bluuebunny/crossref_metadata_2025 huggingface dataset:
miR‐106b‐25 Dysregulation in Complex Regional Pain Syndrome Contributes to T Cell Dysfunction
Authors: Wickman Jason , Shenoda Botros , Van Duyne Rachel , Klase Zachary , Ajit Seena
Published: May 2022

Background

Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder with no effective treatments. Growing evidence implicates aberrant immune regulation in the skin and T cell dysfunction in CRPS pathology. MicroRNA (miRNA) show promise in identifying biomarkers and disease mechanisms. miRNA cluster miR‐106b‐25 is dysregulated in CRPS patient whole blood and serum derived small extracellular vesicles (sEVs) compared to healthy controls. miR‐106b‐25 members are predicted to target several immune genes related to T cell function including CD69. We hypothesize that miR‐106b‐25 cluster plays a role in T‐cell dysfunction by regulating members of CD69 signaling pathway. Here we examine miR‐106b‐25 mediated signaling in sEVs, whole blood, and skin from the tibia fracture model (TFM) of CRPS.

Methods

Mouse TFM was generated, and pain hypersensitivity was confirmed by von Frey and dynamic weight bearing. Serum, whole blood, and hind limb skin were collected from TFM and control mice. sEVs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. miRNA from sEVs were assessed through HTG EdgeSeq. T cells were isolated from single cell suspensions of hind limb skin. Whole blood samples were obtained from CRPS patients or healthy controls. miRNA and gene expression changes in blood and T cells were assessed by qPCR. Luciferase reporter assay was used to confirm miR‐25 binding to CD69. Jurkat cells were transfected with miR‐25 mimic or inhibitor to assess regulation of gene expression by qPCR and flow cytometry. T cell phenotypes and expression of CD69 and other residency markers were determined by flow cytometry.

Results

Characterization of sEVs showed no difference between TFM and control mice. miRNA analysis of TFM sEVs confirmed dysregulation of 30 miRNA previously associated with CRPS patients including miR‐106b‐25. There was an inverse correlation of miR‐25 and CD69 in blood samples from CRPS patients compared to controls. Reporter assay confirmed miR‐25 binding to CD69. miR‐25 nucleofection in Jurkat cells downregulated CD69 as measured by flow cytometry. Analysis of miR‐106b‐25 and target gene expression in TFM whole blood and skin T cells and T cell flow cytometry experiments are ongoing.

Conclusions

miRNA signatures in CRPS patients and TFM mice show common alterations including miR‐106b‐25. miR‐25 can regulate CD69 expression in vitro. miR‐25 may contribute to localized T cell dysfunction in the skin in CRPS.
Decreasing Phosphatidylcholine on the Lipid Droplet Surface Selectively Recruits Proteins with Amphipathic Alpha Helices
Authors: Long Alex , Paetz Olivia , Renier Tim , Vuong Sunny , Kharbanda Kusum , Listenberger Laura
Published: May 2022

Lipid droplets are ubiquitous, intracellular fat‐storage organelles. Each lipid droplet is surrounded by a phospholipid monolayer primarily composed of two phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have previously shown that lipid droplets accumulating in a rat model of alcoholic fatty liver disease have a decreased PC:PE ratio compared to healthy control animals. Moreover, altering the phospholipid composition of the lipid droplet surface changes the amount of specific lipid droplet proteins, suggesting a mechanism for fat accumulation in alcoholic fatty liver disease. Using subcellular fractionation and immunoblotting, we investigated the abundance and lipid droplet association of multiple proteins in AML12 cells cultured under conditions that alter the PC:PE ratio and mimic the lipid droplet structure in alcoholic fatty liver disease. With this approach, we measured the relative binding of proteins that interact with lipid droplets through a variety of structural motifs, including amphipathic alpha helices, lipid anchors, and hairpin loops. Our results indicate that the amphipathic alpha helix‐containing proteins perilipin 1, perilipin 2, and CIDE‐C are significantly more likely to associate with lipid droplets with decreased PC:PE ratios. This same change in PC:PE levels did not change association of rab 18, a protein that interacts with lipid droplets through a lipid anchor, and decreased lipid droplet association of a protein (DGAT2) that trafficks to lipid droplets as they bud from the ER. Our results support a model where increased binding of the amphipathic alpha helical domains of some lipid droplet proteins contributes to lipid droplet overaccumulation in alcoholic fatty liver disease. This work builds on our understanding of alcoholic fatty liver disease and the mechanisms that facilitate protein binding to lipid droplets.
Endogenous Pink1 Regulates Dendritic Architecture and Spinogenesis
Authors: Chu Charleen T. , Fricklas Gabriella , Nigam Aparna , Lizama Britney N. , Wills Zachary P. , Johnson Jon W. , Otero P. Anthony
Published: May 2022

