bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA‐21 Ablation Attenuates Acetaminophen‐Induced Hepatoxtoxicity in Male Mice
Authors: Huffman Alexandra M. , Syed Maryam , Rezq Samar , Anderson Christopher D. , Smith Stanley V. , Harmancey Romain , Yanes Cardozo Licy L. , Romero Damian G.
Published: April 2020

Introduction

Acetaminophen (APAP) induced acute liver failure (ALF) is recognized as the most common cause of ALF in Western societies, occurring in approximately 40% of all cases. The disease is characterized by coagulopathy, hepatic encephalopathy, multi‐organ failure, and death. Micro‐RNAs are small, non‐coding RNAs capable of altering gene expression at the post‐transcriptional level. MicroRNA‐21 is dynamically expressed in the liver and several target genes of microRNA‐21 are highly expressed in the pericentral hepatocytes. We hypothesize that microRNA‐21 genetic ablation attenuates hepatotoxicity following acetaminophen intoxication.

Methods

Eight‐week old microRNA‐21 knockout (miR21KO) or wild‐type (WT) C57BL/6N male mice were injected with acetaminophen (APAP, 300 mg/kg BW) or saline. Animals were sacrificed 6 or 24 hours post‐injection, and serum and livers were harvested.

Results

WT animals showed significantly higher hepatic injury markers serum ALT (20,077±2,084 U/L vs 13,364±1,667 U/L, p<0.05, n=11) and AST (29,125±3,485 U/L vs 20,282±2,445 U/L, p<0.05, n=11) than their miR21KO counterparts after 6 hours of APAP treatment, while no significant difference was observed in animals treated for 24 hours. miR21KO mice presented attenuated liver necrosis compared with WT animals (59.8±5.8 vs 42.5±7.9 %, p<0.05, n=10) 24 hours post‐APAP treatment. Moreover, miR21KO mice had decreased hepatic DNA fragmentation analyzed by TUNEL assay than WT animals (58.26±4.5 vs 42.60±4.9 %, p<0.05, n=8) after 24 hours of APAP treatment. MicroRNA‐21 ablation did not modify hepatic GSSH/GSH or APAP‐Cysteine adducts at either 6 or 24 hours post treatment. MicroRNA‐21 ablation upregulated mRNA expression of autophagy (Beclin‐1, Map1LC3a, Pdcd4, p62) and decreased inflammation (PAI‐1) markers compared with WT mice 24 post APAP treatment. MicroRNA‐21 ablation exacerbated hepatic autophagy marker LC3A/B‐II protein expression at both 6 (2.8 vs 1.5‐fold) and 24 (2.2 vs 1.2‐fold 1.2) hours post APAP, correlating with increased Map1lc3a/b mRNA levels.

Conclusion

Our results suggest that miRNA‐21 mediates APAP‐induced hepatotoxicity. MicroRNA‐21 inhibition could be a novel therapeutic approach to abolish or mitigate APAP‐induced hepatotoxicity. The beneficial effect of microRNA‐21 is due to an upregulation of hepatic autophagy. Specifically, microRNA‐21 inhibition could be particularly useful when APAP intoxication is detected at its late stages and the only available therapy, NAC, is minimally effective.

Support or Funding Information

Supported by NIH grants R21 DK‐113500 and P20 GM‐121334.
Characterization of Highly Antibiotic Resistant Bacteria found in an Urban Waterway
Authors: VanRyn Alexis , Krantz Allyson , Carlson Clayton D.
Published: April 2020

Pseudomonas are a group Gram‐negative Gammaproteobacteria with numerous different species that are a risk of causing bacterial infections. The goal is to measure how many Pseudomonas are present in different urban waterways, to evaluate if they are resistant to antibiotics and the degree of resistance they show, and to determine the mechanisms of resistance that they utilize. The research was conducted on urban waterways in the suburban Chicago region. Pseudomonas Isolation Agar (PIA) is a selective media that enriches for Pseudomonas. Water from the urban waterways was grown on PIA plates containing antibiotics, which allowed the level of resistance to be measured and evaluated based on qualitative growth. An antibiotic panel was chosen that provides a broad set of antibiotics which will each interact with the bacteria differently. Growth assays at varying concentrations of antibiotic allow for the degree of resistance to be evaluated. Whole genome sequencing of highly antibiotic resistant Pseudomonas allows for a bigger picture look at which genes make the bacteria resistant. We have found massive resistance in the waterways in several of the antibiotics. The hope of this work is to characterize potentially dangerous microbes that live in urban waterways.
The Effects of Ciproxifan on Liver in Rats with Cerebral Ischemia
Authors: Yegin Bengi , Donmez Dilek Burukoglu , Oz Semih
Published: April 2020

Ischemia and reperfusion occur in some tissues or organs and may develop the severity of dysfunction. It causes permanent functional or structural damage and occurs many events leading to cell death. It is possible to see the damage not only in the tissues where the blood flow is stopped but also elsewhere in the body. Ciproxifan belongs to a novel chemical series of histamine H3‐receptor antagonists. Ciproxifan has been shown to cause vigilance and consideration in animal studies. We aimed to find out whether ciproxifan has an effect on liver tissue in rats with ischemia‐reperfusion injury.

