bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissection Tutorial Video Series: a BlueLink Resource
Authors: Prall Cristina R. , Alsup B. Kathleen , Sullivan Alyssa , Fox Glenn M.
Published: April 2020

First year medical and dental students at the University of Michigan do not receive formal dissection training as part of their foundational human gross anatomy courses. As a result, many students do not know proper dissection techniques when they begin their dissection on the first day of lab, and often resort to learning by trial and error, or by observing faculty and teaching assistants as labs progress. In an attempt to help students feel more prepared and alleviate some of the anxiety associated with the first day in the gross labs, a series of short videos were produced to discuss and demonstrate the different aspects of dissection. The videos were provided as an adjunct to the first year medical and first year dental curricula on the BlueLink website, as well as on the BlueLink YouTube channel. Feedback data is collected from University of Michigan medical and dental students regarding the usefulness of this video series via surveys. In addition to University of Michigan students, these videos could be a useful resource to students at other institutions that also might not provide formal dissection instruction. As of November 2019, the views for each video ranges between 800 and 1900 views, with an average of 1300 views per video in the series.
Acute in vitro Nephrotoxicity of Three Brominated Flame Retardants
Authors: Barnett Lillie , Cummings Brian S.
Published: April 2020

Brominated flame retardants (BFRs) are organohalogens commonly added to commercial products such as computers, electronics, textiles and furniture to reduce their flammability. BFRs have significant environmental persistence and are reported to be detected in human blood and breastmilk. In particular, tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), and polybrominated diphenyl ethers (PBDEs) occupy nearly 20 percent of the global flame retardant market and are suggested to have adverse effects on humans and wildlife. As such, the mechanisms of BFR‐induced toxicity are actively being explored under the U.S. EPA Toxic Substance Control Act. We previously determined the effects of TBBPA, HBCD, and BDE 47 on cell viability in rat (NRK 52E), adult human (HK‐2 and RPTEC), and human embryonic (HEK‐293) kidney cells after 48 hours, and demonstrated that these compounds induce concentration‐dependent effects on MTT staining. Assessment of the mechanism of cell death using annexin V staining and propidium iodide (PI) staining showed that all three compounds induce apoptosis in these cells. We subsequently performed RNAseq and pathway analysis in these cells to identify key signaling pathways involved in the mechanism of BFR‐induced toxicity. Furthermore, we explored how BFR nephrotoxicity impacts neighboring cells by treating human fibroblasts with conditioned media from BFR‐treated kidney cells and measuring morphological, molecular, and functional changes in fibroblast activation using RT‐PCR, wound healing and high content image analysis. Ultimately, these data will aid future studies concerning the molecular mechanisms of BFR toxicity in kidney cells and how this impacts kidney epithelia‐fibroblast crosstalk using chronic exposure at environmentally relevant doses.
ClC‐K2 Cl− channel determines acid‐base transport and Cl− reabsorption in intercalated cells of the collecting duct
Authors: Zaika Oleg , Tomilin Viktor , Pochynyuk Oleh
Published: April 2020

Intercalated cells (ICs) of the collecting duct play a critical role in regulation of systemic acid‐base homeostasis. In addition, ICs are capable of performing trans‐cellular Cl‐ reabsorption particularly during volume depletion. While the major apical membrane transport systems, such as pendrin in base secreting B‐type and V‐ATPase in acid‐secreting A‐type of ICs, are well characterized, little is known about mechanisms and contribution of the basolateral membrane in both processes. Kidney specific ClC‐K2 (ClC‐Kb in humans) Cl‐ channel is expressed in the basolateral membrane of the distal nephron segments, including the collecting duct. Loss‐of‐function mutations in ClC‐Kb or its auxiliary subunit, barttin manifest as Bartter’s syndrome, an autosomal recessive tubulopathy leading to urinary salt wasting, hypotension, hypokalemia, and metabolic alkalosis. We and others previously demonstrated that ClC‐K2 expression in the collecting duct is restricted to ICs. Here, we generated ClC‐K2 deficient mice using cre‐loxP strategy to investigate the functional role of the channel in acid‐base and Cl‐ transport. We further combined BCECF‐sensitive intracellular pH (pHi) measurements with fluorescent AQP2‐based identification of principal and intercalated cells to assess ClC‐K2‐dependent pHi changes in different cell types. Successful ClC‐K2 deletion (approximately 80%) was verified with immunofluorescent microscopy and patch clamp electrophysiology in freshly isolated collecting ducts. As expected, ClC‐K2 inhibition with NPPB (100 uM) had no effect in principal cells of WT mice, whereas it induced rapid intracellular acidification in B‐type and alkalization in A‐type of ICs. NPPB failed to significantly affect pHi in collecting ducts from ClC‐K2 deficient mice. Extracellular Cl‐ removal to drive basolateral Cl‐ exit via ClC‐K2 had no effect on pHi in principal cells, but caused alkalization in B‐type and acidification in A‐type of ICs. Importantly, Cl‐ removal did not induce pHi changes in both A‐ and B‐type of ICs from ClC‐K2 deficient mice. This suggests that ClC‐K2 mediates trans‐cellular Cl‐ reabsorption and determines apical acid‐base transport by controlling intracellular Cl‐ concentration. Moreover, application of Angiotensin II (Ang II, 500 nM) increased ClC‐K2 single channel activity and augmented respective ClC‐K2‐dependent pHi changes in B‐ but not A‐type of ICs to favor apical Cl‐/HCO3‐exchange. We propose that this, at least partially, contributes to Ang II‐driven metabolic acidosis observed in the clinic. Overall, we demonstrate that ClC‐K2 activity is essential for regulation of acid‐base secretion and Cl‐ reabsorption in both A‐ and B‐type of ICs of the collecting duct.
Optimizing a biosensor‐based assay for ubiquitination activation
Authors: Broadberry Roma Lucette , Berndsen Christopher
Published: April 2020

