bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatic Lipidomic Analysis of Dietary Different Type of Fats and Fructose Interactions in the Pathogenesis of Nonalcoholic Fatty Liver Diseases
Authors: Shi Hongxue , McClain Craig , Song Ming
Published: May 2022

Objective

Dietary nutrient interactions are key determinants to the development of nonalcoholic fatty liver disease (NAFLD). Fat and fructose are two major components of Western diet. This study aimed to determine the combined effects of different type of dietary fats and fructose on the development of NAFLD in a murine model.

Methods

Male adult C57BL/6J mice were assigned to six groups and fed with diets (AIN‐93G based) enriched with different types of fat, such as saturated fat (SF‐beef tallow), n‐6 polyunsaturated fat (n6USF‐corn oil), or n‐3 polyunsaturated fat (n3USF‐menhaden fish oil), respectively, with or without fructose supplementation through 10% fructose (w/v) in the drinking water ad lib for 20 weeks. 42% of calories was derived from the fat. Metabolic phenotypes were characterized by liver histology, plasma ALT and AST, glucose tolerance test, hepatic mRNA expression of genes involved in lipid metabolism, and lipidomic analysis of hepatic fatty acid composition.

Results

While both SF and n6USF fed mice developed obesity and hepatic steatosis with or without fructose, SFA fed mice were characterized with hepatomegaly, liver injury and fibrosis, which were further exacerbated by fructose; conversely, plasma triglyceride levels were lower compared to controls. Interestingly, n6USF fed mice exhibited steatosis without hepatomegaly, and this was associated with markedly elevated plasma triglyceride and free fatty acid levels, which were blunted by fructose. Moreover, fructose and n6USF led to markedly elevated glucose intolerance tests compared to controls. Hepatic lipidomic analysis revealed that total monounsaturated fatty acids, but not total SFA, exhibited a stepwise decrease from SF to n6USF and n3USF fed mice across most of the lipid pools, which parallels the hepatic triglyceride content, irrespective the presence of fructose. In line with this, hepatic mRNA levels of lipogenesis genes, including Srebf1, Scd1, Fasn, Acacaand Acacb, displayed a similar pattern of alterations (SF>n6USF>n3USF), suggesting that increased lipogenesis contributes to hepatic steatosis in SF and n6USF fed mice. Of note, fructose play a mild, but significant role in the alterations of specific individual fatty acids in certain fatty acid pools, despite the predominant role played by the dietary fats.

Conclusion

Our data suggest that excess energy from SF intake results in fat storage in the liver, likely due to impaired triglyceride secretion; whereas excess energy from n6USF intake is stored in the periphery. Both effects were exacerbated by fructose supplementation. n3USF is beneficial even in the presence of fructose.
Small Molecule Modulation of the Atypical Kinase/ATPase COQ8A
Authors: Murray Nathan , Lewis Adam , Asquith Christopher , Rincon Pabon Juan , Fang Zixiang , Ptak Naomi , Smith Robert , Bingman Craig , Gross Michael , Henzler‐Wildman Katherine , Pagliarini David
Published: May 2022

Small molecule manipulation of the protein kinase‐like (PKL) superfamily has emerged as a remarkably effective therapeutic strategy, with more than 70 new drugs approved in the past 20 years, and has aided mechanistic studies of PKL function. However, such tools are still lacking for many PKL members, including the growing number of atypical kinases and pseudokinases. Among these are the ABC1/ADCK proteins, which are conserved throughout all domains of life and are directly tied to human diseases. ADCKs have been implicated in various aspects of prenyl‐lipid biology, with the most established being a role for COQ8 in coenzyme Q (CoQ) biosynthesis. While the importance of these proteins is clear, they are largely understudied and a mechanistic understanding of their function is lacking. To address this, we developed a series of small molecule probes to manipulate COQ8 activity in vitroand in cells. Here, we first present a biophysical investigation into the activation of COQ8A by the CoQ precursor mimetic 2‐propylphenol (2‐PP) using 1H‐13C HMQC NMR and hydrogen‐deuterium exchange mass spectrometry. We found that 2‐PP modulates the highly conserved KxGQ domain to increase COQ8A nucleotide affinity and ATPase activity. Second, we present the development of a custom small molecule inhibitor of COQ8A. Guided by crystallography, activity assays, and cellular CoQ measurements, we repurposed a 4‐anilinoquinoline scaffold to selectively inhibit COQ8A within mitochondria. Our newfound chemical tools promise to lend new mechanistic insights into the activities of these widespread and understudied proteins, and to offer potential therapeutic strategies for human diseases connected to their dysfunction.
Regulation of SIRT2 Deacylase Activity Through Self‐Dimerization
Authors: Yang Jie , Fusco James M. , Dwyer Shannon L. , Nicely Nathan I. , Weiser Brian P.
Published: May 2022