Approval for the animal study was obtained from the ethical committee with file number 556, on October 27, 2016. All surgical procedures were performed under anesthesia (50 mg/kg ketamine hydrochloride +10 mg/kg xylazine). 32 Wistar Albino male rats were used in 4 groups. Control and Sham groups were given dimethyl sulfoxide for 7 days/single dose. 10 mg/kg ciproxifan were administered to the Low Dose group and 30 mg/kg ciproxifan to the High dose group for 7 days/single dose. No surgical procedure was applied to the control group. Sham, Low Dose, and High Dose groups were administered cerebral ischemia induced by common carotid artery technique for 15 minutes. Twenty‐four hours following reperfusion. Rats were sacrificed and liver samples were taken.

Examination of all rat liver revealed that the liver of control rats was observed in the normal histological structure. Also; hepatocyte cells, sinusoidal structures, and vena centralis were observed normal the normal histological structure. Necrotic cells with pyknotic nuclei, eosinophilic cytoplasm, and partial sinusoidal dilatation are seen in rat liver tissues of the Sham group. In the Low Dose and High Dose, groups were observed hepatocyte cells, sinusoidal structures, and vena centralis structures together with a nearly normal liver structure.

As a result, we have observed that ciproxifan has a protective effect on the liver tissue. It was observed that administered of different doses did not affect the result.

Support or Funding Information

ESOGU‐BAP
Intermittent hypoxic training regulates the microglia phenotype by increasing melatonin receptor expression.
Authors: Bhandari Sabita , Ryou Myoung-gwi
Published: April 2020

Microglia is a resident immune cell in the brain. Microglia could be polarized into two phenotypes: pro‐inflammatory (M1) and anti‐inflammatory (M2). The pro‐inflammatory phenotype of microglia is known to harmful in ischemic stroke disease processes. An intervention, therefore, that could shift the phenotype of microglia toward the anti‐inflammatory phenotype could be beneficial in cerebral protection against ischemic stroke. Intermittent hypoxic training (IHT) protects the microglia against hypoxia and reoxygenation stress and increases the M2/M1 ratio. The involvement of the melatonin, an antioxidant, was proposed from the many laboratories. We studied the function of melatonin in the polarization of microglia in the IHT.

Methods

EOC20, mouse microglia, have been assigned to the control, oxygen‐glucose deprivation (OGD), IHT, OGD + IHT. The cell viability of each group was assayed by using Calcein‐AM assay. The phenotype of EOC20 cells was determined with immunocytochemistry staining CD 68 (M1) and CD 206 (M2) and quantified by flow cytometry. The involvement of melatonin (30 μM) and melatonin receptor was determined by using melatonin and melatonin receptor antagonist, Luzindole (50 μM). Protein contents of melatonin receptor 1B, Akt, Erk1/2 were measured by immunoblot. Two factors (treatment and protocol) ANOVA and Tukey post hoc test was used to find statistical significance. P<0.05 was considered a significant value.

Result

Cell viability in the OGD group was significantly decreased comparing to the control, IHT, OGD+ IHT groups. Meaningful elevation in the melatonin 1B receptor and phosphorylation of Akt and Erk1/2 were detected, but those protein contents were blunted with Luzindole treatment. M2/M1 ratio was altered with melatonin treatment, control group 0.32 and melatonin group 2.7.

Conclusion

Microglia was activated and polarized to the pro‐inflammatory phenotype after OGD and reoxygenation. However, IHT was able to mitigate cell death and shift the phenotype to the anti‐inflammatory phenotype. IHT‐induced favorable effects including phenotype of microglia were diminished by inhibiting melatonin 1B receptor with Luzindole. Results in the current study proposed that polarization of microglia influenced by the activation of the melatonin pathway.
Monosynaptic Excitatory Drive of Kölliker‐Fuse Neurons by Noradrenergic Locus Coeruleus Neurons in Mice
Authors: Varga Adrienn G. , Reid Brandon T. , Levitt Erica S.
Published: April 2020

The opioid epidemic is still a national crisis with constantly increasing fatal overdoses. The primary cause of death from opioid overdose is respiratory failure, which is the result of muopioid‐receptor mediated inhibition in respiratory centers of the brain. Recently, we have shown that the pontine Kölliker‐Fuse nucleus (KF) is highly susceptible to inhibition by opioids and may be a compelling target to counter the respiratory side effects of opioids. The ideal strategy to reverse an opioid overdose only affects respiratory depression without causing pain or withdrawal. This approach has to rely on targeting non‐opioid respiratory pathways. Prior work has identified direct projections between the KF and the main noradrenergic arousal center of the brain, the locus coeruleus (LC). In an effort to explore non‐opioid‐mediated excitation of respiratory neurons, we studied how modulation via LC neurons may affect KF neuronal activity. We selectively expressed channelrhodopsin2 in the LC of Th‐Cre hemizygous mice. Whole‐cell patch clamp recordings combined with optogenetic stimulation of LC neurons, and immunohistochemistry confirmed channelrhodopsin2 expression in Th+ LC cells. Next, we optically stimulated LC axon terminals in coronal KF slices and recorded light‐evoked EPSCs in the presence of picrotoxin. Since light‐evoked EPSC onsets had fixed latencies with minimal jitter, followed a similar time course and resulted in no failure, our results indicate the presence of excitatory monosynaptic drive between the LC and KF. KF neurons receiving monosynaptic drive from the LC were then tested for opioid‐sensitivity. Application of a mu‐opioid agonist to KF neurons showing consistent eEPSCs resulted in a hyperpolarizing outward K+ current in voltage clamp recordings in >90% of the cases. Our results demonstrate that the majority of opioid‐sensitive KF neurons receive monosynaptic excitatory drive from noradrenergic LC neurons. This work provides exciting novel evidence for an excitatory pathway between the LC and the respiratory brainstem.