Ubiquitination is a post‐translational modification which regulates protein degradation, DNA damage signaling, and inflammatory response. Three transfer enzymes catalyze the reaction’s pathway: E1 activating enzyme, E2 conjugating enzyme, and E3 ligating enzyme, respectively. Despite the identification of these enzymes’ structure and function, a complete understanding of their catalytic mechanisms remains unclear. Clarification of the ubiquitination mechanism could serve medicinal strategies due ubiquitin’s (Ub’s) involvement with target protein degradation. Existing assays for ubiquitination are either too imprecise, expensive, or radioactive for common usage, thus we are trying to develop a cheap, continuous assay which can make ubiquitination research more accessible. Our approach depends on the detection of ATP as E1 cleaves ATP into pyrophosphate (PPi) and AMP for Ub‐activation. Previous fluorescent assay designs for Ub‐E1 activation were attempted and examined including a terbium‐norfloxacin assay and a chloroacetaldehyde assay, but each showed limitations. The terbium‐norfloxacin assay detected ATP fluorescence, but interfered with the protein pathway, while the chloroacetaldehyde assay used organic sensors to modify ATP, but with little success. We have since turned to adapting a genetically‐encoded nucleotide biosensor, Perceval, toward measuring ATP hydrolysis of E1 in the ubiquitination pathway. Developed in the Yellen Lab at Harvard Medical School, Perceval was designed to report real‐time changes in cellular energy as a result of its competitive binding of ATP and ADP. We have worked to tailor the Perceval model towards measuring solely the ATP hydrolysis of ubiquitination in vitro, starting with E1 activation. After optimizing Perceval’s purification, we showed Perceval has a high affinity for ATP without the ADP competitive binding aspect, and ensured AMP and PPi do not interfere with the biosensor. We similarly tested solutions containing different Ub protein substrates and E1s, and found that Perceval’s affinity for ATP is not interrupted by critical components of the ubiquitination pathway. When directly applied to ubiquitination, fluorescence readings were taken at single‐time points in the ubiquitination pathway and over a continuous duration. Preliminary data suggest Perceval could be a potential biosensor for measuring Ub activation in real time. Future directions will look to further optimize assay conditions as well as test other biosensors for comparison.

Support or Funding Information

James Madison University Department of Chemistry and Biochemistry, NSF REU CHE‐1757874, Thomas and Kate Jeffress Memorial Trust, 4‐VA Organization
Inhibition of Sirtuin‐1 Ameliorates Vascular Remodeling and Hypertension in SHR
Authors: Arifen Nahida , Li Yuan , Srivastava Ashok K. , Anand-Srivastava Madhu
Published: April 2020

Sirtuin‐1 (SIRT1), class III histone deacetylase, has been shown to be overexpressed in hearts from spontaneously hypertensive rats (SHR). We recently showed that vascular smooth muscle cells (VSMC) from SHR exhibit enhanced expression of SIRT1 as compared to age‐matched Wister Kyoto (WKY) rats and contribute to the upregulation of Giα proteins implicated in the pathogenesis of hypertension. The present study was undertaken to investigate the role of upregulated SIRT1 expression in the pathogenesis of hypertension and associated vascular remodeling in SHR and to explore the underlying molecular mechanisms involved in this response. For this study, a selective inhibitor of SIRT1, EX‐527 (5mg/kg of body weight) was injected intraperitoneally into 8‐week‐old adult SHR and age‐matched WKY rats twice per week for 3 weeks. The blood pressure (BP) and heart rate was measured twice a week by the CODA™ non‐invasive tail cuff method. Treatment of SHR with Ex‐527 attenuated high BP by 50 mmHg and inhibited the augmented heart rate. The overexpression of Giα proteins in heart, VSMCs and aorta was also attenuated to the control levels by EX‐527. In addition, inhibition of SIRT1 also attenuated the enhanced levels of superoxide anion, NADPH oxidase activity, enhanced phosphorylation of c‐Src and ERK1/2/AKT in VSMCs isolated from EX‐527 treated SHR. Furthermore, the decreased expression of endothelial nitric oxide synthase (eNOS) in VSMCs and aorta from SHR was also restored to control levels by EX‐527. The hyperproliferation and hypertrophy exhibited by VSMC from SHR, the enhanced expression of cell cycle proteins cyclin D1 and cdk4 and Gqα /PLCβ1 proteins were also attenuated by EX‐527 treatment. These results suggest that the overexpression of SIRT1 contributes to the development of high BP and vascular remodeling in SHR and its inhibition improves hypertension and vascular remodeling. It may thus be suggested that inhibitors of SIRT1 may have the potential to be used as therapeutic agents in the treatment of cardiovascular complications associated with hypertension.