Mutations in the gene for PTEN‐induced kinase 1 (PINK1) are linked to recessive Parkinson’s disease (PD) and PD with dementia (PDD). We previously discovered that overexpression of PINK1 promotes dendritic complexity through interaction with valosin containing protein (VCP) and activation of protein kinase A (PKA). Furthermore, treatment with a small molecule inhibitor of endogenous PINK1 degradation confers protection against the severe dendritic retraction elicited by 1‐methyl‐4‐phenylpyridinium (MPP+), a neurotoxin that causes parkinsonism. The objective of this study was to delineate the role of endogenous PINK1 in regulating neuronal structure and function. We hypothesized that PINK1‐deficient cortical neurons would show simplified dendritic architecture resulting in reduced synaptic input. Primary embryonic neuron cultures from Pink1‐/‐ and control mice were studied using fluorescence microscopy and electrophysiology, with ongoing studies of human PINK1‐mutated iPSC‐derived neurons. We found that loss of PINK1 expression results in diminished dendritic complexity, reduced spine density, and altered electrophysiological function. Moreover, similar changes in dendritic architecture were observed in vivo using Golgi silver‐stained mouse brain sections. We conclude that PINK1 plays a previously underappreciated role in regulating neuronal structure. Ongoing studies are aimed at understanding the signaling pathways involved, and whether these changes are developmental and/or reflect decreased resilience against neurodegenerative stressors.
Elucidating the mechanism of potentiated Hsp104 NBD2 variants against proteotoxicity
Authors: Tabassum Zarin , Gao Allan , Tariq Amber , Lin JiaBei , Shorter James
Published: May 2022

A hallmark of several neurodegenerative disorders is aberrant protein aggregation. For example, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) involve cytoplasmic aggregation of the RNA‐binding proteins TDP‐43 or FUS. Parkinson’s Disease (PD) involves the formation of toxic, cytoplasmic a‐synuclein oligomers. Resolving these protein aggregates, thus restoring them to their functional forms, is a key therapeutic goal in these diseases. Hsp104 is a hexameric AAA+ disaggregase originally found in yeast that has been found to dissolve infectious amyloid fibers (prions), suggesting that Hsp104 can be targeted to eliminate toxic aggregates in neurodegenerative diseases. However, the activity of Hsp104 against these substrates has limits, especially since TDP‐43, FUS, and a‐synuclein are not natural Hsp104 substrates. Potentiating mutants of Hsp104 can be therapeutically beneficial in that they rescue TDP‐43, FUS, and a‐synuclein aggregation and toxicity in yeast, whereas wild‐type Hsp104 is ineffective under the same conditions. Point mutations in the middle domain (MD) of Hsp104 strongly potentiate its activity but display off‐target toxicity. Nucleotide‐binding domain, NBD1 and NBD2 mutants safely potentiate Hsp104 to counter proteotoxicity in ALS‐FTD and PD without significant off‐target effects. Interestingly, several potentiating NBD1 variants have been tested but such mutations have been sparse in NBD2. NBD2 is critical for hexamerization and the site N566 is the only NBD2 site so far that can be potentiated. We suggest that NBD1 and NBD2 have different mechanisms in their ability to potentiate Hsp104 and set out to characterize the effects of N566 mutants.
Salty Enzymes: Protein Splicing Dependence on Salt Concentrations
Authors: Nguyen Thuy T. , Wall Diana A. , Mills Kenneth V.
Published: May 2022

Halophiles are organisms that thrive in very high salt concentrations. Enzymes from halophiles tend to have a negative surface charge and a low isoelectric point to allow for stable folding. We are interested in converting a halophilic enzyme that requires high salt for activity and proper folding into one that is functional at more moderate salt concentrations. Our model enzyme is DNA polymerase II from Halobacterium salinarum, which contains an intein. Inteins are intervening peptide sequences within proteins that are spliced out from their flanking polypeptides, or exteins, which then ligate to form a functional protein. We have induced mutations to the intein that facilitate a splicing reaction at lower salt concentrations. Changing negatively charged amino acids on the surface of the intein to corresponding neutral or positive charged residues observed in homologous inteins in non‐halophilic extremophiles induces the splicing reaction at lower salt concentrations, especially when coupled with a change of the C‐terminal Gln to the more highly conserved Asn. As few as two residue changes can dramatically alter the salt‐dependence of the activity, showing that subtle changes to surface residues can result in conditional folding and activity.
Deletion of MyD88 in T‐Cells Worsens Cardiac Pathology Through Enhanced T‐Cell Survival and Induction of Cardiac Fibrosis
Authors: Bayer Abraham L. , Smolgovsky Sandra , Ngwenyama Njabulo , Aronovitz Mark , Kaur Kuljeet , Alcaide Pilar
Published: May 2022

Heart failure (HF) is a leading cause of death worldwide, associated with cardiac and systemic inflammation. However, no anti‐inflammatory therapies have shown success. Damage associated molecular patterns (DAMPs) released in the heart activate myeloid cells through the adaptor “Myeloid differentiation primary response 88” (MyD88). This signaling cascade promotes cardiac antigen presentation to activate helper T‐cells. Once activated, T‐cells then infiltrate the heart and bind to VCAM‐1 on cardiac fibroblasts, (CFB), transforming them to myofibroblasts which coordinate cardiac fibrosis. MyD88 is also expressed in T‐cells, yet whether it is pro or anti‐inflammatory is less explored, and evidence suggests is context dependent. To investigate the role of T‐cell MyD88 in HF, we reconstituted Tcra‐/‐ mice, normally protected from HF induced by transaortic constriction (TAC), with WT or Myd88‐/‐ Type 1 helper T‐cells (Th1) in the onset of TAC. Surprisingly, we found that mice receiving Myd88‐/‐ Th1 cells showed increased cardiac T‐cells and fibrosis compared to mice receiving WT cells. We hypothesized that T‐cell MyD88 regulates cardiac T‐cell recruitment and survival, and as a result subsequent cardiac fibrosis. We found that Myd88‐/‐ Th1 cells exhibited increased survival in vitro determined by real time imaging of propidium iodide incorporation, as well as in vivo by performing competitive survival studies in which Tcra‐/‐ mice received equal WT CD45.1 Th1 cells and Myd88‐/‐ CD45.2 Th1 cells. Myd88‐/‐ Th1 cells also induced higher CFB α‐smooth muscle actin staining when cultured together, as well as bound more frequently to CFB and immobilized VCAM‐1. Together these data demonstrate that MyD88 limits T‐cell mediated cardiac fibrosis by regulating T‐cell survival, as well as by dampening T‐cell/fibroblast interactions. We identify novel role for T‐cell MyD88 in cardiac inflammation that may be modulated in HF.
A Tale of Two Semesters: Flipped Biochemistry Curriculum in the Time of COVID
Authors: Miller Keith R.
Published: May 2022