Support or Funding Information

Funding: This work was supported by National Institutes of Health Grants R01 DA47976 (E.S.L.). A.G.V. was funded by the UF Breathing Research and Therapeutics Training Program (T32 HL134621) and the Center for Respiratory Research and Rehabilitation.
Examining the Role of p190RhoGAP on the Organization of the Actin Cytoskeleton in Breast Epithelial Cells
Authors: Robinson Courtney Rae , Kitt-Hopper Khameeka
Published: April 2020

Angiogenesis (i.e. blood vessel formation) and metastasis of tumors are two of the most dangerous aspects of malignant cancer cells. Metastasis requires spatial and temporal coordination of protein complexes known to affect cell adhesiveness and migration/proliferative properties. These protein complexes include E‐cadherin, Rho GTPases (e.g., Rac1 and RhoA), and the actin cytoskeleton. Rho GTPases perform different functions in the cell and have been shown to affect cancer progression, wound healing, and inflammation through regulation of cadherin and actin. For Rho GTPases to function, additional proteins increase or decrease their activity in the cell. These include guanine‐nucleotide‐exchange factors (GEFs) and GTPase‐activating proteins (GAPs). The current study aims to examine p190RhoGAP (GTPase activation protein (GAP) for RhoA) on cell adhesion and migration dynamics of normal Human Mammary Epithelial cells (HMECs) and the metastatic adenocarcinoma breast cancer cell line, MCF‐7. Cells were examined under endogenous conditions, as well as transfected with p190RhoGAP wild‐type and a catalytically inactive p190 RhoGAP‐R1283A mutant that locks RhoA into a constitutively active state. Overexpression of wild‐type and catalytically inactive p190RhoGAP had no major effect on actin stress fiber and cortical actin at cell‐cell contacts. However, HMEC cells overexpressing p190 RhoGAP‐R1283A showed reduced cytosolic RhoA protein, suggesting that loss of RhoA activity affects RhoA protein levels. Although no significant change in wound closure was observed, transwell cell invasion assays show a decreased rate of cell invasion in the presence of the chemo‐attractant, epidermal growth factor (EGF) in MCF‐7 cells. These data indicate a role for p190RhoGAP in the regulation of RhoA during cell migration and invasion. Loss of p190RhoGAP catalytic activity could affect the production of RhoA protein, leading to a decrease in the formation of actin structures (i.e., lamellipodia and filopodia) and a decrease in cell migration and invasion. Future work aims to determine how p190RhoGAP controls spatiotemporal regulation of protein complexes involved in metastatic cancer.

Support or Funding Information

Saint Mary's School of Science Research Program and the Robert W. and Beverly J. Summers Scholarship
Role of cystic fibrosis transmembrane conductance regulator in FHC colon epithelial cell dysfunction and colon cancer
Authors: Grant Garett J. , Mogan Cara A. , Liou Theodore G. , Helms My N.
Published: April 2020

Objective

To determine the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in colon cancer development, with a focus on cell migration, EGFR signaling, and inflammation using model FHC colon epithelial cells.

Background

Our clinical observations indicate that individuals with cystic fibrosis (CF) over the age of 40 are 4–8 times more likely to get colon cancer than the general population. Moreover, the likelihood of getting colon cancer increases to over 30 times the general population’s risk in CF patients who have had an organ transplant. The mechanism(s) leading to increased colon cancer in CF patients is not fully understood, but dysfunctional Cl− and Na+ transport are highly implicated. Chronic inflammation has also been shown to play an important role in the development and progression of many cancers. We hypothesize that dysregulated ion transport in CF patients leads to chronic inflammation and increased incidence of colon cancer via altered epidermal growth factor receptor (EGFR) signaling.

Methods

We performed scratch assays on cultured human colon epithelial cells (FHC) and measured the rate of cell migration every 24 hours for 5 days in the presence of either CFTR activating or inhibiting compounds. EGFR signaling was analyzed using standard immunohistochemical labeling techniques and commercially available reagents in similarly treated FHC cells.

Results

Cells treated with 500 μM IBMX and 5 μM forskolin (CFTR activators) had significantly decreased rates of cell migration (26 μm2/24 hours) compared to the control group (51 μm2/24 hours) n=3; p<0.05. Control and colon cells treated with 500 μM IBMX and 5 μM forskolin showed low levels of EGFR labelling (restricted to cell periphery), whereas cells treated with 5 mM CFTR(inh)‐172 (CFTR inhibitor) showed more robust EGFR signal throughout the cytoplasm in 3 independent observations.

Conclusion

Our data indicates that suppressed CFTR activity impacts colon epithelial cell migration and EGFR localization in FHC cells. Additional research on CFTR and EGFR signaling is needed to better understand and improve treatment options for both colon cancer and CF patients.