Support or Funding Information

Supported by grant from Canadian Institutes of Health Research (CIHR)
Prenatal Cannabinoid Exposure Mediated Cognitive Deficits in the Offspring: Elucidation of the Mechanism and Identifying Therapeutic Targets
Authors: Das Pinky Priyanka , Bloemer Jenna , Du Yifeng , Setti Sharay E. , Heslin Ryan T. , Smith Warren D. , Dityatev Alexander , Reed Miranda N. , Suppiramaniam Vishnu
Published: April 2020

Cannabinoid is currently one of the most commonly used recreational drugs in the world with an approximate user of 22.2 million. As more and more states in the US are legalizing the use of cannabinoid, the rate of cannabinoid use is increasing specially in pregnant women. The active ingredient of cannabis is delta‐9‐tetrahydrocannabinol (THC) which can cross blood placental barrier and may cause enduring alterations in synaptic circuitry of the fetal brain. The aim of our study is to investigate the mechanism of learning and memory deficits in case of prenatal cannabinoid exposure (PCE) in the adolescent offspring. An osmotic pump filled with either vehicle or the cannabinoid receptor full agonist WIN55,212‐2 (2 mg/kg body weight/day) was implanted subcutaneously in Gestational Day‐3 to deliver the drug at a constant rate until the pups were born. Open field test was performed to see the effect of PCE on locomotor activity which revealed no abnormality in the general motor activity. Contextual fear conditioning test indicated a significantly reduced freezing behavior in the PCE group compared to the vehicle exposed group. Hippocampus based spatial memory test Morris water maze showed that during the trial period, PCE animals exhibit no preference for any particular quadrant while control animals had an increased preference for the quadrant where the hidden platform was present during the training period. Electrophysiological experiments were performed on the Schaffer collateral pathway of acute hippocampal slices to measure synaptic plasticity. Impairment in both long term potentiation (LTP) and long‐term depression (LTD), which are important parameters of synaptic plasticity, were observed. These impairments were associated with downstream changes in the glutamatergic signaling. An increase in cannabinoid receptor type 1(CB1) expression followed by reduced neural cell adhesion molecule (NCAM) and its active form polysialylated NCAM (PSA‐NCAM) expression have been observed in the hippocampus of PCE animals. Interestingly, restoration of PSA‐NCAM activity restored the LTP deficits observed in PCE animals. Thus, based on the previous studies and our current data, it can be postulated that, the observed cognitive deficits in the PCE animals might be partly due to an imbalance in glutamate receptors mediated neurotransmission influenced by NCAM/PSA NCAM. Understanding this specific NCAM/PSA‐NCAM interdependent signaling cascade will lead to an identification of a specific therapeutic target to improve gestational cannabinoid exposure mediated memory deficits.
TGF‐β Signaling Regulates SLC8A3 Expression and Oxidative Stress in Mouse Developing Dopaminergic Neurons
Authors: Chleilat Enaam , Krieglstein Kerstin , Roussa Eleni
Published: April 2020

Calcium homeostasis is a cellular process required for proper cell function and survival, and maintained by the coordinated action of several transporters, among them members of the NCX family, such as SLC8A3, the isoform 3 of the Na+/Ca2+ exchanger (NCX3). Transforming growth factor beta (TGF‐β) signaling belongs to signaling cascades defining development and survival of several neuronal groups and known to regulate several channels and transporters. We have generated a conditional knockout mouse with cell‐type specific deletion of TGF‐β signalling from Engrailed 1 (En1) expressing cells, i.e. in cells derived from rhombomere r1. Subsequently, we have investigated regulation of SLC8A3, and elucidated the underlying molecular mechanisms. The results show that SLC8A3 expression is significantly downregulated in developing dopaminergic and serotonergic neurons from mutant mice and that low SLC8A3 abundance prevents the expression of the anti‐apoptotic Bcl‐xL. We also show that TGF‐β signaling regulates SLC8A3 expression via the canonical (Smad‐dependent) and MAPK signaling pathways and chromatin immunoprecipitation has revealed that TGF‐β signaling directly regulates transcription of Slc8a3 through binding of Smad4 to the Slc8a3 promoter. Moreover, expression of the lipid peroxidation markers malondialdehyde (MDA) and 4‐hydroxynonenal (4HNE) was upregulated in dopaminergic neurons lacking TGF‐β signaling. These results suggest that TGF‐β signaling regulates SLC8A3 expression in developing dopaminergic and serotonergic neurons thereby preventing oxidative stress.

Support or Funding Information

Deutsche Forschungsgemeinschaft (DFG) [Kr1477/10‐3]
Raf promotes dimerization of the Ras G‐domain
Authors: Packer Morgan Renee , Parker Jillian A. , Chung Jean K. , Li Zhen-lu , Lee Young Kwang , Guterres Hugo , Cookis Trinity , Hossain Amin , Donnelly Daniel P. , Agar Jeffrey N. , Makowski Lee , Buck Matthias , Groves Jay T. , Mattos Carla
Published: April 2020

The Ras/Raf/MEK/ERK pathway is a major regulator of cell proliferation and is highly implicated in human cancers. Ras dimerization was first proposed thirty years ago, and it is known that Ras dimerization is essential for Raf activation, yet the mechanism through which this occurs has remained elusive. Attempts to detect Ras dimers on supported membranes have led to the conclusion that Ras does not dimerize in the absence of other factors. Here we show that the Ras binding domain of Raf (Raf‐RBD) is the key element prompting Ras dimerization. A small amount of Ras/Raf‐RBD dimer is seen in solution, while the addition of Raf‐RBD to Ras on supported lipid membranes produces robust levels of dimers. On the membrane, Ras/Raf‐RBD binding and Ras dimerization appear to be concerted events that lead to a high‐affinity signaling complex. Molecular dynamics simulations of Ras and Raf‐RBD alone, in a complex, and in the dimer reveal that the proteins become increasingly connected with higher levels of allosteric networks. This is apparent in the increasing connectivity between the helices involved in dimerization, helices 4 and 5, developing more consistent communication with the phosphate binding loop in the Ras active site. These data reveal a novel mode of regulation of the mitogenic Ras/Raf/MEK/ERK pathway.