Social distancing and online education demands have had an immediate impact on the delivery of biochemistry curriculum to meet the student, faculty, and institutional needs. During the Fall 2020 and 2021 semesters, one model of hybrid online flipped learning was applied to an introduction to biochemistry lecture course. Normal classroom lectures were turned into voice‐over PowerPoint videos. Students were expected to have watched the lecture videos before coming to class. After gaining experience with the flipped classroom and social distancing model, student learning shifted from passive to active engagement by applying their knowledge during in person classroom activities including question response, board problems, 3D molecular modeling kits, and think pair share exercises. Students self‐reported that they liked the flexibility of watching the lecture videos before class and the ability to rewind and pause the videos. Students also commented in student evaluations that the in‐class activities and practice problems were helpful in applying their understanding. Compared to the previous six years, the withdrawal rate heavily decreased in Fall 2020 using this flipped learning style decreasing from a usual 30% to 10%. In addition, no students earned a D or an F for Fall 2020 and Fall 2021 in the introduction to biochemistry course compared to previous years where 10% of the class normally would be in this grade range. This preliminary assessment of the Fall 2020/2021 grade distributions and withdrawal number supports that flipped learning and focus on application of the lecture material in class can be a powerful combination for improved understanding. This is especially relevant to the threshold concepts identified by the ASBMB. Out of class recorded lectures can introduce topics and in class time can be spent focusing on the more difficult concepts and understanding in biochemistry.
From Alignment Laser to 3D Structures: Determining Structures of GPCRs in a Compact X‐ray Light Source
Authors: Diaz‐Figueroa Gabriela , Messerschimdt Marc , Woo‐Kim Jong , Olson Tien
Published: May 2022

The Compact X‐ray Light Source (CXLS) at Arizona State University produces diffraction patterns with intense X‐ray by Inverse Compton Scattering (ICS). In ICS, a photon acquires energy from an electron to produce an X‐ray. With this system, we can study G‐Protein Coupled Receptors (GPCRs), which are peripheral membrane proteins targeted by 60% of FDA approved drugs. When GPCRs are activated, a series of reaction can occur. For example, opening and closing of ion channels, activation or deactivation of enzymes, and protein hydrolysis. The structural characterization of GPCRs can lead to optimization of drug development, by assessing a better targeting mechanism that will lead to fewer side‐effects and better drug biding. We conducted a laser alignment in the CXLS using a YAG screen, which is a fluorescent screen that helps determine the size and intensity of the beam. Furthermore, we provide the crystallization process of GPCRs using Lipidic Cubic Phase (LCP) and describe its potential for structure determination using the CXLS and CXFEL.
Gq Signaling in the Placental Syncytiotrophoblast Layer During Preeclampsia
Authors: Opichka Megan A. , Livergood M. C. , Sigmund Curt D. , McIntosh Jennifer J. , Grobe Justin L.
Published: May 2022

Preeclampsia is a pregnancy‐associated cardiovascular disorder characterized by hypertension and proteinuria after 20 weeks of gestation. Generalized cellular ‘stress’ (oxidative, mitochondrial, etc.) within the syncytiotrophoblast (STB) layer of placenta is theorized to drive its pathogenesis. Hormones implicated in preeclampsia such as angiotensin, endothelin, and vasopressin signal via receptors that typically couple to the Gq second‐messenger cascade, and Regulator of G protein Signaling‐2 (RGS2) buffers this signaling. We have published that RGS2 expression is decreased in human preeclamptic placenta, and reducing RGS2 expression in placenta is sufficient to cause development of key features of this disorder in mice. Unpublished in situ hybridization data indicate that in both humans and mice, RGS2 is present in the STB layer – and that RGS2 expression in the STB layer is reduced during preeclampsia in humans. Collectively, these findings lead us to generally hypothesize that excess Gq signaling specifically within the STB layer, whether due to excessive hormonal activation or loss of RGS2‐mediated buffering, causes STB‐Stress and thereby contributes to the pathogenesis of preeclampsia. Therefore, the objective of this study was to test whether artificial, exogenous activation of Gq signaling only within STB cells is sufficient to induce placental and maternal phenotypes of preeclampsia in mice.CAG‐FLEX‐hM3Dq+/+ (Gαq DREADD, Jax 026943) or C57BL/6J dams were bred with Gcm1‐Cre+/‐ sires (Jax 026625) or non‐transgenic littermates to elicit STB‐restricted hM3Dqexpression in ≍50% or 0% of placentas. The synthetic DREADD ligand clozapine N‐oxide (CNO) or saline vehicle was administered (2 mg/kg/d, ip) mid‐gestation (GD 12.5‐14.5). Placental and maternal phenotypes were compared between Cre– and Cre+ placentas within a dam, CNO‐ vs saline‐injected double‐transgenic dams, and double‐ vs single/non‐transgenic dams treated with CNO. Activation of Gq in the STB layer caused maternal proteinuria (n=4‐9, 49±6 mg/day vs 28±4 mg/day; p=0.01). Labyrinth vascularization, assessed by percentage of CD31+ area, was reduced with Gq activation (n=5, Cre+ 21±1%, Cre– 27±2%; p=0.01), while other regions remained unaffected (Decidua: Cre+ 14±4%, Cre– 14±3%; Junctional Zone: Cre+ 11±4%, Cre– 12±3%). Possible reductions in placental mass (n=5 Cre+ 0.12±0.01g, Cre– 0.15±0.01g; p=0.06), and placental vascular endothelial growth factor protein levels (n=6‐7 117±67 ng/g vs 151±12 ng/g; p=0.06) were noted, but no obvious trend in fetal growth restriction was observed (n=5 Cre+ 0.21±0.06g, Cre– 0.29±0.04g). These data support the pathological significance of excess Gq signaling specifically within the STB layer. Ongoing studies are aimed at further characterizing maternal phenotypes (blood pressure, circulating factors) in these models, and the synergistic effects of simultaneous STB‐specific deletion of RGS2 upon sensitivity to Gq stimulation in this cell type.
The Visible Ape Project: a free, comprehensive, web‐based anatomical atlas designed to raise public awareness about apes
Authors: Barger Nicole , Martín José S. , Boyle Eve K. , Richmond Marli , Diogo Rui
Published: May 2022