Support or Funding Information

Funding: NIH R01 HL137033‐01 awarded to MNH.
Structure‐Activity Relationships for Cumyl‐Containing Synthetic Cannabinoids to Induce Hypothermic, Cataleptic and Analgesic Effects in Mice
Authors: Baumann Michael H. , Garibay Nancy , Partilla John S. , Brandt Simon D.
Published: April 2020

Synthetic cannabinoids (SCs), also known as “spice” or “K2”, are an evolving class of new psychoactive substances that pose public health risks. SCs exert their psychoactive effects by stimulating cannabinoid‐1 receptors (CB1) in the brain, yet few studies have systemically evaluated structure‐activity relationships (SAR) for newly‐emerging compounds. Here, we investigated the pharmacology of SCs which possess a fixed cumyl ‘head group’ but a variable N‐alkyl ‘tail group’ which ranged from 3 to 7 carbons in length. Cumyl‐PINACA, which contains a 5‐carbon pentyl tail, served as a reference compound. In vitro CB1 binding was assessed by displacement of [3H]SR141716 in mouse brain membranes. In vivo effects of subcutaneously (sc) administered drugs were examined in male C57Bl/6J mice bearing surgically‐implanted telemetric temperature transponders. Mice were subjected to a ‘triad’ test battery which assessed temperature, catalepsy, and analgesia every 30 min post‐injection for 2 h. Temperature was measured via a handheld receiver. Catalepsy was measured using the bar test, with a cut off of 60 sec, whereas analgesia was measured using a hot plate set at 52°C, with a cut off of 45 sec. Cumyl‐PINACA was the most potent inhibitor of CB1 binding with an IC50=1.8 nM. Decreasing N‐alkyl chain length to a 3‐carbon propyl markedly reduced binding affinity (IC50=44.8 nM), as did increasing chain length to a 7‐carbon heptyl (IC50=124.0 nM). Consistent with the in vitro findings, cumyl‐PINACA was the most potent compound to induce cannabinoid‐like effects in the triad test battery, with ED50s ranging from 0.06–0.09 mg/kg, sc. Importantly, all cumyl compounds induced hypothermic, cataleptic, and analgesic effects with a rank order of potency that matched their IC50s for inhibition of CB1 binding. THC exhibited ED50s in the triad test ranging from 18.7–30.3 mg/kg, sc. Overall, our data reveal that certain cumyl‐containing SCs are more potent CB1 agonists than THC. Furthermore, N‐alkyl chain length is a critical determinant for the pharmacological effects of cumyl SCs, with a 5‐carbon pentyl chain conferring optimal activity. More research examining the SAR for newly‐emerging SCs is needed to inform stakeholders involved with responding to the abuse of these compounds.

Support or Funding Information

This work was supported generously through IRP, NIDA, NIH.
Effect of H9, a formula of nine medicinal herbs derived from Osuyubujaijung‐tang, on trastuzumab pharmacokinetics in mice
Authors: Choi Young-Hee , Chae Hee-Sung , Chin Young-Won
Published: April 2020

Recently, the resistance of trastuzumab as an established monoclonal antibody therapy for women with breast cancers overexpressing human epithelial growth factor receptor 2 (HER2) has been issued and the activation of phosphoinositide 3‐kinase (PI3K) pathway was identified as a main mechanism of resistance. As the combinations of western and oriental medicines have emerged accompanying the improvement of efficacy and/or safety in chemotherapies, the cases for combinations of trastuzumab and oriental medicines has been reported. H9 is a formula of nine medicinal herbs derived from Osuyubujaijung‐tang, a traditional Korean prescription for Soeumin constitution, targeting HER2‐PI3K/Akt pathway and consequently showing anticancer effects against breast cancer and lung cancer in vitro and in vivo. The synergistic effect of trastuzumab and H9 combination was reported in vivo mice model of lung cancers. Regarding that the current drug interaction issues of therapeutic proteins including monoclonal antibody with small molecules, the effect of H9 on pharmacokinetic interaction of trastuzumab in aspect of systemic exposure and tissue distribution was investigated in this study. Prior to evaluation of pharmacokinetic interaction of trastuzumab with H9, the analytical method of trastuzumab using ELISA was validated.

In the validation of ELISA analytical method, 0.1–20 mg/mL of trastuzumab were analysed showing below 13.8% of precision and 84.3–112% accuracy values, respectively. In pharmacokinetics, H9 did not change any pharmacokinetic parameters of trastuzumab and also there was no difference on tissue distribution of trastuzumab to liver, heart, lung, and spleen in trastuzumab and H9 combinations. These results provided an evidence for the development of this combination therapy in clinical levels after the further investigations of dosage regimen adjustments.

Support or Funding Information

This study was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF‐2016R1C1B2010849 and NRF‐2018R15A2023217).
Opioid sensitivity of projection‐specific Kölliker‐Fuse neurons
Authors: Bateman Jordan , Reid Brandon , Maletz Sebastian , Levitt Erica
Published: April 2020