Support or Funding Information

NSF‐MCB‐1517295
Relationship Between Single Nucleotide Polymorphisms and Dysregulation of the HPA axis in Predisposition to Depression
Authors: Hennel Devon B. , Cohen Justin R. , Cohen Brian D.
Published: April 2020

There is currently a gap between what is known about genetic susceptibility to depression and how the condition presents itself. It is known that individuals with diseases that cause too much or too little of the stress hormone cortisol are typically diagnosed with depression. This led our lab and others to the hypothesis that genetic factors related to cortisol action may put someone at an increased risk for developing depression. We used allele specific PCR to study the frequency of single nucleotide polymorphisms in genes associated with the function of different components of the hypothalamic‐pituitary‐adrenocortical axis (HPA axis) to determine if dysregulation is associated with increased risk. Additionally, saliva samples were taken to quantify cortisol levels. Thus far, we have found significant relationships between the glucocorticoid receptor (GR) SNPs rs41423247 and rs56149945 and scores on the Beck Depression Inventory. The GR SNP rs10052957 and the mineralocorticoid receptor SNP rs5522 showed significant relationships with the State and Trait Anxiety Inventory. Understanding how these changes in the HPA axis relate to altered function may be an important part of determining how to better diagnose and treat depression. Through the use of genomic data, this research could provide the means to improve current treatments of depression and be the next step to personalized clinical care.
Evidence for Reactive Neurogenesis in the Forebrain of the Leopard Gecko (Eublepharis macularius)
Authors: Austin Laura E. , Graham Chloe A. , Vickaryous Matthew K.
Published: April 2020

Neurogenesis is the ability to generate new neurons from resident progenitor populations. Although best understood as a normal physiological process, in several species of teleost fish and salamanders neurogenesis is also capable of replacing neurons lost or damaged due to injury – so called reactive neurogenesis. While reactive neurogenesis does not appear to resolve brain injuries in mammals, for reptiles less is known. Here, we investigated reactive neurogenesis in the lizard Eublepharis macularius, the leopard gecko. To initiate reactive neurogenesis, we administered geckos with a single dose of the neurotoxin 3‐acetylpyridine (3AP). Using the cell death markers Fluoro‐Jade and the TUNEL assay, we determined that neuronal loss occurs within 4 days following 3AP administration. Cell death is largely restricted to the medial and dorsomedial cortices, areas of the forebrain widely accepted to be the reptilian homologs of the mammalian hippocampus and neocortex, respectively. As evidenced by a decrease in the number of cells expressing the mature neuronal marker NeuN, cell death within these regions appears to selectively target neurons. Remarkably, by 30 days following 3AP administration the medial and dorsomedial cortices appear to be structurally restored with a pattern of NeuN expression that closely resembles the uninjured brain. These data provide the first evidence that the leopard gecko is capable of reactive neurogenesis, a phenomenon otherwise rare among amniotes (reptiles and mammals).

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC), Discovery Grant 400358)
Characterization of the Transcription Termination Factor Responsible for Polarity in Archaea
Authors: Wu Wenrun
Published: April 2020

The absence of a nuclear compartment permits prokaryotic ribosomes to bind nascent RNAs to initiate translation before a transcript is completely synthesized. Transcription and translation are normally tightly coupled in prokaryotes (Archaea and Bacteria) and uncoupling translation from transcription serves as a regulatory signal, permitting premature transcription termination when translation fails or is inhibited. The resultant decrease in downstream gene expression, termed polarity, is a major regulatory pathway to ensure coordinated gene expression. While the importance and widespread use of polar repression is well documented, to date, the factor responsible for polarity in archaea cells has not been explicitly identified.

In bacteria, the transcription termination factor rho is responsible for polarity, but there are no rho homologues encoded in archaeal genomes. FttA (Factor that terminates transcription in Archaea), a recently identified archaeal termination factor, appears to potentially fulfill the requirements to direct polarity in archaea, but other factors may be responsible. Our primary objective, employing an unbiased genetic selection, is to identify the transcription termination factor(s) responsible for polar repression in Archaea.

We have established and will present our findings resultant from a three‐tiered genetic selection for spontaneous mutations that impact the efficiency of polar repression in archaeal cells. The selections were carried out in specialized strains of Thermococcus kodakarensis, a marine, hyperthermophilic archaeon with a facile genetic system and robust laboratory growth.

Briefly, genomic modifications were introduced to place a non‐essential gene with a premature translation stop codon directly upstream and within the same operon of a reporter or essential (selection) gene. In wide‐type strains, the early stop codon permits a polarity factor to recognize uncoupling of transcription and translation and terminate transcription before productive expression of the downstream essential gene. However, when the activity of a polarity factor(s) is hindered, these selective genes can be expressed at high levels. For selection, the final genes of the tryptophan and histidine synthesis pathway were chosen, constructing auxotrophic mutant strains which can only develop in the absence of tryptophan and histidine if there is some disruption in polarity‐ allowing them to become prototrophic. The spontaneous mutations resultant in reduced polar repression are easily identified through whole‐genome sequencing and we present evidence for roles of multiple factors in mediating polarity in archaeal cells.
SPARC: Deep Learning for Stomach Segmentation with Contrast Enhanced Magnetic Resonance Imaging of the Gastrointestinal Tract
Authors: Wang Xiaokai , Lu Kun-Han , Choi Minkyu , Cao Jiayue , Jaffey Deborah , Powley Terry , Liu Zhongming
Published: April 2020

Contrast‐enhanced magnetic resonance imaging of the gastrointestinal tract (gMRI) enables non‐invasive assessment of gastric emptying and motility and provides imaging‐based evaluation of pharmacological or bioelectrical treatment of gastric disorders. For both purposes, robust image processing is required to segment the gastrointestinal tract, separate its compartments, capture its dynamics, and quantify its physiology or pathology. Due to the complex anatomy and dynamics of the gastrointestinal tract, conventional image processing methods are hard to generalize across animals or species, are semi‐automatic, time‐consuming and in need of manual correction.

