bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic Central Nervous System Leptin Infusion Improves Cardiac Function and Metabolism after Ischemia/Reperfusion Injury
Authors: Omoto Ana Carolina Mieko , do Carmo Jussara M. , Nelson Benjamin , Flynn Elizabeth , Aitken Nikaela , Wang Zhen , Mouton Alan , Li Xuan , Moak Sydney , Dai Xuemei , Hall John E. , da Silva Alexandre A.
Published: May 2022

Important adverse changes in cardiac energy metabolism occur after ischemia/reperfusion injury (I/R), contributing to the worsening of cardiac function and development of heart failure. We recently showed that leptin, via its actions on the central nervous system (CNS), improves left ventricular (LV) function in a model of heart failure induced by permanent ligation of the left anterior descending coronary artery (LAD). In the present study, we examined whether the CNS‐ effects of leptin protect against myocardial I/R injury, and if chronic intracerebroventricular (ICV) leptin infusion improves cardiac substrate utilization, assessed by markers of myocardial fatty acid (FA) and glucose oxidation, NAD+/NADH redox state and plasma levels of β‐hydroxybutyrate (β‐HOB). Male Wistar rats were instrumented with an ICV cannula in the brain lateral ventricle. After recovery and baseline assessment of cardiac function by echocardiography (ECHO), myocardial I/R was induced by temporary (60 min) ligation of the LAD. Vehicle (saline, 0.5 µL/hr) or leptin (0.62 µg/hr) was infused chronically for 28 days starting 20 min after reperfusion using osmotic minipumps connected to the ICV cannula. ECHO assessment of cardiac function was performed weekly. At the end of week 4, +dP/dtmax was accessed by LV catheterization. Hearts and plasma samples were collected for evaluation of CD36, PPAR‐δ, PDK4 and p‐PDH1/PDH1 by western blot and cardiac NAD+/NADH ratio and plasma levels of β‐OHB were measured by ELISA. Our results showed that ICV leptin treatment improved cardiac function as evidenced by increased ejection fraction 4 weeks after I/R (46±3 vs. 26±3 %) and +dP/dtmax (10387±1686 vs. 5022±442 mmHg/s) when compared with vehicle‐treated rats. ICV leptin infusion also significantly increased cardiac protein expression of CD36 (1.4±0.1 vs. 1.0±0.1 au), PPAR‐δ (1.8±0.1vs. 1.0±0.01 au), PDK4 (1.8±0.1 vs. 1.0±0.1 au) and p‐PDH1/PDH1 (2.2±0.3 vs. 1.0±0.2 au) when compared with vehicle‐treated animals. In addition, ICV leptin infusion increased cardiac NAD+/NADH ratio (6.8±2 vs. 1.02±0.3 pmol/µL) and reduced plasma levels of β‐OHB (18.2±3 vs. 62.8±7.0 nmol/µL). These results demonstrate that chronic ICV leptin infusion improves cardiac function following I/R injury and suggest that leptin’s CNS‐mediated cardioprotective effects may involve improved myocardial FA oxidation and NAD+/NADH redox state, reducing the reliance on ketone bodies (β‐OHB) as an energy source.
The Regulation of Glial Glutamate Transporters after in‐vitro Ischemic Stroke
Authors: Gill Simran
Published: May 2022