Opioid‐induced respiratory depression is the primary cause of death in individuals who overdose on opioids. Respiration is controlled by a highly interconnected neural network located in the pons and medulla. The Kölliker‐Fuse nucleus (KF), located in the dorsolateral pons, is an essential part of this network and projects to core respiratory nuclei in the ventrolateral medulla. Opioids injections into the KF disturb respiratory rate, volume, and pattern. Deletion of opioid receptors from KF neurons attenuates morphine‐induced respiratory depression. These studies suggest that the KF is a critical site in opioid‐induced respiratory depression. Mu opioid receptor agonists hyperpolarize a population of neurons in the KF due to activation of G protein‐mediated inwardly rectifying conductance. However, we do not know the projection target of the opioid‐sensitive KF neurons. Given the extensive projections from the KF to medullary respiratory rhythm generators, we hypothesize that opioid‐sensitive KF neurons project to respiratory rhythm generators. Fluorescent beads (FluoSpheres 580/605, ThermoFisher) were injected into the Bötzinger complex (BötC), pre‐Bötzinger complex (preBötC), and rostral ventral respiratory group (rVRG) of mice. Whole‐cell voltage‐clamp recordings were made from retrograde‐labeled KF neurons contained in brain slices. Mu opioid receptor‐mediated outward currents were identified in KF neurons that project to the preBötC, and rVRG, but not in KF neurons that project to the BötC. Opioid‐sensitive connections between the KF and respiratory rhythm generators may be a possible mechanism by which opioids reduce respiratory rate.

Support or Funding Information

R00, DA038069
Evolutionary Potential and Morphological Integration of the Shoulder Girdle Among Primates
Authors: Agosto Elizabeth R. , Auerbach Benjamin M.
Published: April 2020

The morphology of the shoulder girdle is highly varied among primates, possibly reflecting an array of functional demands placed through the range of locomotor and behavioral movements exercised in the upper limb. Variation in the shoulder girdle, and especially the scapula, is hypothesized to be the result of directional selection for different functional demands. Evolutionary analyses of the shoulder girdle often assess this anatomical region and its traits individually, which implicitly models the evolution of the girdle as independent of other traits. However, the primate shoulder girdle has developmental and functional covariances with the basicranium, pelvis, humerus, and vertebral column that we have recently demonstrated have potential to influence its evolution through correlated responses to directional selection. Whether these relationships are similar across primate taxa and how these relationships may affect morphological variation among primates is unknown.

Here we evaluate the strength of the covariance and evolutionary potential among four anatomical regions: shoulder girdle, basicranium, pelvis, and humerus. We obtained cranial and postcranial linear dimensions from Alouatta, Colobus, Homo, Hylobates, and Papio. Measurements were mean standardized prior to analysis. We estimated morphological integration using Wagner's variance of the eigenvalues (VE), and estimated evolutionary potential using Hansen and Houle's evolutionary flexibility (EF) with 1000 simulated random selection gradients. EF captures how closely the evolutionary response in a group of traits follows the direction of the selection. We evaluated differences in VE and EF among taxa by performing a pairwise comparison of the bootstrapped values for each. Analyses were performed using the R statistical environment.

Our results show that patterns of morphological integration and evolutionary flexibility among anatomical regions differ across primate taxa, with humans having the greatest evolutionary potential and Papio the lowest. Broadly, the patterns revealed among taxa do not strictly correspond to the functional demands of the upper limb experienced by each; taxa with different locomotor demands on the upper limb have the same kinds of covariance or evolutionary flexibility. Thus, evolutionary hypotheses of primate shoulder girdle morphological variation that evaluate functional demands alone may not account for the effect of these relationships. We conclude that different trait covariances among anatomical regions may manifest as morphological differences in the primate shoulder girdle, which has important implications for models of shoulder evolution.

Support or Funding Information

This research is supported by a National Science Foundation Doctoral Dissertation Improvement Grant (NSF BCS‐1825995).
Assessing Undergraduate Competencies in Biochemistry and Molecular Biology via an Annual Exam
Authors: Tyler Ludmila , Carastro L. Michael , Zeidan Quira , Wolfson Adele J. , Tansey John T. , Offerdahl Erika G. , Del Gazio Moore Victoria , Martin Debra , Loertscher Jennifer , Kennelly Peter J. , Kanipes Margaret I. , Dries Daniel R. , Dean Diane M. , Bailey Cheryl
Published: April 2020

Assessment of student learning can both provide a measure of individual achievement and support data‐driven decisions about educational programs. The annual American Society for Biochemistry and Molecular Biology (ASBMB) Certification Exam is an important component of the undergraduate accreditation program offered by the Society. Exam development and scoring have resulted in a community of scientist‐educators dedicated to the improvement of biochemistry and molecular biology (BMB) education. The exam assesses student competence in four BMB concept areas: energy and metabolism; structure and function; information storage and transfer; and quantitative reasoning. Undergraduate students nearing completion of their ASBMB‐accredited program are eligible to take the exam. A community of volunteers, aided by the organizational efforts of ASBMB staff, has come together to implement all aspects of the exam process. Over 50 volunteers from diverse institutions across the country helped score the exam in 2019, and additional volunteers are being recruited for 2020. This scoring community primarily consists of BMB faculty members, but also welcomes postdoctoral scientists and advanced graduate students. In preparation for the exam, scoring teams review and suggest revisions for proposed exam questions and answer keys. Several scoring teams of 3–5 members are assigned to each concept area, and each exam question is scored independently by one or more teams. Student responses are marked as “highly proficient”, “proficient”, or “not yet proficient”. Scoring is statistically analyzed for inter‐rater reliability. Any discrepancies are resolved cooperatively. Students’ overall exam scores are categorized as “Pass with Distinction”, “Pass”, or “Not Passing”. Since the exam was first administered in 2014, nearly 4,000 students have participated. Historically, the “Pass with Distinction” rate has ranged between 10% and 18% and the “Pass” rate 35% to 55%. Scorers value the opportunity to contribute to this national effort to support quality BMB education. Many have served in this role for multiple years, and 98% of the 2019 scorers report they plan to score again in 2020.