Here, we reported a deep learning algorithm to segment the gastrointestinal tract based on 3‐dimensional (3D) contrast‐enhanced gMRI. Specifically, we constructed a 3D U‐net with an encoder‐decoder architecture in which convolutional layers were connected in a U‐shape. The encoder took the gMRI images as its input and learned a representation of the input; the decoder used the learned representation to generate pixel‐wise classification for segmentation of the stomach and small intestines.

To train and validate the U‐net, we used gMRI images (from 54 rats) segmented with a semi‐automatic processing pipeline as described in a prior paper. We used 1,047 3D volumes from 48 rats for training the model and used 139 volumes from 6 rats for validating the model. We found that the model was able to extract meaningful features to achieve accurate pixel‐wise classification on new image sets. The segmentation results were more accurate, consistent and generalizable than were previous algorithms. Quantitative evaluation suggested that the segmentation was highly accurate for the stomach (DICE=0.982±0.011) and reasonable for the small intestines (DICE=0.918±0.042). Importantly, this algorithm significantly reduced the processing time from >2 minutes to <1 second and thus was expected to be well suited for real‐time assessment.

In conclusion, our model results in high segmentation accuracy and fast processing speed. It has the advantage over conventional methods and holds the unique promise to enable real‐time gMRI‐based assessment of gastrointestinal physiology. Future studies are desirable for using this technique to guide neuromodulation or pharmacological treatment of gastric disorders, e.g. gastroparesis.

Support or Funding Information

National Institutes of Health’s SPARC ‐ Stimulating Peripheral Activity to Relieve Conditions ‐ program (OT2OD023847)
Liver Diseases Contribute to Functional Platelet Aggregation Defects
Authors: Bacher Cassandra , Farooqui Ambar , Siddiqui Fakiha , Daravath Bharathi , Hoppensteadt Debra , Iqbal Omer , Fareed Jawed , George Magdalena , Van Thiel David
Published: April 2020

Introduction

The role of platelets in hemostasis involves their adherence to sites of vessel injury, aggregation to form plugs. Agents that physiologically activate platelets in vivo include adenosine diphosphate (ADP), epinephrine, collagen, thromboxane A2 (TxA2) and serotonin. Vascular and platelet dysfunction represent complications of hepatic disease. The purpose of this study is to investigate the effect of various agonists on platelet aggregation profile of patient with hepatic disease.

Materials and Methods

Icteric Samples from the clinical lab from Loyola University medical center were randomly selected for this study (n=25). Blood samples from the healthy donors were included to prepare platelet rich plasma (PRP) and platelet poor plasma (PPP). PRP and PPP from various donors were separated to make pools. Four different pools were made namely Normal PRP, Icteric PRP, Icteric PRP + Normal PPP 1:1, Normal PRP + Icteric PPP1:1. Agonists such as ADP, arachidonic acid (AA), epinephrine and collagen were used to activate the platelets. The final assay concentration for ADP is 20 uM, AA is 500 ug/ml, collagen is 190 ug/ml and epinephrine is 10 ug/ml.

Results

All results were compiled in terms of percent aggregation of platelets and represented as mean ± standard deviation. Normal PRP showed platelet aggregation response reaching a peak aggregation of 93.± 27.70 with ADP agonist. Icteric PRP, and two different pools did not show any significant platelet aggregation with ADP. Varying degrees of the inhibition of platelet aggregation was noted with epinephrine, collagen and arachidonic acid in comparison to normal PRP.

Discussion

On the basis of these results it is concluded that while the normal PRP showed platelet aggregation responses to various agonists’ icteric plasma, when mixed with normal PRP produced inhibition of platelet aggregation. This data suggests that the icteric plasma contains an endogenous substance which produces the inhibitory effect on agonist induced platelet aggregation.
Yeast Bax Inhibitor (Bxi1p/Ybh3p) is a calcium channel in E. coli.
Authors: Kalhorn John C. , Raffa Amanda P. , Strakosha Aleksander , Mullin James , Mello Nicholas , Austriaco Nicanor
Published: April 2020

Human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is the founding member of the evolutionary conserved TMBIM superfamily of proteins that share sequence homology within the transmembrane Bax inhibitor‐containing motif (TMBIM). Mechanistically, BI‐1/TMBIM6 and all the other mammalian TMBIM proteins appear to be involved in the maintenance of calcium homeostasis, and the crystal structure of a bacterial TMBIM protein, BsYetJ, suggests that the protein is a pH‐sensitive calcium leak. The budding yeast, Saccharomyces cerevisiae, has a single TMBIM family member (YNL305C) named Bxi1p/Ybh3p. To determine the function of Bxi1p/Ybh3p, we overexpressed Bxi1p‐EGFP in E. coli to determine if it is a calcium channel. We show that bacterial cells expressing Bxi1p‐EGFP are more permeable to calcium than controls. Thus, our data suggests that yeast Bax inhibitor (Bxi1p) is a calcium channel in E. coli, lending support to our proposal that Bxi1p is a bona fide member of the TMBIM family of proteins. Further, we use our bacterial system to show that gadolinium is an inhibitor of Bxi1p in vivo, suggesting a path forward to identifying other small‐molecular inhibitors of this clinically‐important and highly conserved superfamily of proteins. Finally, parallel experiments revealed that the human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is also a calcium channel in bacteria that can be inhibited by gadolinium.