Based at a Howard University, the Visible Ape Project (www.visibleapeproject.com) aims to provide free, publicly accessible anatomical educational resources suitable for classrooms from K‐12 and beyond. It is designed to comprehensively illustrate anatomy and anatomical variation within and between extant ape species. It contains photographs, MRI, CT scans, and 3D models to explore homologies and variations in soft and hard tissues across hylobatids, orangutans, gorillas, chimpanzees, bonobos, and humans. These include detailed, labeled 3D artist renderings of the musculoskeletal system as well as 3D models of the skeletal and central nervous system based on radiological imaging of apes. They can be manipulated or downloaded and 3D printed to explore anatomy interactively, promoting active learning. Additional educational materials are available, including information about non‐human ape species, lesson plans, and a glossary of evolutionary and anatomical terms.

The primary goals of the project are to promote human and ape evolutionary anatomy, broadly, and to integrate and disseminate to communities underrepresented in anthropology and evolutionary biology, specifically. Accordingly, the VAP incorporates outreach activities to disseminate science and promote awareness of apes, forming partnerships with veterinarians and conservationists in Africa and Asia. Additionally, we include many resources in Spanish. The site has been accessed by individuals from many countries across the globe, including many Spanish speaking countries, and nearly all US states. In this presentation, we will introduce the website to illustrate how this accessible, evolving resource can support research and education in human and comparative anatomy.
Investigating the Therapeutic Potential of Cdk8 Small Molecule Inhibitors to Prevent Pathological Cardiac Remodeling and Heart Failure
Authors: Oppman Alexis M. , Gardner Richard N. , Martins Ines , Minerath Rachel A. , Hall Duane D. , Grueter Chad E.
Published: May 2022

Pathological cardiac hypertrophy represents a major risk factor for heart failure (HF). Cardiac hypertrophic responses are coordinated in part through altered transcriptional dynamics involving chromatin structure and accessibility, transcription factor (TF) activation, and enhancer utilization. Mediator is a multiprotein complex that integrates signal‐dependent TFs with basal transcriptional machinery to regulate downstream gene expression. Cyclin‐dependent kinase 8 (Cdk8) is a Mediator kinase demonstrated to have a complex role in transcription regulation, driving both transcription activation and inhibition in context‐specific ways. Previous studies have demonstrated that Cdk8 protein expression is increased in human and mouse HF. Cardiomyocyte‐specific Cdk8 overexpression leads to eccentric cardiac hypertrophy consistent with HF, suggesting that Cdk8 activity may play a significant role in the progression of HF. To investigate the therapeutic potential in regulating Cdk8 activity, we used Cdk8 selective inhibitors SenexinA and CCT251545 in an in vitro neonatal rat cardiomyocyte hypertrophy model, resulting in altered regulation of hypertrophic transcriptional programs and blunted cardiomyocyte hypertrophy. To further assess Cdk8 kinase activity in regulating hypertrophic responses, Cdk8 inhibitor CCT251545 was used in an in vivo angiotensin II‐induced hypertensive mouse model. It was hypothesized that Cdk8 inhibition by CCT251545 in vivo would lead to reduced hypertrophic responses. Results indicate that CCT251545 is effective at inhibiting Cdk8 activity, leading to blunted hypertrophic responses with altered gene expression. These studies demonstrate a role for Cdk8 activity in transcriptional regulation of genes associated with pathological cardiac hypertrophy, which can ultimately lead to heart failure.
Differential Expression of Cardiac Fat Long Non‐Coding RNAs in Coronary Artery Disease
Authors: Flinn Brendin , Burgess Tristan , Chowdhury Nepal , Gress Todd , Santanam Nalini
Published: May 2022