Human sirtuins are a family of nicotinamide adenine dinucleotide (NAD+)‐dependent enzymes that are responsible for removing acyl modifications from lysine residues. The deacylase activity of sirtuin isoform‐2 (SIRT2) is involved in the formation and proliferation of cancers and is thought to regulate the progression of neurodegenerative diseases. The acyl substrates of SIRT2 are chemically diverse and range from small acetyl modifications to much larger, fatty acyl groups such as myristoyl. Here, we show that recombinant SIRT2 self‐associates to form dimers in a substrate‐dependent manner that regulates its deacylase activities. SIRT2’s Kd for self‐association was determined to be 98 nM, which is within range of its cellular concentration. SIRT2 alone readily forms dimers, but the enzyme undergoes a transition to monomer when bound to myristoyl substrate. SIRT2’s oligomeric transition from dimer to monomer upon myristoyl substrate binding slows its demyristoylase reaction by adding an additional step to its reaction mechanism. In contrast, SIRT2 appears to remain dimerized when performing its deacetylase reaction which enhances its activity. We propose that SIRT2 abundance in cells may regulate its oligomeric state and deacylation kinetics, and additionally, its oligomeric states may be pharmacologically targeted to modulate acyl substrate selectivity. Finally, sirtuin isoforms 3 and 6 (SIRT3 and SIRT6) do not oligomerize at concentrations relevant to cellular activity, indicating that this mode of regulation is not inherent to all human sirtuins.
Nanoplastic effects on human vascular endothelial cells: A comparison of primary cells (HUVEC) and immortalized cells (hy926) after exposure to polystyrene nanoplastics
Authors: Simmons Timothy , Vandergon Thomas
Published: May 2022

Humans are being exposed to nanoplastics primarily through inhalation and ingestion. Once in the thoracic or digestive tract, the nanoplastics come in contact with the epithelial cell layer. This layer has been known to uptake nanoplastics leading to oxidative stress and ROS generation. The next cell layer beneath the epithelial layer is the capillary endothelium. There is currently very little information known about what happens once the endothelial layer is exposed to nanoplastics. Research on endothelial stress response has involved two types of cell lines, primary and immortalized cell lines. The primary cell line comes directly from human/animal tissues, Human Umbilical Vein Endothelial Cells (HUVEC), while the immortalized cell line is a fusion of two cell types that is a functional equivalent of the endothelial layer (hy926 cells). I found that both endothelial cell models were found to uptake polystyrene nanoplastics and both were similar in their responses to polystyrene nanoplastics. Both cell lines showed no significant difference in the amount of Caspase‐3 activity, mitochondrial degradation, or Reactive Oxygen Species (ROS) generation when exposed to 10, 20, and 40 μg/ml of polystyrene nanoplastics compared to controls. Our data suggest that at these polystyrene concentrations, either model system can be used as an accurate model for measuring endothelial cell layer responses to nanoplastics.
Peptide Ligand Interaction with Maltose‐Binding Protein Tagged to the Calcitonin Gene‐Related Peptide Receptor: Inhibitory Roles of Receptor N‐Glycosylation
Authors: Lee Sangmin
Published: May 2022

Calcitonin gene‐related peptide (CGRP) and adrenomedullin (AM) are peptide hormones and their receptors play a critical role in migraine progression and blood pressure control, respectively. CGRP and AM receptors are structurally related since they are the complex of the calcitonin receptor‐like receptor (CLR) with the different types of receptor activity‐modifying protein (RAMP); the CLR complex with RAMP1 is the CGRP receptor while the CLR complex with RAMP2 is the AM receptor. The extracellular domain (ECD) of CLR and RAMP provides a binding site for peptide ligands as reported in crystal structures. Several crystal structures of the CGRP and AM receptor ECD used maltose‐binding protein (MBP) as a tag protein to facilitate crystallization. Unexpectedly, the recent crystal structures of CGRP receptor ECD showed that the N‐terminal MBP tag located in proximity of the bound/mutated peptide ligands. The objective of the current study is to investigate potential chemical interaction between peptide ligands and the MBP tagged to the CGRP receptor ECD. Peptide ligand interaction with purified CGRP receptor ECD was evaluated with fluorescence polarization/anisotropy peptide binding assay with a fluorescent peptide probe. The results of the current study provided evidence that MBP N‐terminally tagged to the CGRP receptor ECD formed chemical interaction with mutated peptide ligands. Interestingly, N‐glycosylation of the CGRP receptor ECD was predicted to prevent MBP docking to the mutated peptide ligands. The N‐glycosylation of CLR ECD N123 was the most critical for inhibiting MBP interaction with the mutated peptide ligands. The MBP tag protein interaction was also dependent on the sequence of the peptide ligands. An endogenous CGRP fragment did not appear to interact with the MBP tag. In contrast to the CGRP receptor, the MBP tag was not involved in peptide ligand binding at AM receptor ECD. Here, I provided evidence that N‐glycosylation of the CGRP receptor ECD inhibited the MBP tag protein interaction suggesting an additional role of N‐glycosylation in the MBP‐tagged CGRP receptor ECD. This study reveals the importance of using a tag protein‐free CGRP receptor ECD for the accurate assessment of peptide ligand affinity.
ADAR Dysfunction in Brain Aging and Alzheimer's Disease
Authors: McEntee Cali M. , Cavalier Alyssa N. , LaRocca Thomas J.
Published: May 2022