Support or Funding Information

Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.
γ‐Tocotrienol Protects Against Mitochondrial Dysfunction, Energy Deficits, Tissue Damage, and Decreases in Renal Functions After Renal Ischemia.
Authors: Nowak Grazyna , Megyesi Judit , Che Yingni
Published: April 2020

Renal ischemia is encountered in multiple clinical settings and frequently results in acute kidney injury (AKI) due to insufficient energy production to sustain renal functions. Ischemia‐induced mitochondrial dysfunction and ATP depletion result in disruption of the primary functions of the kidney in many affected hospitalized patients who suffer AKI, but are left without an effective treatment. This study tested whether γ‐tocotrienol (GT3), a member of the vitamin E family, promotes mitochondrial function, reduces ATP deficits and tissue damage, improves renal functions, and diminishes AKI after renal ischemia/reperfusion injury in mice. Mice were treated with vehicle or GT3 (200 mg/kg) 15h before or immediately after bilateral kidney ischemia, and end points were assessed in kidneys harvested at 24h, 72h, and 168h of reperfusion. GT3 treatment prior to and after ischemia reduced decreases in mitochondrial state 3 respiration and accelerated recovery of this function after ischemia. GT3 prevented the decreases in activities of NADH:Ubiquinone Oxidoreductase (complex I) and Ubiquinol:Cytochrome c Oxidoreductase (complex III) of the mitochondrial electron transport chain regardless of whether it was administered before or after ischemia. GT3 treatment also blocked ischemia‐induced decreases in ATP synthase (FOF1‐ATPase) activity and ATP content in renal cortical tissue. Furthermore, GT3 reduced renal tissue injury (decreased renal tubular necrosis and cast formation) and accelerated morphological regeneration of renal proximal tubules after ischemia. Kidneys of mice treated with GT3 displayed morphological improvements at 72h after ischemia by exhibiting fewer necrotic proximal tubules and inflammatory cells, while demonstrating increased tubular regeneration. GT3 treatment ameliorated increases in plasma creatinine levels and accelerated recovery of renal function after ischemia. Lastly, 89% of mice receiving GT3 and 70% of those receiving vehicle survived 72h after ischemia. In conclusions, GT3 administration improves mitochondrial respiratory functions and activity of FOF1‐ATPase, and prevents ATP deficits in ischemic kidneys. This is associated with improved kidney morphology, ameliorated decreases in renal functions, and increased survival after AKI.

Support or Funding Information

This research was supported by Arkansas Science & Technology Authority; Grant 15‐B‐27.
A novel integrated PK/PD model to assess combination therapy: Determination of the synergistic antitumor efficacy between doxorubicin and sorafenib
Authors: Choi Young-Hee , Zhang Chao , Liu Zhenzhen , Tu Mei-Juan , Yu Ai-Ming
Published: April 2020

Understanding pharmacokinetic/pharmacodynamic (PK/PD) relationship is essential to identify optimal dosage regimens to achieve desired therapeutic outcomes. Existing PK/PD model for combination therapy lacks consideration of quantitative contributions from individual drugs while the “interaction factor” (y) may be arbitrarily assigned to one drug or the other. As such, distinct estimation and prediction results will be obtained, which will be confusing. In this study, we developed a novel integrated PK/PD model to characterize and predict combination therapy by considering contributions (e.g., a and β) from individual drugs (A and B, respectively), and established a new formula to define the “combination factor” (δ) concerning synergistic, additive, or antagonist effects. Doxorubicin (Dox; drug A) and sorafenib (Sor; drug B) were used as model drugs to validate this new approach. Dox (i.v.) and Sor (p.o.) PK data were obtained in BALB/c mice, and both were fit well to two‐compartment PK models. Physiological xenograft tumor growth was described by Simeoni model, and inhibition of tumor growth was attributable to the exposure to single drug (k2A×CA(t) or k2B×CB(t)), or combination (a×k2A×CA(t) + β×k2B×CB(t)). This PK/PD model was able to describe all experimental data, and reveal a greater antitumor potency for Dox (k2A = 13.8 L/mg/day) than Sor (k2B = 0.0267 L/mg/day), as well as a clear synergism (d = 1.24) for Dox plus Sor combination therapy with current dosage regimen. Interestingly, model simulation showed that increase of Sor doses for combination therapy preferably produces greater degrees of tumor suppression. Moreover, combination therapy with 2‐fold higher of tested Sor dose and half of Dox dose may lead to optimal synergistic antitumor effect. These results indicate that this new PK/PD model can be utilized to evaluate combination therapy in a definite manner.