Coronary artery disease (CAD) is characterized by the buildup of atherosclerotic plaques within the coronary arteries, leading to serious complications. CAD affects 7.2% and 4.2% of males and females, respectively. Epicardial adipose tissue (EAT) interacts with the heart and vasculature, but its mechanistic role in CAD is not completely understood. A more comprehensive view of non‐coding RNA (ncRNA) expression in EAT is needed to better characterize its role in CAD. To conduct this characterization, in an IRB‐approved investigation, we isolated the microRNA (miRNA), long non‐coding RNA (lncRNA), and messenger RNA (mRNA) from the EAT and subcutaneous adipose tissue (SAT) of male and female CAD patients undergoing coronary artery bypass graft as well as from non‐CAD patients undergoing aortic valve replacement. Differential expression of these RNAs between tissues, disease status, and sex was determined using next generation sequencing (mRNA) and microarrays (miRNA and lncRNA). Functional and pathological associations of the differentially expressed RNAs were determined using Ingenuity Pathway Analysis, GO, KEGG, and TAM 2.0. DIANA‐LncBase was used to identify experimentally verified miRNA‐lncRNA interactions. NGS showed differential expression of mRNAs associated with cardiovascular function in EAT but not in SAT. miRNA microarray showed that ncRNA expression within the EAT of CAD patients is distinct from its expression in non‐CAD patients. 14 miRNAs were differentially expressed in EAT from patients with CAD compared to those without CAD. Similarly, lncRNA RT2 PCR array showed a downregulation of three lncRNAs: IPW, TINCR, and linc00853, between the EAT of CAD compared to non‐CAD male patients but not in the EAT of female patients. DIANA‐LncBase showed high confidence pairings of IPW with three miRNAs that were upregulated in the EAT of patients with CAD: hsa‐miR‐24‐3p, hsa‐miR‐26b‐5p, and hsa‐29c‐3p. This suggests that IPW may serve as a ‘sponge’ for these three miRNAs in the EAT of patients without CAD, but its downregulation in CAD reduces that interaction. TAM 2.0 showed these three miRNAs to be associated with functional pathways such as vascular inflammation, apoptosis, adipogenesis, etc. Overall, these findings both reveal potential ncRNA biomarkers for CAD and elucidates possible pathological roles of EAT in CAD mediated by ncRNAs.
Matrix Metalloproteinase‐9 Promotes Membrane Protrusive Activity in Ovarian Cancer Cells
Authors: Cook Nelson A. , Finnegan Katie D. , Lines Caleb L. , Reyes Sam J. , Ellerbroek Shawn M.
Published: May 2022

Matrix Metalloproteinase‐9 (MMP9) is an extracellular endopeptidase associated with ovarian cancer development and progression. To further understand MMP9 contributions to metastatic behavior of an established ovarian cancer cell line (OVCA433), we utilized CRISPR to generate seven cell clones null for MMP9 expression. Analysis of the pooled clones revealed MMP9‐null cells exhibited identical phenotype and growth compared to parental OVCA 433 cells. Cell adhesion and transwell migration towards type I collagen was also not impacted by MMP9 loss. MMP9‐null cells were significantly delayed, however, in EGF‐driven non‐directional filamentous actin‐based membrane protrusions on a variety of matrix substrates. Further, over or re‐expression of MMP9 in OVCA433 or MMP9‐null cells, respectively, was sufficient to drive both lamellipodial and filopodial membrane protrusions on glass. These results indicate MMP9 directly contributes to ovarian cancer cell protrusive activity.
The Hypertension Induced by Clozapine is Related with Increased Renal Sodium Transporters in Mice
Authors: Ren Zhiyun , Wang Weiwan , Zhang Chenyang , Ye Min , Wang Xiaoyan
Published: May 2022

Introduction

Hypertension is caused by multi‐factors, and the pathogenesis is linked to, generally, the increases in the protein expressions and functions of renal sodium transport proteins along the nephron. One of the major side effects of clozapine (common antipsychotic drug) is hypertension. The mechanism is unknown.

Hypothesis

the pathogenesis of the clozapine‐induced hypertension is associated with increases of renal sodium transport proteins.

Methods

we examined the blood pressures (BP) and protein expressions of major sodium transport proteins profiled along the renal tubules in the C57Bl6/J male mice (9 weeks old, n=4‐5/group) treated with clozapine at three doses of 5,10 and 20 mg/ kg for 1 week via intraperitoneal injection.

Result

blood pressures (tail‐cuff, BP98A, softron, Japan) were higher in clozapine at 20mg/kg/day than vehicle (SBP: 130±1.64 vs 109±1.39, DBP: 87 ±4.12 vs 69±2.10 mmHg, n=5/group) and not altered at 5 & 10 mg/kg/day. There were no differences in BW, serum/urine creatinine and electrolytes except for a slight increase in serum sodium at dose of 20 mg/kg (152.8±0.71 vs 150.6±0.40, mmol/L). In renal homogenates, total protein abundance of NaPi2 (western blotting,199±26% of vehicle) NKCC2(184±20), NCC(213±14),and ENaC‐α(217±47),were higher at 20 mg/kg than vehicle. No increases were found in protein expressions of NHE3, αNKA, ENaC‐β and ENaC‐γ. The expressions of renin, AT1R and ACE at 20mg/kg were similar between groups while ACE2 was decreased (42±10). No increases were found in protein expressions of pNKCC2 in 5&10mg/kg(100±50,45±15); NCC was increased at 5 mg/kg (175±9); α NKA was increased in 5mg/kg (216±5) and 10mg/kg (163±2) groups.

In plasma membrane enriched fraction (17k), NKCC2(204±43) and ENaC‐α(381±28)were higher at 20 mg/kg than vehicle. In intracellular membrane enriched fraction (200k), NKCC2(521±127) was higher than vehicle. Phosphorylated NKCC2 was increased in renal homogenates(331±35),17k(427±87) 200k(521±127) but WNK4 was not changed.