Stroke is the 3rd leading cause of death in the United States but adequate treatment is still lacking. Moreover, the pathological mechanisms involved are still not fully understood, highlighting the need for further studies. In this work, we employed an in vitro model of ischemic stroke to study the regulation of excitatory amino acid transporters (EAATs), which are key proteins that control the extracellular concentration of glutamate in the brain and thereby help maintain brain homeostasis. Glutamate dysregulation is a fundamental feature of many neurological disorders, leading to neuronal death through a mechanism known as excitotoxicity. This makes EAATs key players in mitigating glutamate imbalance in the brain and thereby have a major role in excitotoxic outcomes following ischemia. Our goal was to understand how the activity and expression of EAATs are modulated following different severities of ischemic insults. Glia cultures were subjected to oxygen‐glucose deprivation (OGD) for 30‐minutes, 1‐hour and 2‐hours. Our results demonstrated that increasing lengths of insult result in decreased levels of glutamate transport, suggesting an inability of these transporters to effectively clear excess glutamate. The expression of glial transporters EAAT1 and EAAT2 are differentially regulated: EAAT1 is downregulated after ischemia, with a more profound effect after 1‐hour OGD, whereas EAAT2 expression is up regulated after 30‐minutes and 1‐hour OGD before decreasing below control levels after 2‐hour OGD. These results suggest that these transporters may work together, likely as a compensatory mechanism. We also investigated the effects of Clavulanic Acid (CA), a compound that enhances EAAT2, which is the main subtype of EAAT in the CNS. CA pretreatment was only able to rescue EAAT activity and significantly increase EAAT2 levels in control conditions and after 30‐minutes of OGD, suggesting that this strategy only offers benefit in mild ischemic insults. Furthermore, we investigated reactive gliosis, which can be defined by the activation of glia cells in response to a pathology. Previous work has shown that reactive gliosis is a phenotype associated with ischemia, however we wanted to further define and evaluate this type of astrocytic response. In specific, we wanted to determine how reactive gliosis is affected following different ischemic insults. Results show that reactive gliosis, which was determined by an increased expression and fluorescence of GFAP, increases with OGD insult, with a significant difference seen after 2‐hours of OGD. This suggests the reactive phenotype is more prominent as the severity of ischemia increases. These findings advanced our knowledge on EAAT regulation following different severities of ischemic stroke in‐vitro. Future studies will investigate potential mechanisms associated with their differential regulation, such as transport reversal, internalization, and degradation pathways.
Understanding the regulatory effects of a biofilm regulator (BifR) and the implication of its interactions in Burkholderia thailandensis
Authors: Nkwocha Elaine , Grove Anne
Published: May 2022

Biofilm formation plays a key role in the pathogenesis of chronic infections. In pathogenic Burkholderia species, biofilm formation is a severe complication, and it makes bacteria resistant to host defenses and antibiotics. BifR (Biofilm regulator) is a recently described redox‐sensitive member of the multiple antibiotic resistance regulator (MarR) protein family that has been found to repress biofilm formation in Burkholderia thailandensis. BifR appears to regulate the expression of other proteins critical to biofilm formation, but its overall functions are poorly understood. BifR is encoded in the same operon with an efflux pump (emrB) and divergently oriented to a gene (ecsC) that codes for a protease (LasA) that has been shown to be implicated in virulence by its contribution to elastin degradation in Pseudomonas aeruginosa. Previously, BifR was shown to repress the expression of emrB‐bifRand ecsCand to repress the expression of these genes further under oxidizing conditions. However, the substrate of the efflux pump (EmrB) is unknown. To determine the substrate of EmrB, we carried out antibiotic sensitivity tests on the Wild Type (WT), emrB‐bifRmutant and bifRmutant strains. Following a 24‐hour incubation at 37 oC, our results showed that the bifR mutant was resistant to novobiocin at concentrations of 50 ug/ml, 75 ug/ml and slightly resistant at 100 ug/ml. The bifRstrain was also more resistant to ampicillin at concentrations of 50 ug/ml, 75 ug/ml and 100 ug/ml. Similar results were obtained for the related β‐lactam, penicillin G. This indicates that novobiocin and β‐lactam antibiotics are substrates for EmrB. We also conducted onion tissue maceration and tested for the survival of Caenorhabditis elegans after infection with the three strains. Our results showed that after 72 hours of incubation of the onions at 30 oC, the bifRmutant strain was more virulent compared to the WT.Meanwhile, with a 70 % survival of the C. elegansafter 12 hours of incubation at room temperature, the emrB‐bifRmutant strain was more virulent. This study reveals that BifR plays a role in virulence and that the role may be host specific.
Divalent cation driven liquid‐liquid phase separation of disordered acidic proteins
Authors: Mayfield Joshua E. , Newton Alexandra C. , Dixon Jack E.
Published: May 2022