Neuroinflammation is a central mechanism of brain aging and neurodegenerative disease, but why neuroinflammation develops with aging is incompletely understood. Adenosine deaminase RNA specific (ADAR) proteins are potential suppressors of neuroinflammation through their adenosine‐to‐inosine (A‐to‐I) editing and subsequent inhibition of double‐stranded RNA (dsRNA, an innate immune activator). However, the role of ADARs in brain aging and/or neurodegenerative disease (e.g., Alzheimer’s disease, AD) is unclear. To address this, we looked for age‐related changes in ADAR expression and function (indicated by A‐to‐I editing) in multiple, brain‐specific transcriptomic datasets. We found evidence of age‐related declines in ADAR expression in: 1) astrocytes (glial cells linked with immune/inflammatory signaling in aging and AD) in old vs. young mice; 2) induced astrocytes from old vs. young human subjects; and 3) brain tissue of AD patients vs. healthy age‐matched controls. We also found declines in A‐to‐I editing related to reduced ADAR expression, suggesting that ADAR and ADAR activity decline with age and disease. We followed up on these observations via in vitro experiments in primary human astrocytes, in which we suppressed ADAR via siRNA knockdown or with an ADAR inhibitor (8‐Azaadenosine). These treatments increased levels of the proinflammatory cytokine interleukin 6 (IL‐6) and intercellular adhesion molecule 1 (ICAM‐1, a marker of pro‐inflammatory astrocytes), and this was associated with accumulation/changes in the cellular distribution of dsRNA (assessed via immunofluorescence). Preliminary data showed that ADAR knockdown tended to increased levels of protein kinase R (PKR, a key pro‐inflammatory dsRNA sensor), and that dsRNA colocalizes with PKR. To identify related mechanisms, we are currently performing RNA‐seq to profile whole‐transcriptome changes associated with ADAR inhibition in astrocytes. Taken together, our data suggest that ADAR may be an important modulator of neuroinflammation in aging, and ultimately neurodegenerative diseases.
The TSPO ligand 3,17,19‐androsten‐5‐triol attenuates NASH by increasing FXR expression and autophagy
Authors: Li Yuchang , Sottas Chantal , Chen Liting , Garza Samuel , Li Lu , Papadopoulos Vassilios
Published: May 2022

Translocator protein (TSPO; 18 kDa) is a ubiquitous high‐affinity cholesterol‐ and drug‐binding protein located in the outer mitochondrial membrane. 3,17,19‐androsten‐5‐triol (19‐Atriol) is a ligand binding at the cholesterol recognition amino acid consensus domain of TSPO. Our previous study suggested that TSPO deficiency ameliorated methionine‐choline‐deficient induced non‐alcoholic steatohepatitis (NASH) through downregulation of bile acid synthesis (Li et al., iScience 24: 102457, 2021). We examined the role of cholesterol binding to TSPO in NASH using the 19‐Atriol on high‐fat‐fructose‐cholesterol diet (HFFC)‐induced NASH in rats. After HFFC‐feeding for 13weeks, the rats were subcutaneously administered daily 19‐Atriol (20mg/kg) for 2 weeks (weeks 13‐15). The ratio of liver/body weight and serum triacylglycerol content were significantly reduced in 19‐Atriol‐ compared to vehicle‐treated rats. Immunoblot analyses indicated that liver inflammation (TNFα) and fibrosis markers (COL1A1, ACTA2) were reduced following 19‐Atriol treatment. Farnesoid X receptor (FXR), a bile acid receptor, was upregulated in response to 19‐Atriol treatment in HFFC‐fed rats. In search of the mechanism mediating the effects of 19‐Atriol we observed an increase in autophagy flux and the levels of phosphorylated transcription factor EB (TFEB), a major regulator of autophagy and lysosomal biogenesis. Taken together these results suggest that blocking cholesterol binding to TSPO by 19‐Atriol leads to the attenuation of NASH by increasing FXR expression levels and activating of autophagic degradation as was the case in TSPO‐deficient rats.
Effects of Methanolic Extract of Moringa oleifera on an In Vitro Model of Diabetic Nephropathy
Authors: Tsotakos Nik , Mitra Debashree , Castaneira Ryan , Morris Daniel , Byler Josiah J.
Published: May 2022

Diabetic nephropathy (DN) is a severe diabetic complication and a leading cause of chronic kidney disease and renal failure worldwide. The high prevalence of end stage renal disease (ESRD) due to diabetes, coupled with the lack of efficient treatments, prompts the need for newer therapeutic strategies that overcome the drawbacks of the existing ones. In recent years, Moringa oleiferahas been identified as a plant with multiple nutritional and medicinal advantages. Importantly, previous studies have reported antioxidant and anti‐hyperglycemic properties of Moringa leaf extracts in diabetic rats. The growing interest in the potential use of nutraceuticals over conventional drugs along with the recent evidence of the beneficial effects of M. oleifera extract led us to investigate the effects of the methanol extract of M. oleifera on an in vitro model of human glomerular epithelial cells (HGEC).

HGEC were chronically cultured in the presence of physiological (5mM) or high glucose (25mM) levels and treated with different concentrations of methanolic extract of M. oleifera(MOE). We evaluated the effect of MOE on the production of advanced glycation end‐products (AGE), cell viability, and response to oxidative stress. Our results indicate that MOE can effectively inhibit AGE formation in vitro and provide a cytoprotective effect to cells following an oxidative challenge. However, in line with prior data indicating that HGEC chronically exposed to high glucose are primed for apoptosis, these cells showed reduced viability following exposure to MOE compared to cells exposed to normal glucose. These results suggest that acute, high doses of MOE might not be beneficial following chronic exposure of kidney cells to glucose, but the possibility of a protective effect of MOE when administered early is worth exploring.
Sexually Dimorphic Responses to Growth Hormone and Luteinizing Hormone within the Somatotropic and Reproductive Axes of Tilapia
Authors: Celino‐Brady Fritzie , Breves Jason , Seale Andre
Published: May 2022