Additional mice were treated with clozapine at 20mg/kg/day via osmotic mini‐pump for 1 week (mini‐pump) or administrated by intraperitoneal injection for 2 weeks (2wks). Increases of renal NKCC2 (mini‐pump:180±21, 2wks:153±18) and NCC (mini‐pump:171±22, 2 wks:160±23) were also found in those models.

Conclusions

increases of renal sodium transport proteins, mainly pNKCC2 and NKCC2, contribute to the dose‐related increase of BP in mice treated with clozapine and may play an important role in the pathogenesis of the clozapine‐induced hypertension.
Erythrocyte Fatty Acids and Association with Cardiometabolic Risk Factors in Free‐Living Elderly
Authors: Jehi Tony , Sabate Joan , Bitok Edward , Rajaram Sujatha
Published: May 2022

Aging significantly increases the incidence of various cardiometabolic disease risk factors including diabetes, insulin resistance, hypertension, obesity, and dyslipidemia. These factors, in return, exacerbate the risk of cardiovascular disease (CVD), the major cause of morbidity and mortality among the elderly in the U.S. Unsaturated fats, specifically n‐3 polyunsaturated fatty acids (n‐3 PUFAs), have been shown to be inversely related to CVD risk factors, while saturated fat seems to increase the risk for CVD. Not much is known on the association between different fatty acids and cardiometabolic factors, especially in the elderly population.

In a subset of the Walnuts and Healthy Aging (WAHA) study, a dual center, randomized controlled trial, we wanted to assess the possible associations between various red blood cell (RBC) membrane fatty acids, biomarkers for long‐term intake of fatty acids, and select cardiometabolic risk factors. Our study encompassed 192 participants with a mean age of 69 years from the Loma Linda, CA center only. The RBC membrane fatty acids and cardiometabolic risk factors (blood pressure, blood lipids, and fasting glucose) were measured at baseline and at the end of one year.

RBC membrane n‐3 PUFAs were inversely associated with total plasma cholesterol (ß = ‐3.83; p= 0.02), triglycerides (ß = ‐7.66; p= <0.01), and fasting glucose (ß = ‐0.19; p=0.03). N‐6 PUFAs were positively associated with systolic blood pressure (ß=1.10; P=0.02). Mono‐unsaturated fatty acids were positively associated with TAG (ß = 4.16; P=0.03). Total saturated fatty acids were not associated with any of the cardiometabolic risk factors.

RBC membrane fatty acids are good long‐term markers of dietary intake of fatty acids and can be used for diet‐disease relationship studies. Among elderly participants, increased n‐3 PUFAs in RBC may be predictive of lowered cardiometabolic risk.
Manipulation of Left Ventricular Loading Conditions is Necessary to Identify Diastolic Dysfunction in Swine with Repetitive Pressure Overload‐Induced HFpEF
Authors: Gilligan Alexandra K. , Nsumbu Dorcas , Tajlil Arezou , Canty John M. , Weil Brian R.
Published: May 2022

Objective

Diagnosis of heart failure with preserved ejection fraction (HFpEF) is often complicated by the fact that many patients exhibit normal left ventricular (LV) mechanics at rest with a reduction in diastolic compliance that is only revealed when loading conditions are altered (e.g., during exercise stress). In an effort to identify parameters of diastolic function that could be utilized to detect alterations in compliance without manipulation of LV loading conditions, we utilized invasive hemodynamic analysis and speckle‐tracking echocardiography (STE) to assess diastolic strain and stiffness in swine with repetitive pressure overload (RPO)‐induced HFpEF.

Methods

Swine (n=13) were subjected to 2‐weeks of RPO via daily intravenous administration of phenylephrine (PE; 400 ug/min; 1 hour/day) through an indwelling jugular vein catheter. Indices of LV relaxation as well as LV minimum and end‐diastolic pressure were obtained invasively before and after RPO at rest and during transient PE‐induced alterations in loading conditions. STE (Vivid 9; GE) was also performed throughout a single cardiac cycle to collect circumferential diastolic strain data, which were used alongside echocardiography‐derived measurements of chamber radius and wall thickness as well as catheter‐derived measurements of LV pressure to calculate regional and global circumferential subendocardial diastolic stiffness before and after RPO.

Results

Compared with baseline measurements, 2‐weeks of RPO did not significantly increase the time constant of LV relaxation (Tau; Figure A) or decrease the rate of LV pressure change during isovolumic relaxation (dP/dtmin; Figure B). Circumferential subendocardial stiffness derived via STE across a single cardiac cycle at rest was also unaffected by RPO (Figure C), despite a significant reduction in diastolic compliance that was revealed by construction of two‐point end‐diastolic pressure‐volume curves (Figure D) after manipulation of loading conditions (Figure E). Interestingly, single‐cycle STE‐derived stiffness measurements made during acute pressure overload were also similar at baseline and after RPO due to persistent elevations in LV minimum diastolic pressure during PE infusion at both timepoints.

Conclusions

Although reductions in LV diastolic compliance are evident after chronic RPO when loading conditions are manipulated, abnormalities in LV mechanics are not exhibited at rest even when STE is used to assess diastolic stiffness throughout a single cardiac cycle. These findings highlight the challenges associated with identification of myocardial stiffening at rest in patients with HFpEF and reinforce the importance of studying LV mechanics under variable loading conditions.
Additive Effects of Diabetes and Lower‐Limb Amputation on Osteoarthritis with Comparison to Diabetic and Healthy Controls
Authors: Ngo Wayne , Finnerty Caitlyn , Finco MG , Holley Bethany , Menegaz Rachel A.
Published: May 2022

Individuals with type II diabetes and individuals with lower‐limb amputation each have increased risks of developing osteoarthritis compared to the general population. Despite the high co‐occurrence of type II diabetes with lower‐limb amputations, the additive effects of these conditions are unclear. In order to better manage the risk of developing osteoarthritis in these populations, a better understanding of how diabetes and amputation might compound osteoarthritis risk is needed.