Support or Funding Information

This project is supported by the American Society for Biochemistry and Molecular Biology, through the Education and Professional Development Committee.
Mechanosensitivity of TRPV Channels: Implications for Vasopressin Neuron Activity
Authors: Brown Emily , Brown Colin H. , Fronius Martin
Published: April 2020

Vasopressin is synthesised by magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and is secreted from the posterior pituitary gland in response to increased plasma osmolality. Vasopressin maintains body fluid balance by promoting renal water retention. Mechanosensitive TRPV channels facilitate vasopressin neuron responses to plasma osmolality and express various TRPV subunits, including ΔN‐TRPV1, TRPV2 and TRPV4. Most TRPV channels express more than one type of TRPV subunit but the subunit composition of the TRPV channels expressed by vasopressin neurons is unknown, as is the potential contribution of any changes in composition to vasopressin neuron function. Here, we tested the hypothesis that a change in TRPV channel composition alters the mechanosensitivity of ΔN‐TRPV1‐containing channels. To test this hypothesis TRPV and vasopressin expression in the SON of female Sprague Dawley rats was visualised using immunohistochemistry and will also be visualised with RNAscope. Xenopus oocytes and HEK293 cells were used for heterologous expression of TRPV channels and membrane current recorded via electrophysiology. TRPV2 was expressed on ~33% of SON vasopressin neurons and TRPV4 was expressed on ~40%. However, the TRPV1 antibody caused non‐specific staining and instead RNAscope will be used. Xenopus oocytes expressing ΔN‐TRPV1, TRPV2 and TRPV4 were recorded via two‐electrode‐voltage‐clamp (TEVC). However, TRPV channels in Xenopus oocytes do not appear to be functional because there was no change in membrane current during application of TRPV antagonists and agonists, independent of injected RNA amount (1–20ng) and incubation time (1–4 days). Transfection of TRPV channels into HEK293 cells with co‐expressed fluorescent protein; ΔN‐TRPV1‐GFP, TRPV2‐mCherry and TRPV4‐CFP was performed. Maximum ΔN‐TRPV1‐GFP expression occurred from 10–20 h, however increased cell death was observed after 24 h. In conclusion, vasopressin neurons express TRPV channels but Xenopus oocytes appear to lack specific cytoskeletal proteins for mechanical gating such as beta‐tubulin. HEK‐293 cells express the required cytoskeletal proteins for TRPV trafficking, membrane tethering and gating and we are in process of testing whether ΔN‐TRPV1‐GFP transfection in HEK‐293 cells forms mechanosensitive channels.
Structural and Functional Insights into Aldosterone Synthase Interaction with Ligands and Redox Partner Proteins for Enhanced Treatment of Hypertension
Authors: Brixius-Anderko Simone , Scott Emily
Published: April 2020

Primary aldosteronism is the most common form of secondary hypertension and is often associated with cardiac disease. Primary aldosteronism develops due to excessive amounts of the steroid hormone aldosterone, the most potent human mineralocorticoid. Aldosterone synthesized by cytochrome P450 11B2 (CYP11B2, aldosterone synthase) regulates blood pressure and mineral homeostasis. CYP11B2 is 93% identical to the cortisol‐producing CYP11B1. As a result, treatment of aldosterone‐derived hypertension is impeded due to the lack of drugs specifically targeting CYP11B2.

Herein a structure/function approach was used to examine CYP11B2 interaction with both 1) drugs and 2) its redox partner protein adrenodoxin. First, the X‐ray structure of CYP11B2 was determined with the drug LCI699. LCI699 was initially developed as CYP11B2 inhibitor but is now the first drug in clinical trials for the treatment of Cushing’s disease as an inhibitor of CYP11B1. LCI699 positioning in the CYP11B2 active site and comparison with similar compounds in CYP11B1 provides valuable clues for enhanced design of drugs for both enzymes. The second half of this study is focused on CYP11B2 interaction with its redox partner protein adrenodoxin. Studies revealed that adrenodoxin promotes CYP11B2 binding of its physiological substrate 11‐deoxycorticosterone and an adrenodoxin/CYP11B2 fusion protein was generated to examine the protein/protein interaction. An X‐ray structure of this complex identified the adrenodoxin binding site on the proximal face of CYP11B2 and specific interactions. Overall these studies improve our understanding of the aldosterone biosynthetic system and support the design of selective drugs for primary aldosteronism.

Support or Funding Information

The research was funded by a postdoctoral fellowship from the American Heart Association (19POST34430199, SBA) and startup funds from the University of Michigan (EES).
Exercise Preconditioning Prevents Chemotherapy‐Induced Alterations to Transport Proteins Required for Mitochondrial Iron Uptake and Metabolism in the Diaphragm
Authors: Smuder Ashley J. , Huertas Andres Mor , Morton Aaron B.
Published: April 2020