Support or Funding Information

This study was supported by grants R01CA225958 and R01GM133888 from the National Institutes of Health
Consumption of B. longnum changes expression of miR‐652 in the brains of mice
Authors: DeVries Laura D. , Carlson Clayton D.
Published: April 2020

The purpose of this research is to investigate the relationship between the microbiota of the gastrointestinal system and relative gene expression of miRNAs and mRNAs in the brain. This research utilizes C57BL/6 mice as a control group and BALB/c mice, a strain of mice that demonstrate abnormal social and developmental characteristics, as an experimental group. The mice are divided into four groups, a control group for each mouse strain and a probiotic group for each mouse strain. The control groups are fed a bacterial media as a negative control and the experimental groups are fed Bifidobacterium longum. This bacterial species is a well‐characterized probiotic shown to change behavior and improve sociability of BALB/c mice. The mice were fed probiotic bacteria or media control daily for three weeks. To determine the effects on gene expression in the brain the mice were sacrificed and the RNA was extracted from whole brains. Both general and neurological PCR arrays were performed to determine changes in the gene expression of brain‐specific miRNAs. The results of these PCR arrays reveal that the relative gene expression of miR‐652 in the BALB/c mice is substantially more similar to the miRNA’s expression in C57BL/6 only following the B. longum probiotic treatment. qPCR was completed on miR‐652 and on Dab1, an mRNA target of this miRNA. The final goal of this study is to further understand the relationship between changes in gut microbiota and its impacts on neurological gene expression and brain function.
Depot‐Specific Response of White Adipose Tissue to MicroRNA‐21 Ablation in Polycystic Ovary Syndrome
Authors: Rezq Samar , Huffman Alexandra M. , Syed Maryam , Tiwari Suman , Basnet Jelina , Yanes Cardozo Licy L. , Romero Damian G.
Published: April 2020

Background and aim

Polycystic ovary syndrome (PCOS) is associated with obesity and insulin resistance. However, the molecular mechanisms that mediate this phenotype remain elusive. MicroRNA‐21 (miR‐21) is upregulated in PCOS and may be involved in the pathogenesis of the disease. We aim to study the role of miR‐21 in the deleterious metabolic effects associated with PCOS.

Methods

Three week‐old miR‐21 knockout (miR21KO) or wild type (WT) female mice were treated with DHT (8 mg/silastic tube) or vehicle for 90 days (n=12/grp). Body composition was measured by EchoMRI. Energy expenditure (EE), oxygen consumption (VO2), and carbon dioxide production (VCO2) were measured by indirect calorimetry. Glucose homeostasis was measured by oral glucose tolerance test (OGTT). Gene expression was quantified by RT‐qPCR in subcutaneous (SC), retroperitoneal (RP) and mesenteric (M) white adipose tissue (WAT). Adipocyte size was evaluated by histological analysis.

Results

DHT increased body weight (25.07 ± 0.52 vs 21.79 ± 0.47 g, p<0.05), fat mass (4.60 ± 0.46 vs 1.98 ± 0.12 g, p<0.05) and adipocyte size (944 ±12 vs 513 ± 2 μm2, p<0.05), impaired OGTT (29.70 ± 2.21 vs 22.33 ± 1.06 AUC, p<0.05), and didn’t significantly change EE, VO2 or VCO2. Insulin signaling markers Irs1 and Slc2a4 expression was downregulated by DHT in all 3 WATs while Akt2 expression was upregulated. Adipogenesis markers Pparg and Adipoq were upregulated by DHT exclusively in the SC WAT. Inflammation marker Ccl2 was upregulated in all three WATs while Ikbkb was only upregulated in M WAT. Extracellular matrix (ECM) remodeling makers Mmp9 and Timp1 were upregulated by DHT only in RP and M WATs. Immune cells infiltration markers CD14, CD68, Itgam, Itgax, and CD86 were upregulated by DHT only in RP and M WATs.

DHT‐treated miR21KO mice showed attenuated increase in body weight (23.84 ± 0.99 vs 25.07 ± 0.52 g, p<0.05) with no change in fat mass or OGTT. Additionally, DHT‐treated miR21KO mice showed a trend to reduced EE, VO2 and VCO2 compared with DHT‐treated WT. Gene expression data showed lower expression of the insulin signaling marker Akt2, higher expression of ECM remodeling markers (Mmp9 and Timp1) and an exacerbated inflammatory and immune responses in M (Il‐18 and CD14) and SC (Il‐18 and CD86) WATs. Moreover, DHT‐treated miR21KO mice had exacerbated adipocyte hypertrophy compared with DHT‐treated WT controls (1,408 ± 16 vs 944 ±12 μm2, p<0.05).

Conclusion and significance

These findings suggest that adipose tissue miR‐21 may have a protective role in PCOS and ameliorate some of the DHT‐mediated molecular changes observed in WAT in a fat depot‐specific manner. WAT‐specific modulation of miR‐21 levels could be a novel therapeutic approach for PCOS treatment.

Support or Funding Information

Supported by National Institutes of Health grants P20 GM‐121334 (DGR and LLYC) and R21 DK‐113500 (DGR).
Beta‐cell‐specific Loss of the Inhibitory G protein, Gαz, has Sex‐dependent Effects on Development and Pathophysiology of Type 1 Diabetes
Authors: Peter Darby , Fenske Rachel , Wienkes Haley , Reuter Austin , Carbajal Kathryn , Kimple Michelle E.
Published: April 2020