We measured hip and knee joint space, as indicators of osteoarthritis, in four groups of individuals: 1) lower‐limb amputees with diabetes, 2) lower‐limb amputees without diabetes 3) diabetic controls, and 4) healthy controls. We hypothesized lower‐limb amputees with diabetes would have the most impaired musculoskeletal health, followed by amputees without diabetes, diabetic controls, then healthy controls.

30 total CT scans of males (42‐79 years; BMI 19.7 ‐ 48.9 kg/m2) were obtained from the New Mexico Decedent Image Database. 10 scans were identified for amputees, diabetic controls, and healthy controls. Half of the lower‐limb amputees had diabetes while half did not, to differentiate effects of diabetes and amputation on musculoskeletal health. 3D Slicer software was used to measure hip and knee joint spaces as indicators of osteoarthritis. Comparisons between groups were assessed using Kruskal‐Wallis with Dunn’s post hoc tests.

Amputees with and without diabetes showed significantly narrower hip (p=0.01) and knee (p=0.08) joint space bilaterally compared to diabetic and healthy controls. This result suggests amputees could be at a higher risk of developing lower‐limb osteoarthritis compared to diabetic and healthy individuals, which is in line with prior work demonstrating the prevalence of osteoarthritis in the amputee population.

In agreement with our hypothesis, box plots showed trends of amputees with diabetes having the most narrowed joint space, followed by amputees without diabetes, then diabetic controls, and healthy controls. While not statistically significant, these trends suggest amputees with diabetes are at increased risk of developing osteoarthritis compared to amputees without diabetes. Perhaps the aggressive management of blood glucose and post‐amputation physiotherapy treatments could help reduce joint deterioration in these patients. Future work will focus on increasing sample size to assess if these findings are generalizable to a larger population.

Increased risks of osteoarthritis can lead to pain, limited mobility, and decreased quality of life. This study can potentially inform clinical standards of care for patients with amputations. Earlier interventions such as proactive musculoskeletal screenings and targeted exercises may reduce risks of developing osteoarthritis, leading to improved clinical outcomes.
DNA Replication Checkpoint Activates Respiration in Budding Yeast
Authors: Nagar Shreya , Mehta Riddhi , Kaur Pritpal , Vancura Ales
Published: May 2022

In addition to DNA damage checkpoint (DDC), eukaryotic cells have also DNA replication checkpoint (DRC) that is distinct from the DDC and specifically signals slowly progressing or arrested replication forks. We have showed previously that DDC activates respiration to increase ATP production and to elevate dNTP levels, which are required for efficient DNA repair and cell survival upon DNA damage. The underlying mechanism involves DNA damage‐induced downregulation of histone expression and altered chromatin structure, leading to increased transcription of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain (ETC), and oxidative phosphorylation. Here we show that similarly to DDC, activation of DRC also induces respiration. However, unlike DDC, DRC does not affect histone gene expression and chromatin structure. DRC induces respiration by inducing transcription of RNR1‐4, upregulating synthesis of dNTPs, and elevating mtDNA copy number. Fitness of rrm3∆ and sgs1∆, mutants with defects in DNA replication, requires checkpoint kinase Dun1, but does not require ETC and oxidative metabolism. However, in the absence of Dun1, ETC is required for viability of rrm3Δ and sgs1Δ cells. In addition, inactivation of ETC in dun1Δ cells results in a synthetic growth defect. Together, our data show that when Dun1p function is compromised, rrm3Δand sgs1Δ cells depend on oxidative metabolism.
Down‐Draft Tables Effectively Reduce Formaldehyde Levels in Anatomical Dissection Laboratories
Authors: Dixon Crissy M. , Peterson Kerry D. , Dorius Geoffrey T. , Morton David A. , Delucchi Danielle M.
Published: May 2022