Divalent cations are essential to cellular processes through interaction with biological macromolecules, involvement in enzyme‐mediated catalysis, and as secondary messengers in signaling cascades. Recently, we have discovered divalent cation driven liquid‐liquid phase separation (LLPS) underlies endoplasmic reticulum Ca2+ stores through disordered acidic calcium binding protein calsequestrin‐1 (CASQ1). CASQ1 interacts with divalent cations to enter a LLPS state via complex coacervation. CASQ1 LLPS propensity is positively regulated by FAM20C‐dependent phosphorylation that induces an order‐to‐disorder transition accompanied by dramatic structural expansion. These events increase intracellular Ca2+ stores and regulate cellular stress response. Proteome wide analysis of disordered acidic proteins suggests divalent cation driven LLPS may be an emerging mechanism extending beyond the ER. Particularly, these proteins are highly enriched in the nucleus and cytosol where they accumulate in a number of biological condensates alongside other polyanions like RNA to regulate gene expression. We hypothesize divalent cation driven LLPS is a widespread mechanism driving both protein‐protein and protein‐nucleic acid interactions.
Fusion gene transcripts to predict prostate cancer clinical outcome
Authors: Luo Jianhua , Liu Silvia , Yu Yanping
Published: May 2022

Prediction of the clinical outcomes of prostate cancer remains a challenge. Recently, we discovered a panel of 8 fusion genes that occurred in aggressive prostate cancer. In order to make the fusion gene test clinically ready as a predictor, we have modified to test into a semi‐quantitative Taqman QRT‐PCR. In the funding period, Two hundred seventy‐one prostate cancer samples with clinical follow‐up were collected from University of Pittsburgh Medical Center. In addition, 194 prostate cancer samples from University of Wisconsin, Madison and 108 prostate cancer samples from Stanford University were collected. Taqman QRT‐PCRs were performed on these samples. Significant numbers of samples were found positive for some of these fusion genes. The expression of MAN2A1‐FER, SLC45A2‐AMACR, MTOR‐TP53BP1 fusions are associated with prostate cancer recurrence in the UPMC cohort. Cross‐validation showed that fusion gene model predicts up to 91% clinical outcomes of prostate cancer accurately. When cohorts of UPMC, Stanford and Wisconsin were combined, the accuracy is 74%. The combination of fusion with Gleason appeared to improve the overall accuracy from 77% (Gleason) to 92% (Gleason+fusion) in the UPMC cohort, and from 71% (Gleason) to 82% (Gleason+fusion) when all three cohorts are combined. When fusion combined with both pathology stage and Gleason, the accuracy was improved a little further: 93% accuracy in the UPMC cohort and 83% when all three cohorts are combined. In summary, fusion transcript prediction model may have a role in prostate cancer prognosis prediction and guiding the management of prostate cancer patients.
The Role of MUC1 in Lactation‐Associated Bone Mineral Conservation
Authors: Ray Evan C. , Al‐Bataineh Mohammad , Lam Tracey , Kinlough Carol C. , Marciszyn Allison L. , Barbour Jenna , Tourkova Irina , Blair Harry C. , Kleyman Thomas R. , Hughey Rebecca P.
Published: May 2022

To provide bone minerals to nursing offspring, lactating females dramatically up‐regulate their own renal and gastrointestinal transport processes to maximize conservation of these minerals. This includes increased expression of Ca2+ and Mg2+‐selective Trp channels in the kidney’s distal convoluted tubule (DCT) and in the duodenum. The transmembrane glycoprotein, Mucin 1 (MUC1) is expressed in both of these epithelia and interacts with Trp channels, enhancing apical localization of the channels in vivo. Human polymorphisms in MUC1 are associated with altered blood Mg2+ and with differences in bone density. We observe that MUC1‐/‐ animals exhibit hypomagnesemia, but only mild reduction in blood Ca2+, and no change in daily urinary Ca2+ or Mg2+ excretion, in spite of reduced apical localization of TrpV5 in the kidney’s DCT, and TrpV6 in the duodenum. We hypothesized that MUC1‐/‐ dams would exhibit reduced ability to conserve Ca2+ and Mg2+ during lactation. We find that lactating females have reduced blood Ca2+ and increased urinary Mg2+ concentrations compared to wild‐type lactating dams. Additionally, pups from MUC1‐/‐ dams exhibit reduced bone mineral density compared to pups from control dams. These findings suggest an important role for MUC1 in minimizing excretion of Ca2+ and Mg2+ during lactation, an extreme physiologic stressor requiring conservation of these minerals.
Comparative Analysis of Annexin A1 (ANXA1) Protein Expression in Different Cancer Types
Authors: Olascuaga‐Castillo Karyn , Rubio‐Guevara Susana , Cáceres‐Andonaire Elena , Caballero‐Aquiño Olga , Hilario‐Vargas Julio , Altamirano‐Sarmiento Dan , Morgado‐Díaz José Andrés , Berezovski Maxim
Published: May 2022