Sexual dimorphism in body size is a common phenomenon among vertebrates, including teleost fishes. The hormonal underpinnings of this phenomenon, however, have not been fully clarified and may be associated with interactions between the endocrine factors that control growth and reproduction. Given their well‐described patterns of sexually dimorphic growth, we used Mozambique tilapia (Oreochromis mossambicus) as a model to examine how the activities of luteinizing hormone (LH) are modulated by growth hormone (GH), and conversely, how LH affects targets of GH. We hypothesized that GH would induce factors within the GH/insulin‐like growth factor (GH/IGF) axis that favor growth, while LH would attenuate growth‐promoting factors and stimulate factors that favor reproduction. We analyzed gonad morphology and gene expression in hypophysectomized tilapia injected with ovine GH (oGH) and LH (oLH) either alone or in combination. Gonadosomatic index (GSI) was reduced in females following hypophysectomy; the combined administration of oGH and oLH restored GSI to control levels. Hypophysectomy regressed the ovary and the combined treatment of oGH and oLH restored ovarian structure. Gonadal gh receptor (ghr2) and estrogen receptor β (erβ) were higher in females, whereas igf1, igf3, erα, and androgen receptor ß (arß) were higher in males of control fish. The inhibitory effects of hypophysectomy were most pronounced on igf3transcripts, and these levels were restored by both oGH and oLH alone, or in combination, in males. In females, the combination of both hormones was required to recover gonadal igf3 levels. A hypophysectomy‐induced decrease in arβ was restored by a combination of oGH and oLH in females, while there were no effects of oGH or oLH on arβ in males. In muscle, ghr2 was more responsive to oGH in males, while igf2 was more responsive to oLH in females. oGH and oLH both inhibited a hypophysectomy‐induced increase in hepatic igf2 in males. Our results indicate that genes associated with growth and reproduction exhibit sexually dimorphic responses to GH and LH in tilapia, and therefore, provide insight into the endocrine bases of sexually dimorphic growth in fishes.
Targeting Estrogen Non‐Genomic Signaling in a Three‐Dimensional (3D) in VitroModel of Inflammatory Breast Cancer
Authors: Negrón‐Figueroa Dalissa , Peterson‐Peguero Esther A.
Published: May 2022

Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer with a low overall 5‐year survival rate of ~30‐60%. To this date the molecular signature associated with IBC has not been fully characterized, which lead to a lack of effective targeted therapeutics, especially for those patients that account for the 20‐40% of triple‐negative (TNBC) IBC cases. Several studies established that molecular signature of IBC shows important differences from the other breast cancer subtypes, such as overexpression of ErbB receptors, RhoC GTPase and Cox‐2, E‐cadherin aberrant expression, and loss of WISP3, among others. However, these molecular alterations do not explain completely the aggressive and rapid IBC progression. Recently, several studies have shown that estrogen can exert non‐genomic effect in triple‐negative IBC and other TNBCs, mediated by the expression of alternate estrogen receptors, including ERα36 and GPR30. Estrogen non‐genomic signaling refers to the rapid action of these alternate estrogen receptors that upon estrogen binding activates protein‐kinase cascades in the cytoplasm. We hypothesize that estrogen can elicit a specific non‐genomic signaling cascade that promotes the acquisition of aggressive oncogenic phenotypes in IBC and that targeting this pathway can be an effective anticancer therapy for IBC. To test this hypothesis, we used pharmacological inhibitors of the Epidermal Growth Factor Receptor (EGFR) alone and GPR30 alone or in combination. The first approach was to determine if inhibiting estrogen non‐genomic signaling by targeting one of the alternate receptors, GPR30, affects cell proliferation in a 3D colony formation assay using Matrigel. Preliminary observations showed that after G15 treatment (GPR30 inhibitor), there was a slight reduction in cell proliferation compared to E2, but when combined with IRESSA (EGFR inhibitor) there was a further decreased on proliferation when compared to IRESSA alone. Ongoing experiments include the effects of Icaritin (modulator of ERα36) and knockdown of ERα36 using siRNA experiments to test the effects in cell proliferation, migration and invasion. Also, we are in the process of determining the half‐maximal inhibitory concentration (IC50) of G15, IRESSA and IRESSA + G15 by performing a dose response curve (Alamar Blue cell viability assay) of SUM149 cell line, to observe if there is an additive effect when combined the pharmacological inhibitors of EGFR and GPR30. This study will provide the foundation for further characterization of the role estrogen non‐genomic signaling in the progression of IBC and to test its potential as a therapeutic target.
Engineering a Better FLUture with Neuraminidase‐Targeted Monoclonal Antibodies
Authors: Cegielski Evelyn , Samtani Smriti , Thompson Anna , Lane Jim
Published: May 2022