Cadaver dissections are an essential anatomical teaching tool in health sciences. However, these dissection courses increase staff and students to formaldehyde exposure. The Occupational Safety and Health Administration (OSHA) previously set standards designed to protect individuals from excess formaldehyde and individuals working in the laboratories were protected through ventilation. However, older buildings that host cadaveric dissections may not provide the ventilation necessary to adequately keep formaldehyde levels below a safe threshold. Constructing or renovating a building's ventilation system is expensive. The estimated cost to retrofit the ventilation system in the University of Utah's anatomy lab was three million dollars. This study sought to determine a cost‐effective solution to reduce formaldehyde exposure; thereby creating a safe environment for laboratory occupants. Using formaldehyde‐monitoring badges strategically placed above each cadaver and on each dissector, dissections were conducted for 200‐230‐minute intervals. The badges were analyzed and showed formaldehyde Permissible Exposure Levels (PELs) were above OSHA limits (0.34‐1.91ppm). Dissection gurneys were then fitted with paired side vents measuring 64 x 4 x 4 inches. These vents were attached to the Python Portable Floor Sentry‐Fume Extractor (Model #SS‐450‐PYT; Sentry Air Systems) measuring 19.5 x 22.5 x 20 inches, which directed air surrounding the embalmed body through the formaldehyde filtration system. Following the installation of down‐draft tables, and using the same experimental method, data from eight new badges were analyzed. These samples resulted in PEL levels between 0.06‐0.13 ppm, well below the OSHA limit. With the use of down‐draft tables, formaldehyde PEL levels were reduced by an average of 0.63ppm; a statistically significant decrease (T‐test of 0.036). The cost to retrofit 10 gurneys into down‐draft tables was approximately 55,000 dollars, saving the University more than 2.9 million. This study suggests the use of modified down‐draft tables provide an affordable and safe environment for anatomy dissection personnel, as well as a cost‐ and time‐effective alternative to retrofitting ventilation in facilities not specifically designed for dissection.
Loss of Endothelial CFTR Drives Barrier Failure and Edema Formation in Lung Infection and Can Be Targeted by CFTR Potentiation
Authors: Erfinanda Lasti , Zou Lin , Gutbier Birgitt , Lei Disi , Reppe Katrin , Teixeira Alves Luiz Gustavo , Kneller Laura , Schneider Bill , Michalick Laura , Fatykhova Diana , Schneider Paul , Liedtke Wolfgang , Sohara Eisei , Mitchell Timothy J. , Hocke Andreas , Hippenstiel Stefan , Mall Marcus A. , Witzenrath Martin , Kuebler Wolfgang M.
Published: May 2022

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS), a potentially fatal lung disease characterized by hyperinflammation and endothelial barrier failure. Infectious and inflammatory stimuli can cause rapid downregulation of cystic fibrosis transmembrane conductance regulator (CFTR), and inhibition of CFTR has been proposed to increase lung microvascular endothelial permeability in vitro. Here, we identify loss of lung microvascular endothelial CFTR as important pathomechanism in lung barrier failure in pneumonia‐induced ARDS, delineate the molecular signaling pathway underlying this effect and identify CFTR potentiation as novel therapeutic strategy in ARDS.

CFTR was downregulated in endothelial cells following Streptococcus pneumoniae(S.pn.) infection in human and murine lung tissue. Isolated perfused lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular Cl‐ and Ca2+concentrations ([Cl‐]i, [Ca2+]i). Inhibition of the Cl‐ sensitive with‐no‐lysine kinase 1 (WNK1) replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i while WNK1 activation attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4‐/‐) developed less lung edema and protein leak than their wild‐type littermates following infection with S. pn. Conversely, lungs of heterozygous Wnk1‐deficient mice (Wnk1+/‑) showed spontaneous leak. The CFTR potentiator ivacaftor prevented CFTR loss and reduced endothelial leak in response to pneumolysin (PLY), a key S.pn. virulence factor, or plasma from COVID‐19 patients in vitro, and prevented lung edema and protein leak after S. pn.infection in vivo.

Lung infection causes rapid loss of CFTR that promotes lung edema formation through intracellular Cl‐accumulation, inhibition of WNK1 and subsequent disinhibition of TRPV4, resulting in endothelial Ca2+ influx and vascular barrier failure. Ivacaftor prevents CFTR loss and may thus present a promising therapeutic strategy in ARDS due to severe pneumonia including COVID‐19.
Induction of ALOX5 during Polarization of M1 Macrophages by Multi‐walled Carbon Nanotubes
Authors: Lim Chol Seung , Ma Qiang
Published: May 2022

Fibrogenic carbon nanotubes (CNTs) induce the polarization of M1 and M2 macrophages in mouse lungs. Polarization of the macrophages differentiates the production of proinflammatory and pro‐resolving lipid mediators (LMs) to mediate acute inflammation and its resolution in a time‐dependent manner. In the present study, we examined the molecular mechanism by which multi‐walled CNTs (MWCNTs) induce M1 polarization in vitro, with focus on induction of arachidonate 5‐lipoxygenase (ALOX5), which is required to produce proinflammatory LMs, such as leukotriene B4 (LTB4). Treatment of J774A.1 macrophages with MWCNTs at a concentration of 2.5 µg/mL increased the expression of ALOX5 mRNA by 2.0‐fold at 1‐day and 2.5‐fold at 3‐day post‐exposure. At a concentration of 10 µg/mL, MWCNTs increased the ALOX5 mRNA expression by 3.5‐fold at 1‐day and 5.0‐fold at 3‐day post‐exposure. Immunoblotting shows that MWCNTs significantly increased the ALOX5 protein expression in macrophages. Luciferase reporter assays with the mouse ALOX5 promoter of 2 kilobase upstream of translation start codon demonstrate that the ALOX5 promoter activity increased more than 5‐fold over background. This activity was significantly elevated following 5’‐deletion analyses of ALOX5 promoter and further enhanced by MWCNT treatment, implicating a transcriptional mechanism in the induction of ALOX5. MWCNTs preferentially induced the expression of CD68, a cell surface marker of M1, by 2.5‐fold and induction was persistently high to 3‐day post‐exposure. Moreover, both the expression and activity of the inducible nitric oxide synthase, an intracellular marker of M1 macrophages, were increased by MWCNTs, indicating induction of M1 polarization. Consistent with this notion, MWCNTs increased the capacity of the macrophages to produce proinflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β), and proinflammatory LMs, such as LTB4 and prostaglandin E2 (PGE2), as measured by enzyme‐linked immunosorbent assays. Taken together, these results support that persistent exposure to MWCNTs polarizes macrophages to M1 cells with induction of ALOX5 and elevated production of proinflammatory LMs to boost the acute inflammatory response to fibrogenic nanoparticles.