐negative soil bacteria, but this bacteria species has not been previously linked to antibiotic production or pathogen inhibition. Therefore, research continued with repeated, successful screenings against B. subtilis, as well as with efforts to support the identification of the producer through the following biochemical tests: urease (positive), catalase (positive), nitrate (positive, reduction beyond nitrite), gelatin (negative), and citrate utilization (negative). Future work will involve further characterization of the isolate through additional biochemical testing as well as attempts to isolate and purify the antibiotic.
Docking of inhibitors to the structure of the human K‐Cl cotransporter
Authors: Delpire Eric
Published: April 2020

Following the release of the cryo‐electron microscopy structure of the human K‐Cl cotransporter, hKCC1, we used a ligand‐protein docking program (Autodock Vina from the Scripps Research Institute) to assess the binding of KCC inhibitors to the K‐Cl cotransporter. We hypothesized that the inhibitors were likely to utilize the ion permeation pathway and overlap with some of the ion binding sites. We first modeled the binding of ML077 starting from the position of the 3 ions at SCl1, SK, and SCl2. ML077 is a potent inhibitor of KCC function, as it binds rat KCC2 with an EC50 of 560 nM. The top binding energies for the best solutions from these three positions were: −7.6, −7.6, and −8.5 kcal/mol, indicating that the compound clearly fits better in the lower portion of the permeation pathway. We observed that the ring structures of the inhibitory compound nicely fitted within the hydrophobic groove or cavity which is located at the bottom of the transporter core. Furthermore, the most distal methyl group of ML077 was conveniently positioned 3.6 Å from the side chain of Tyr216. Since this residue also coordinates the placement of K+, this data is consistent with previous report showing that the binding of ML077 is competitive with regard to K+. When we docked the stilbene derivatives DIDS and H2DIDS to the structure, we observed binding modalities with binding energy around −7.5 kcal/mol. The inhibitors were located in the groove with the nitrogen atom or sulfur atom of an isothiocyanate group placed 3.3 – 3.5 Å from Tyr216 and one the sulfonic acid group oriented either towards the Cl− ion at SCl2 or TM8. The second sulfonic acid group orients towards the outside of the cavity, while the second isothiocyanate group nicely fits within a lower portion of the groove. As these conformations do not involve Tyr216 and do not seem to overlap with the bound K+, this docking is consistent with non‐competitive DIDS inhibition with regards to K+. Finally, docking of furosemide to the hKCC1 structure also preferentially placed the loop diuretic within the cavity with a possible hydrogen bond between the amine group of the ligand and the hydroxyl group of Tyr216 (distance 1.8 Å). Involvement of the tyrosine residue in coordinating furosemide binding is also consistent with the competitive inhibition of furosemide with respect to K+. Thus, it seems that the binding of ML077, DIDS and H2DIDS, and furosemide are consistent with some older functional studies. Single site mutagenesis and functional experiments should confirm these predictions.

Support or Funding Information

This work was supported by NIH grant DK93501 and by grant 17CVD05 from the Leducq foundation.
Pattern of distribution of lipocalin 2 in the pancreas of diabetic rat
Authors: Adeghate Ernest A. , D’Souza Crystal , Al-Jaberi Saeeda
Published: April 2020

Lipocalin 2 (LCN2), discovered in 1989, is an innate, inducible glycoprotein and a cytokine implicated in the regulation of inflammation and iron homeostasis. Although, LCN2 was thought to be present only in neutrophils, it has now been reported to be secreted by many other cells including adipocytes, hepatocytes and more recently, osteoblasts. LCN2 induces insulin secretion and suppress appetite by activating melanocortin‐4 receptors in the hypothalamus. Even though LCN2 has been associated with glucose homeostasis, the pattern of distribution and function of LCN2 in the pancreas of an animal model of diabetes mellitus has not been elucidated. The aim of this study was to determine whether LCN2 is present in the pancreas of diabetic and non‐diabetic Wistar rats. Immunofluorescence method was used to identify the pattern of distribution of LCN2 in pancreatic islet cells of Wistar rats. Co‐localization staining was also performed to determine whether LCN2 is located with either, insulin, glucagon, somatostatin, or pancreatic polypeptide (PP) in the islets of Langerhans. Immunofluorescence study shows that a large number of cells in the central region of rat pancreatic islet contains LCN2, where it co‐localizes mainly with insulin. LCN2 does not co‐localize with any other islet peptide. The number of LCN2 and insulin‐containing cells in pancreatic islets decreased significantly (p < 0.05) after the onset of diabetes. In conclusion, LCN2, a resident of pancreatic beta cells, may play a role in the regulation of insulin and glucose metabolism.

Support or Funding Information

This work was supported by UAE University Research Grant # G00002701
Proximal tubular mitochondrial substrate overload is an independent factor in developing kidney disease
Authors: Stadler Krisztian , Noland Robert , Mynatt Randall L. , Kruger Claudia
Published: April 2020

Alterations in mitochondrial function are linked to the development of chronic and diabetic kidney diseases (CKD/DKD). Proximal tubular epithelial cells (PTCs) are highly energy demanding. Their energy need is covered mostly from mitochondrial fatty acid oxidation. It is suggested, but not entirely clear whether derailments in fatty acid metabolism and mitochondrial dysfunction are forerunners of tubular damage. Here we modeled mitochondrial substrate overload ‐ an important aspect of metabolic disease and diabetes ‐ by creating mice lacking the enzyme carnitine acetyl‐transferase (CrAT) in the PTC. CrAT is crucial in serving as a “relief valve”, linking excess acetyl‐CoA to carnitine and allowing transport from the mitochondria. Deletion of CrAT therefore models a scenario of substrate excess where the export of carbons is limited. Mice developed tubular disease and interestingly, secondary glomerulosclerosis. This was accompanied by increased levels of apoptosis and fibrosis markers, increased oxidative stress and abnormal profiles of acyl carnitines/organic acids including those derived from branch chain amino acid and glucose metabolism. These results indicate that during mitochondrial overload in the kidney cortex, all three major forms of metabolic pathways are impacted. When mice with CrAT deletion were placed on a high fat diet, kidney disease was more severe and developed faster. Primary PTCs isolated from the knockout mice displayed energy deficit and impaired respiration before the onset of pathology. Next generation sequencing analysis from cortices at different time points revealed that dysregulation of genes related to mitochondrial function precede development of pathology. Mitochondrial overload also led to the enrichment of genes that are deregulated in CKD/DKD. Importantly, mice with heterozygous CrAT deletion in the PTC also developed kidney disease, suggesting that even moderate impairments in substrate efflux impede PTC function. Our findings support the hypothesis that derailments of mitochondrial substrate metabolism in PTC alone may be causative to CKD. Our results also suggest that tubular injury may be a primary event followed by secondary glomerulosclerosis, raising the possibility that focusing on normalizing tubular cell metabolism could be an important preventative strategy.

Support or Funding Information

This research is supported by NIH grants R01‐DK115749‐01A1 (to KS) and R01‐DK103860 (to RN)