Each year epidemiologists and public officials predict which influenza strain will be most prevalent and design a vaccine to target those specific strains; unfortunately, they are not always accurate. Traditional vaccines cause our bodies to produce antibodies that target two subtypes of the influenza A, that both bind to the surface protein Hemagglutinin (HA). However, HA mutates quickly, leaving the body susceptible to new mutant strains of influenza. Fortunately, another surface protein, Neuraminidase (NA), mutates significantly slower than HA, which would makeaa vaccine that targets NA potentially more effective against a broader spectrum of influenza A and B subtypes. According to Stadlbauer et al., one H3N2 infected donor naturally created monoclonal antibodies (mAbs) against NA that are protective against various strains of influenza A and B. Our research explored the mAb 1G01 in relation to H3N2 NA using PDB 6Q23. We found that 1G01 targets the active site of NA resulting from attraction due to a difference in charges, thereby blocking binding to its substrate necessary for NA to support virus binding and replication. Therefore, implementing properties of anti‐NA antibodies into current influenza vaccines could be used to improve their protectiveness against more strains of the virus, increasing the predictive reliability of current vaccines, and as a step towards a universal influenza vaccine. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of antibodies in relation to influenza. The visual model will be a valuable tool in developing our story.
Synthesis of MUC1 Peptide Backbone Bearing Sialyl‐Tn Antigen for Structural and Functional Studies With Endogenous Lectins
Authors: Ayyalasomayajula Ramya , Cudic Mare
Published: May 2022

Objective

To synthesize the tumor associated analogs of MUC1 for use in structural and functional studies between sialyl‐Tn (sTn) antigen with macrophage galactose‐specific lectin (MGL).

Mucin 1 (MUC1) is a heavily glycosylated transmembrane protein, altered in both expression and glycosylation pattern in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor‐associated carbohydrate antigens (TACAs), on the cell surface is a well‐known cancer biomarker. Accumulating evidence suggests that TACAs expression is associated with tumor escape from immune defenses through interaction with endogenous carbohydrate binding proteins (lectins). These interactions often lead to creation of a protumor microenvironment, favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C‐type lectin receptor found on antigen‐presenting cells (APCs) which facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor‐associated forms of MUC1 with MGL in tumor immunology, a thorough understanding of this interaction is essential for it to be exploited for cancer vaccine strategies. The specific aim of this research is to synthesize structurally well‐defined chemical probes, mono and multiple glycosylated MUC1 glycopeptide models carrying the Tn or sTn, that allow for the control of the complexity of the chemical space of the multivalent ligands. For this purpose, a concise scheme was developed for the large synthesis of the sTn antigen building block. Thiophenyl glycoside donors, in the presence TfOH/NIS or TMSOTf/NIS as promoter system, were used for the galactosylation and sialylation steps of the amino acid building block synthesis, respectively. We explored the effect of activator, temperature, solvent, and excess equivalent of sialic acid thioglycoside donor on sialylation reaction. The most favorable reaction conditions improved the desired α‐stereoselectivity, decreased the competing 2,3‐elimination reaction on the sialyl donor during activation, leading to overall increase in the reaction yield. This building block was used in the stepwise solid‐phase peptide synthesis of MUC1 glycopeptide models. The secondary structure of synthesized peptides was determined by circular dichroism (CD) spectroscopy.
Proteomics‐informed physiologically‐based pharmacokinetic (PBPK) modeling revealed differential effects of UGT2B17 variability on the pharmacokinetics of diclofenac following intravenous and oral administration
Authors: Ahire Deepak S. , Prasad Bhagwat
Published: May 2022

Uridine 5′‐diphospho‐glucuronosyltransferase 2B17 (UGT2B17) demonstrates dramatic inter‐individual variability (>3000‐fold) in the activity and expression in humans, which is associated with age, sex, genotype, and ethnicity.1We recently found that diclofenac is a UGT2B17 substrate, however, the quantitative contribution of UGT2B17 in the metabolism and pharmacokinetics (PK) of diclofenac is not well studied. Moreover, we hypothesized that the higher intestinal abundance of UGT2B17 could further amplify the magnitude and variability in its in vivo PK. We first investigated diclofenac glucuronide (DG) formation kinetics in human liver microsomes (HLM) and human intestinal microsomes (HIM) that were isolated from human donors containing the deletion genotype and the highest‐expressor of UGT2B17 in our tissue bank. Diclofenac showed 4‐ and 16‐fold difference in maximum DG formation rate (Vmax) in UGT2B17 highest‐expressor versus deletion in HLM and HIM, respectively (Fig. 1). Proteomics‐informed PBPK modeling2 estimated the quantitative contribution (fm) of UGT2B17 in the intestinal and hepatic glucuronidation of diclofenac, which revealed fm of 0.77, 0.24, and 0.0 in the highest, average, and null UGT2B17 expressors, respectively. Interestingly, intestinal fm of UGT2B17 was 0.94, 0.90, and 0.0 in the highest, average, and null expressors, respectively. The organ‐specific changes in the fm were also substantiated by the predicted fraction gut escaped (fg), which was primarily associated with UGT2B17 abundance in the enterocytes. A PBPK model was first developed and validated in the UGT2B17 average expressor and then extended to other populations utilizing UGT2B17 abundance data. Predicted plasma levels (AUC) of diclofenac were 20 % and 100 % higher following an IV dose (50 mg) in UGT2B17 deletion versus the average and the highest expressor subjects, respectively. Similalry, after 50 mg oral dose, ~2‐fold and 8.5‐fold higher AUC was observed in UGT2B17 deletion as compared to that in the average and the highest UGT2B17 expressors, respectively. Sex differences in UGT2B17 abundance explained ~2‐fold higher diclofenac AUC in females after oral administration. In summary, the unique intestinal abundance and the highly prevalent gene deletion of UGT2B17 could likely contribute to the variable bioavailability of its substrate drugs. Similarly, poor metabolism of diclofenac in children and in UGT2B17 deletion subjects could predispose these patients to high GI discomfort.