Doxorubicin (DOX) is an anthracycline antibiotic used in cancer treatment. Unfortunately, the clinical use of this highly efficacious anticancer drug is limited due to the development of respiratory and diaphragm muscle dysfunction in patients. DOX‐induced ventilatory impairment is a debilitating condition that promotes the onset of dyspnea, fatigue and exercise intolerance. While the mechanisms responsible for DOX‐induced respiratory insufficiency are unclear, it has been proposed that DOX myotoxicity develops as a result of increased cellular reactive oxygen species production and mitochondrial iron accumulation. Conversely, endurance exercise training performed prior to DOX administration has been shown to prevent the chemotherapy‐induced diaphragm weakness and mitochondrial dysfunction. While the mechanisms for this protection remain elusive, evidence suggests that exercise may provide protection by attenuating DOX‐induced alterations to proteins required to maintain mitochondrial iron homeostasis. Cause and effect were determined by exposing Sprague‐Dawley rats to a two‐week treadmill preconditioning protocol (5 days/week; 60 min/day; 30 m/min). Twenty‐four hours following the last exercise training period, animals received either saline or DOX (20mg/kg ip) treatment. Our results demonstrate that DOX administration in sedentary animals results in increased expression of the mitochondrial iron importer mitoferrin‐1 and a significant reduction in the cellular iron importer transferrin receptor 1 in the diaphragm. In addition, DOX also stimulated a significant decrease in the expression of sideroflexin 2 and SLC25A28, proteins required for mitochondrial heme biosynthesis. Importantly, exercise training prior to DOX administration prevented the DOX‐induced increase in mitoferrin‐1, and also reduced mitoferrin‐2 below that of sedentary animals treated with DOX. Moreover, diaphragm expression of SLC25A38 was maintained in exercise trained animals receiving DOX. These findings confirm a role for mitochondrial iron accumulation and dysregulation of heme synthesis in the development of DOX‐induced respiratory dysfunction, and demonstrate that exercise preconditioning may ameliorate DOX‐mediated diaphragm muscle pathology through the prevention of mitochondrial iron dysregulation.

Support or Funding Information

This work was supported by NIH R01 HL146443 awarded to AJS
Simvastatin in Endothelial Cell Barrier Regulation: A Novel Role for SAMD4A
Authors: Faraj Reem , Kelly Gabriel T. , Feng Anlin , Roberts Rebecca , Kheshtchin-Kamel Nabia , Wang Ting
Published: April 2020

Introduction

Acute lung injury (ALI) is a critical illness which results in significant morbidity and mortality. Endothelial barrier disruption is a key pathologic feature of ALI and serves as the main cause of life‐threatening microvascular leakage and alveolar flooding. Efforts to develop novel therapeutics have been futile with only a handful of agents available, none of which prevent or reverse endothelial barrier disruption. We believe that the answer may lie in the most unlikely of places: a cholesterol‐lowering drug. Simvastatin confers vascular protection through modulation of the endothelial barrier, attenuation of vascular leak, and promotion of endothelial repair following lung inflammation. Integrin β4 (ITGβ4, gene code: ITGB4), a transmembrane receptor protein, plays a key role in endothelial barrier regulation during ALI, and its upregulation is necessary for the enhanced barrier function seen following administration of simvastatin.

Rationale

The mechanism by which simvastatin up‐regulates ITGβ4 expression has not been elucidated. We sought to define the mechanistic pharmacology that controls the regulation of ITGβ4 mRNA and protein expression by simvastatin.

Methods

In this study, we identified potential RNA binding proteins regulated by simvastatin and inflammatory mediators that target ITGB4 using genomic analysis and molecular biology methods (western blotting and silencing), and further validated with endothelial cell biology approaches. Endothelial barrier function modulation is also investigated by electric cell‐substrate impedance sensing (ECIS).

Results

We have identified that Sterile Alpha Motif Domain‐Containing Protein 4A (SAMD4A), an ITGB4 RNA binding protein, is up‐regulated by LPS or TNFa, and downregulated by simvastatin. Additionally, intracellular SAMD4A levels are negatively correlated with ITGB4 mRNA levels. Silencing of SAMD4A in endothelial cells (ECs) results in increased mRNA expression of ITGB4 and enhances ITGB4‐mediated barrier function in ECs.

Conclusion

Our results suggest that simvastatin upregulates ITGB4 via the suppression of SAMD4A, an ITGB4 RNA binding protein, leading to enhanced endothelial cell barrier function. These findings have re‐defined the pharmacology of simvastatin on ITGB4 regulation. Furthermore, SAMD4A may serve as a novel therapeutic target for ALI via the modulation of EC barrier.

Support or Funding Information

This study is supported by NIH grants HL134610 and T32HL007249.
Optimization of Propargylcholine to Label Newly Synthesized Myelin in the Rhesus Monkey Brain
Authors: Bottenfield Karen R. , Moore Tara L. , Mortazavi Farzad , Rosene Doug L.
Published: April 2020