The mechanisms that underlie the beta‐cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our lab has discovered a role for the unique inhibitory G‐protein, Gαz, in beta‐cell function, survival, and replication in both normal conditions and those of pathological stress. Gαz, when acting as a canonical inhibitory G‐protein, reduces production of cAMP through inhibition of adenylate cyclase. Cyclic AMP acts as essential player in the amplification of glucose‐stimulated insulin secretion, while also mediating pathways that regulate beta‐cell death and replication. Previous work in our lab has shown that non‐obese diabetic (NOD) mice with global loss of Gαz are protected from developing T1D‐like hyperglycemia through increased beta‐cell function, increased beta‐cell replication, and decreased beta‐cell death. In order to determine if Gαz in the beta‐cell was solely responsible for the protected phenotype, we generated a beta‐cell‐specific Gαzknockout mouse by breeding GzFlox/Flox mice with Rat Insulin Promoter (RIP) driven CreHerrmice, on the NOD background for more than 10 generations. Experimental mice were sNP genotyped to ensure preservation of NOD loci. Weekly random‐fed blood glucose measurements were taken beginning at weaning. Loss of beta‐cell Gαz(GαzβKO), only provided a modest protection against diabetes development;
69% of Gαz
βKO
females remained euglycemic over the first 30 weeks of life compared to 59% in Cre+ NOD controls. Consistent with this phenotype, neither pancreases from Gαzβ‐cell‐KO males nor females had increased beta‐cell fractional area as compared to Cre+ controls. Overall, both euglycemic Gαzβ‐cell‐KO males and females had improved glucose tolerance at 12 weeks of age, with this effect persisting at 28 weeks of age only in females. Yet, the mean insulitis score at 28 weeks‐of‐age was dramatically reduced in GαzβKO males only, and this score was strongly correlated with 28‐week glucose tolerance test AUC. The effects of Gαzβ‐cell‐KO on plasma insulin and ex vivo islet insulin secretion are also differentially regulated by sex: effects that appear to correlate with baseline expression of the constitutively‐active splice variant of the Gαz coupled EP3 receptor. Finally, changes in intra‐islet hormone production and secretion in full‐body vs. beta‐cell‐specific Gαz‐KO mice provide a potential mechanism for the incomplete protection from hyperglycemia in Gαzβ‐cell‐KO mice. Overall, our results show a divergent, sex‐dependent role for beta‐cell Gαz in T1D pathophysiology and implicate Gαz as a hub of intra‐islet cell‐cell signaling.
Patent Foramen Ovale Channel Morphometric Characteristics as Risk Factors for Cryptogenic Stroke
Authors: Holda Jakub Piotr , Bugala Kamil , Krawczyk-Ozog Agata , Koziej Mateusz , Sorysz Danuta , Dudek Dariusz , Holda Mateusz Krystian
Published: April 2020

It is still disputable whether the specific morphological properties of the patent foramen ovale (PFO) may contribute to the occurrence of stroke. Therefore, the aim of this study was to evaluate the differences in the morphometric and functional features of the PFO channel in cryptogenic stroke and non‐stroke patients. A total of 106 consecutive patients with cryptogenic stroke and 93 non‐stroke control patients with diagnosed PFO (by transesophageal echocardiography) were analyzed. Multivariate regression logistic analyses indicated PFO channel length change (OR: 2.50 [95%Cl:1.75–3.55], p<0.001), the PFO length/height ratio during the Valsalva maneuver (OR: 0.75 [95%Cl:0.60–0.95], p=0.015), septum primum thickness (OR: 0.34 [95%Cl:0.14–0.80], p=0.013), septum secundum height (OR: 0.91 [95%Cl:0.84–0.98], p=0.013), the presence of an atrial septal aneurysm (OR: 3.38 [95%Cl:1.27–8.97], p=0.014), and large shunt (OR: 2.49 [95%Cl:1.13–5.46], p=0.022) as PFO‐related risk factors for stroke. A MorPFO score was developed (AUC=0.816, SD=0.031), where six factors were included: PFO channel length reduction (≥ 23%) (3 pts), low length/height ratio during the Valsalva maneuver (≤ 2.1) (1 pt), thin septum primum (≤ 1.5mm) (1 pt), short septum secundum (< 5.6mm) (1 pt), presence of an atrial septal aneurysm (1 pt), and large right‐to‐left shunt (1 pt). Patients with scores of 0–2 pts had low‐risk PFO channels, 3–4 pts intermediate‐risk PFO channels, and 5–8 points high‐risk PFO channels. In conclusion transesophageal echocardiography can be used to determine the PFO‐related risk of stroke. MorPFO score may help to identify high‐stroke‐risk PFO channels.
ERK3 Interacts with DGKζ and Alters Signaling Lipids in Lung Cancer Cells
Authors: Myers Amanda , Bedi Shimpi , Popp Katherine , Aldharee Hitham , Long Weiwen
Published: April 2020

Extracellular‐Regulated Kinase 3 (ERK3) promotes migration and invasion in many cancers, including lung cancer. However, the mechanisms by which this atypical Mitogen‐Activated Protein Kinase (MAPK) induces these effects remain poorly understood. The p21‐activated kinase (PAK)‐Rac1 complex, which can be activated by phosphatidic acid, is associated with cell migration and has been shown to activate ERK3. Here, we show ERK3 also binds to phosphatidic acid and treatment of cells with phosphatidic acid results in an increase in ERK3 phosphorylation. Intriguingly, we have also found ERK3 modulates Rac1 activity and PAK phosphorylation, suggesting a positive feedback loop. In ERK3 knockdown cells, total diacylglycerol level was elevated while total phosphatidic acid level was decreased. In a yeast two‐hybrid assay, ERK3 was shown to interact with Diacylglycerol Kinase ζ (DGKζ), an enzyme which converts diacylglycerol to phosphatidic acid. This interaction has been confirmed by co‐immunoprecipitation and appears to occur through the C34 domain of ERK3. By immunofluorescence, these proteins were shown to co‐localize in cells with ERK3 enhancing cytoplasmic localization of DGKζ. Together, these data suggest ERK3 may regulate DGKζ to facilitate phosphatidic acid production, thereby activating the Rac1‐PAK complex and inducing cell migration.

Support or Funding Information

NCI 1R01 CA193264‐01Wright State University start‐up fund