References

Bhatt et al., Drug Metab. Dispos. (2018).

Parvez Md M et al., Drug Metab. Dispos.(2021).
The activation status of integrin β1 determines the ACE‐2 expression and functions in renal epithelial and cancer cells
Authors: Mia Md Saimon , Shrestha Hum N. , Palamuttam Riya J. , Kumar Bony D. , Mathew Sijo
Published: May 2022

Background

Integrins are heterodimeric cell surface receptors that participate in intracellular signaling and regulate multiple cellular functions. These membrane receptors are also crucial for the organ development, structural maintenance, and disease pathologies of the kidney. Integrin β1 is the major integrin subunit present in the kidney. Alteration of integrin β1 expression is a characteristic of many kidney diseases such as kidney cancer and chronic kidney disease. The purpose of this study is to find the importance of integrin β1 in the expression and function of angiotensin‐converting enzyme 2 (ACE‐2) in the kidney. We hypothesized that the expression and function of ACE‐2 depend on the activation status of integrin β1 in the renal epithelial and cancer cells.

Method

Kidney epithelial‐specific integrin β1 KO mice were generated by crossing between ITGBfl/fland γGT‐Cre mice. PCR‐based analysis of genomic DNA was used to confirm the gene deletion. Expression of integrin β1 in the kidney cortex was monitored by qRT‐PCR. Immunoblotting and qRT‐PCR were performed to observe protein expression and mRNA level of ACE‐2 in mouse kidneys. The pharmacological compound, BTT 3033, was used to inhibit integrin β1 in HK‐2 and Caki‐1 cells. Integrin β1 activation status was monitored by immunocytochemistry using a conformation‐specific antibody. SARS‐CoV‐2 spike protein was used to find out the functional importance of the integrin β1‐ACE‐2 complex. Statistical significance of the observed data was carried out using a two‐tail t‐test.

Results

Deletion of integrin β1 from the renal epithelial cells reduced expression of ACE‐2 in the mice kidneys (n=3; p<0.05). Immunoblotting and qRT‐PCR showed, expression of integrin β1 (n=3; p<0.05) and ACE‐2 (n=3; p<0.05) were higher in renal cancer cells than renal epithelial cells indicating a positive correlation in their expression patterns. With the treatment of BTT 3033, there was a significant decrease in the activation status of integrin β1 in kidney epithelial (HK‐) and cancer (Caki‐1) cells (n=30; p<0.05). ACE‐2 mRNA levels decreased in HK‐2 and Caki‐1 cells when treated with an integrin β1 antagonist, BTT 3033 (n=3; p<0.05). Treatment of HK‐2 and Caki‐1 with BTT 3033 also decreased the internalization of the SARS‐CoV‐2 spike protein.

Conclusion

Results from this study demonstrate the importance of matrix proteins receptor integrin β1 in maintaining the ACE‐2 expression and function in the kidney. This study will have implications in various chronic diseases where significant changes in the composition of matrix proteins and integrin expressions are observed. Further studies are needed to understand the role of altered matrix composition in ACE‐2 mediated physiological functions.
Innate‐like T‐cells correlate with functional changes post‐myocardial infarction
Authors: Corker Alexa , Broughton Philip , Troncoso Miguel , Deleon‐Pennell Kristine Y.
Published: May 2022

Cardiovascular disease (CVD) is the leading cause of death in the United States. Ischemic heart failure is caused by adverse remodeling of the infarct scar after a myocardial infarction (MI). CD8+ T‐cells facilitate in the remodeling process and influence scar formation highlighting the importance of these cell types post‐MI. Single cell data from our laboratory identified a particular subset of CD8+ T‐cells in the infarct at post‐MI day 5 that expressed innate marker Nk1.1 (innate‐like T‐cells or ILTs). In cancer, ILTs were found to become activated in response to IL12 and IL18, independent of antigen‐presentation. We hypothesized that ILTs in the heart would lead to poor cardiac wound healing and eventual development of heart failure. Paraffin embedded left ventricular tissue from male mice (n=8; 3.75±0.63 months of age) 7 days post‐MI were stained with antibodies CD3 (1:100, #NBP2‐12159), CD8 (1:100, #CLANT280), and Nk1.1 (1:150, #BS‐4682R). Confocal images (10 images/tissue region) were taken at 40X and ImagePro was used to determine colocalization and percent area of CD3+ CD8+ Nk1.1+ cells in the infarct and remote tissue. Images were separated and correlated with echocardiography measurements and infarct size (calculated by triphenyltetrazolium chloride staining). Results indicated that while not significant, fractional shortening (R2=0.398; p=0.093) positively correlated with increased ILTs in the infarct whereas, infarct size (R2=0.528; p=0.064) negatively correlated with increased ILTs. None of the other measurements showed any trend in regard to the number of ILTs (p‐values were greater than 0.214). This data suggests that at post‐MI day 7, ILTs are associated with improved fractional shortening and decreased cardiac injury. Further analysis of additional time points will give us a better understanding of the role ILTs have on the post‐MI remodeling process.
Maternal Western‐style diet and obesity during pregnancy reduces mitochondrial Complex I‐linked oxidative capacity in tandem with decreased oxidative stress in skeletal muscle of adolescent Japanese macaque offspring
Authors: Greyslak Keenan T. , Hetrick Byron , Carey Kat , McCurdy Carrie E.
Published: May 2022