Neuropathology in monkey and human brains often involves loss or damage to myelin and is a major factor impacting normal function as myelin constitutes 35%–40% of the forebrain. Methods to assess alterations in myelin include electron microscopy, histological stains (e.g. Luxol Fast Blue), immunohistochemistry (e.g. for myelin basic protein) and a label free confocal method (SCoRe), as well as diffusion MRI. Each method has advantages but none offer a reliable, validated method to assess microscopic changes in myelin (synthesis or repair) across the entire forebrain from gray matter fascicles to subcortical white matter. Here we present preliminary data that newly synthesized myelin can be detected with in vivo administration of a choline analog, propargylcholine (P‐Cho), that is readily incorporated into all classes of choline phospholipids including major components of the myelin sheath, phosphatidylcholine and sphingomyelin. Importantly, since P‐Cho contains an alkyne moiety, it can be visualized post mortem by conjugation with a fluoro‐labeled azide using click chemistry and detection with confocal microscopy. P‐Cho can be administered by intraperitoneal injection where it enters the circulation and is incorporated into myelin, allowing detection and quantification of newly synthesized myelin. While previous research has demonstrated the use of P‐Cho for assessing remyelination in mice, its efficacy and safety in monkeys has yet to be explored. This is important for our monkey model of normal aging where age‐related myelin pathology accounts for cognitive deficits and for our studies of recovery after damage to the motor cortex. The ability to quantify myelin plasticity in both of these models could provide significant insight into myelin pathology, repair, and maintenance. To test this, we administered P‐Cho daily for 6 days via IP injections (3.5 – 4.0 mg/kg) to 6 young adult and 1 older adult monkey at different time points before euthanasia (1, 2 and 6 weeks). Frozen brain sections were cut, labeled with a fluorescent azide, and the sections evaluated for the presence and specificity of P‐Cho by co‐labeling the tissue with antibodies for axons (SMI‐312) and newly formed actively myelinating oligodendrocytes (BCAS1). Co‐localization of P‐Cho was also performed using confocal reflectance microscopy (SCoRe). Preliminary analyses suggest effective incorporation of P‐Cho into newly synthesized myelin within 1 week of dosing and sustained presence beyond 6 weeks. These results demonstrate a promising new method for investigating remyelination and white matter pathologies related to aging that could also be applied to studies of neurodegeneration, brain injury, and neurological diseases in an animal model highly translatable to humans.

Support or Funding Information

NIH Grants R01‐AG062831, R01‐AG043640; R01‐AG042512; R21‐AG061678; R21‐NS111174, U01‐NS076474
Investigating Human Follicle Stimulating Hormone Receptor and its Partners Using the APEX Assay
Authors: Temple Alexandra Elizabeth , Cohen Brian D.
Published: April 2020

Follicle stimulating hormone (FSH) and its receptor (FSHR) play important roles in reproduction and fertility in both men and women. Fertility problems arise when FSH, FSHR, or any downstream signaling don’t function properly. FSHR is a G‐protein coupled receptor (GPCR) found on the cell surface of granulosa cells in women and Sertoli cells in men. When activated by FSH, FSHR initiates a signaling cascade that can result in different downstream results such as ovarian follicular development and estrogen production in women and sperm production and regulation in men. To better understand how the receptor functions, the interactions between FSH, FSHR and other effector proteins involved in signaling must be identified and understood. One hypothesized mechanism of signaling is through the receptor localization in lipid rafts. The lipid rafts act as a signaling complex where FSHR and other effector proteins colocalize to make for efficient signaling of FSHR. This colocalization in lipid rafts is believed to play an important role and we plan to study the interactions between FSHR, effector proteins, and lipid rafts using the APEX Assay. The APEX Assay has been developed to study these interactions using different GPCRs. This assay utilizes an engineered ascorbate peroxidase (APEX) which is attached to the C‐terminus of FSHR. APEX modifies proteins using biotin phenol and hydrogen peroxide, which creates a biotin‐radical, labeling proteins within a small radius of FSHR because of the short lived nature of the radical. Using mass spectrometry, the biotinylated proteins can be quantitatively analyzed to give a better understanding of the proteins associated with FSHR and the changes in the proteins over time after hormone treatment, as well as lipid raft localization. Using HEK293 cells, we are working on creating a stable cell line expressing an FSHR‐APEX construct. Once the cell line is created, the FSHR‐APEX receptor can be used in the APEX Assay to analyze the proteins that are involved in FSHR signaling. This research will give us a better insight as to what effector proteins FSHR interacts with, when these interactions occur, and if they occur in lipid rafts. This will provide us with a better understanding as to how the receptor signals and initiates downstream signaling and how this might affect fertility in humans.
Neonatal testosterone treatment sensitizes the angiotensin II‐elicited hypertensive response in both male and female adult rats
Authors: Xue Baojian , Beltz Terry G. , Guo Fang , Johnson Alan Kim
Published: April 2020

The sex steroid hormones, testosterone and its metabolite oestradiol, exert a profound influence on sexual differentiation and function of the neural circuits in which microglia are implicated in masculinization of brain and behavior. Our previous studies demonstrated that there were sex differences in the sensitization of hypertension, and activation of microglial cells was necessary for the induction of hypertensive response sensitization. The present study investigated whether neonatal testosterone treatment sensitizes the ANG II‐elicited hypertensive response in male and female rats when they are in adulthood. Subcutaneous testosterone (T, 1.25 mg in 25 μL corn oil) injections were performed on the dorsal surface of the pup on postnatal days 1, 3 and 5. All rats were weaned at 3 weeks of age and weighed one time per week until blood pressure experiments began. At 12 weeks of age, the T injected rats and oil injected control rats in both sexes received a subcutaneous infusion of a slow‐pressor dose of ANG II (120 ng/kg/min) for 2 weeks. Both male and female T injected rats exhibited a significant increase in body weight and had a significantly enhanced hypertensive response to the ANG II infusion when compared to control rats. These results support the hypothesis that early T‐induced masculinization in both males and females sensitizes ANG II‐induced hypertension, and suggest that masculinization disorder induced by neonatal T may predispose individuals to exacerbation of renin‐angiotensin‐elicited hypertension.

Support or Funding Information

NIH HL139575