Background

Exposure to an obesogenic gestational environment during early development increases offspring risk of developing obesity and cardiometabolic diseases. Skeletal muscle of obese and insulin resistant individuals show signs of mitochondrial dysfunction including reduced oxidative capacity and increased oxidative stress. Subsequent oxidative damage is caused by reactive oxygen species (ROS) generated primarily by mitochondrial Complex I (CI). Previous work in our non‐human primate model shows decreased oxidative capacity in fetal skeletal muscle from obesogenic pregnancies, a phenotype we have confirmed is retained into adolescence. The purpose of this study was to evaluate how this reduced oxidative capacity correspond with ROS production in skeletal muscle of lean adolescent offspring.

Experimental Design

Lean (body fat <25%) adult female Japanese macaques were maintained on a control diet (CD) or Western‐style diet (WD) prior to and throughout pregnancy and lactation. Dams chronically consuming WD with body fat >30% were classified as obese and included in this study. Male and female offspring were weaned to CD and moved to independent housing at 7 months of age. Muscle samples were collected from offspring gastrocnemius (gastroc) and soleus at 40 months. Offspring data were analyzed by Student’s t‐test.

Results

Exposure to maternal WD during pregnancy showed moderate but significant reductions in oxidative damage and a modest (but insignificant) decrease in ROS generation in gastroc of offspring from obese pregnancies. In the soleus, ROS production was significantly reduced in these offspring. Both nuclear‐ and mitochondrial‐encoded genes for CI were decreased with maternal obesity/WD pregnancy, indicating transcriptional regulation of CI in exposed offspring. Finally, CI protein abundance was also decreased in primary myotubes from these offspring in lieu of additional metabolic challenges, providing further evidence of lasting metabolic reprogramming in skeletal muscle.

Conclusions

We find that early life exposure to maternal WD‐induced obesity leaves a lasting impression on offspring skeletal muscle metabolism that persists into young adulthood. It is possible that transcriptional suppression of CI activity is a protective adaptation to mitigate oxidative damage caused by mitochondrial ROS. While long‐term consequences of altered oxidation patterns relating to ROS flux require further investigation, the loss of CI content and diminished oxidative capacity in these lean offspring likely drive their increased susceptibility for cardiometabolic diseases – particularly when faced with metabolic stress and environmental insults throughout the lifespan.
Re‐Purposing Drugs as Countermeasures for Chemical Weapon Toxicities: Interactive Pharmacology Training
Authors: Aleksunes Lauren M. , Gray Joshua P. , Andres Jaclynn , Laskin Jeffrey D. , Laskin Debra
Published: May 2022

The risk of a terrorist attack in the U.S. has created challenges on how to effectively treat toxicities that result from exposure to chemical weapons. To address this concern, the U.S. has organized a trans‐agency initiative across academia, government, and industry to identify drugs to treat tissue injury resulting from exposure to chemical threat agents. We sought to develop and evaluate an interactive educational session that provides hands‐on instruction on how to re‐purpose FDA approved drugs as therapeutics to treat toxicity from exposure to chemical weapons. As part of the Rutgers Summer Undergraduate Research Fellowship, 23 undergraduate students participated in a two‐hour session that included: 1) an overview of chemical weapon toxicities, 2) a primer on pharmacology principles, and 3) an interactive session where teams of students were provided lists of FDA approved drugs to evaluate potential mechanisms of action and suitability as countermeasures for four chemical weapon case scenarios. The interactive session culminated in a competition for the best grant ‘sales pitch’. Pre‐ and post‐program self‐assessments using 5‐point Likert rating scales were conducted during the session using Poll Everywhere. From this interactive training, students improved their understanding of 1) the ability of chemical weapons to cause long‐term toxicities (means: pre‐2.2; post‐4.1, p<0.0001), 2) impact of route of administration and exposure scenario on drug efficacy (means: pre‐2.6; post‐4.3, p<0.0001), and 3) re‐purposing FDA‐approved drugs to treat exposure to chemical weapons (means: pre‐1.7; post‐4.0, p<0.0001). Seventy six percent of participants were ‘very likely’ or ‘extremely likely’ to recommend this activity to other students. These findings demonstrated that an interactive training exercise can provide students new insights into drug development for chemical threat agent toxicities.
Alcohol Consumption Pattern and Nutritional Intake: Analysis of the 2017‐2018 National Health and Nutrition Examination Survey
Authors: Zirnheld Kara , Warner Jeffrey , Huirong Hu , Floyd Alison , Kong Maiying , McClain Craig , Kirpich Irina
Published: May 2022

Background

Poor nutrition is often considered a component of alcohol use disorder as heavy drinkers substitute calories from macronutrients with ‘empty’ calories from ethanol. Indeed, the relationship between alcohol use and nutrition has long been of research interest due to the key role of diet in modifying individual susceptibility to alcohol‐induced multi‐organ pathology, such as alcohol‐associated liver disease. The goal of the current study was to evaluate nutritional intake among individuals with various drinking patterns through analysis of the 2017‐2018 National Health and Nutrition Examination Survey (NHANES). We hypothesized that individuals with the greatest alcohol intake would have the highest total caloric consumption, but that these individuals would have the lowest nutrient density in their overall diet.

Methods

Using the 2017‐2018 NHANES, we divided survey participants into the following four drinking categories based on guidelines from the National Institute on Alcohol Abuse and Alcoholism and the United States Department of Agriculture: never drinkers (n=500), non‐drinkers (past‐year abstainers, n=874), moderate drinkers (1 or 2 drinks per day for females or males, respectively, with no binge drinking, n=2658), and heavy drinkers (>1 or >2 drinks per day for females or males, respectively, or binge drinking, n=393). We analyzed 24‐hour dietary recall variables including macro‐ and micronutrient intake, as well as anthropometric measurements. One‐way ANOVA was used to determine significance by p values <0.05.

Results

Compared to never, non‐, and moderate drinkers, heavy drinkers consumed the highest total calories (by roughly 500 calories), coming from higher intake of carbohydrates, protein, fat, and alcohol. Further, heavy drinkers also consumed more of most micronutrients reported by the NHANES (apart from several antioxidant carotenoids such as alpha‐ and beta‐carotene and lutein/zeaxanthin). Interestingly, despite increased alcohol consumption and macro‐/micronutrient intake, heavy drinkers did not have an increased body mass index or waist circumference compared to other drinking groups.

Conclusion

Though poor nutrition has often been associated with excessive alcohol consumption, the results of this study showed that heavy drinkers consumed the most nutrient‐dense diet overall. Despite significant differences in total energy intake in heavy drinkers, anthropometric measurements were comparable across drinking groups. Future research should further investigate the relationship between alcohol use and nutrition in the NHANES and other sources.
The utilization of a three‐dimensional interactive virtual dissection model as an alternative approach in the education of post‐traumatic stress students
Authors: Ara Hosne , Bebee Jordan , Powell Taylor , Juneau Patrick , Mack Samuel , Miketinas Robert , Domingue Natalie , Panchatcharam Manikandan , Miriyala Sumitra
Published: May 2022

Human gross anatomy has evolved as the central course in the curriculum of first year Allied health students. Cadaveric dissection is the primary teaching modality of this course. Traditional teaching methods of dissection, prosection, tutorials and lectures are now supplemented by anatomical models and e‐learning. Although the traditional approach in anatomy instruction provides students with a comprehensive understanding of the three‐dimensional organization of the human body, the cadaveric approach in anatomy education fails to appreciate the profound psychological effects on students with existing psychological disorders. This case report is a presentation of a psychological effect of a cadaveric approach on a post‐traumatic stress disorder (PTSD) student. In this case study, we used a student who served in the United States Army in Afghanistan and was given an honorable discharge due to diagnosis of tinnitus and adjustment disorder. The student was also assigned a disability rating of 40%. This student was diagnosed as PTSD due to exposure of a high‐stress environment. The cadaveric approach to the student’s anatomy education only exacerbated his condition, triggering his stress disorder. Beside this, since the student had a history of losing a relative due to colon cancer, this made abdominal dissection difficult to perform. We considered an alternative approach to accommodate the student, while still maintaining the sufficiency needed to provide the standard of education for a health sciences student. The virtual approach of cadaveric dissections, with benefits impossible for a traditional dissection, also strives to provide students with the opportunity to improve the understanding of the three‐dimensional and spatial relationships of human gross anatomy. This case is presented to those who teach anatomy to identify this modality of education as an alternative approach to sufficient and comprehensive anatomy education.
NBMI Exerts Protection Against Copper Overload in HepG2 and SH‐SY5Y Cell Lines
Authors: Gadde Rajitha , Betharia Swati
Published: May 2022

Copper (Cu) is a naturally occurring essential element required for many biological processes. At low concentrations, Cu plays a vital role by getting incorporated into various metalloenzymes and at higher concentrations can be harmful to hepatic, central, and renal systems. Chronic toxicity with high concentrations of Cu have been characterized in Wilsons’s disease patients, and chelation has been the mainstay of therapy. As the currently approved chelator D‐pencillamine is associated with many adverse effects, the search for other therapeutic interventions is ongoing. N,N′bis‐(2‐mercaptoethyl) isophthalamide (NBMI) is a novel metal chelator and thiol redox antioxidant. NBMI has been shown to chelate harmful heavy metals such as mercury and lead in vitro. We explored whether NBMI displayed cytoprotective effects in HepG2 and SH‐SY5Y cells exposed to copper sulfate (CuSO4). Both the cell types were treated with either vehicle or NBMI (0 ‐ 100 μM) for 24 hours followed by CuSO4 (750 μM) for an additional 24 hours. Cell viability (CellTiter‐Glo® Assay) and degree of oxidative stress (ROS Assay) were determined. Comparisons between multiple groups were performed using analysis of variance (ANOVA) followed by a post hoc Tukey’s test. Results with p‐value <0.05 were considered statistically significant. Exposure of HepG2 and SH‐SY5Y cells to CuSO4 decreased cell viability in a concentration‐dependent manner. In addition, CuSO4 exposure led to an increase in ROS levels in HepG2 cells indicating oxidative stress. Pretreatment with NBMI diminished CuSO4‐induced cell death in both cell types and decreased ROS levels in HepG2 cells. The results imply that NBMI is a new and promising chelator for treating Cu‐overload genetic disorders.