Annexin A1 (ANXA1) is the first member of the Lipocortin family, a calcium‐dependent phospholipid‐binding protein with potent immunomodulatory activity. ANXA1 is related to cellular proliferation, apoptosis, progression, and metastasis in cancer, and its expression is variable depending on the tumor type. Our research will compare the expression of ANXA1 in various forms of cancer and establish if the protein is up‐or down‐regulated.

Methods

We analyzed 1904 immunohistochemistry datasets from The Human Protein Atlas (HPA). The samples represent 20 different forms of cancer from 455 patients. The expression of ANXA1 was compared to the same in tissue samples from 137 healthy subjects, which meant the tissues of origins of primary tumors. The levels of ANXA1 expression were quantified using a visual grading method based on staining intensity (I) and cell fraction stained (F). The Q score (combination of stained cell fraction and staining intensity) was obtained by the product of (F)*(I).

Results

9 of 20 cancer types showed increased protein expression (up‐regulation) comparison to their healthy counterparts. Eight cancer types showed decreased protein expression (down‐regulation). Three types of cancer showed no significant differences.

Conclusion

ANXA1 expression may serve as an important diagnostic marker for such a variety of different cancer types.
Structure‐Function Relationship of the 20‐HETE‐GPR75 pairing: Development and characterization of agonist, partial agonists, and receptor blockers
Authors: Pascale Jonathan V. , Wolf Alexandra , Kulaprathazhe Melissa‐Maria , Ali Samir , Kim Namhee , Froogh Ghezal , Adebesin Adeniyi M. , Falck John R. , Schwartzman Michal L. , Garcia Victor
Published: May 2022

20‐Hydroxyeicosatetraenoic acid (20‐HETE) and its receptor (20HR), GPR75 (Gq), exhibit diverse bioactions that promote the activation of pro‐hypertensive, ‐diabetic and ‐obesity signals. The pharmacological properties associated with analogues that target GPR75 remain unclear. The screening of 20‐HETE and synthetic 20‐HETE analogues using changes of intracellular calcium (iCa2+) in the endothelial cell line, EA.hy926, as a measure of 20HR activation, revealed that the compounds 20‐HETE (10 nM), sodium 20‐hydroxyeicosa‐5Z,14Z‐dienoate (20‐5,14‐HEDE) (10 nM) and sodium 14‐((6‐hydroxyhexyl)oxy)tetradec‐5(Z)‐enoate (5Z‐HOTE) (10 nM) promote significant and comparable elevations in iCa2+. In EA.hy926 cells, 20‐HETE elicited a half‐maximal effective concentrations (EC50) with respect to iCa2+ of 1.228 e‐9 M while 20‐5,14‐HEDE’s EC50 was 6.908 e‐10 M. The water‐soluble derivative of 20‐5,14‐HEDE, SOLAGO, exhibited a marked and leftward shift in the dose‐response with an EC50 of 2.702 e‐10 M. Interestingly, sodium 19(R)‐ and 19(S)‐hydroxyeicosa‐5(Z),14(Z)‐dienoate, analogues of 19(R)‐HETE, an endogenous 20HR blocker (20HRB), demonstrated partial agonist activity; elevating iCa2+to 1.6‐ and 2‐fold over baseline vehicle treatment, respectively. With respect to 20HR blockers, the water‐soluble 20HRBs 2,5,8,11,14,17‐hexaoxanonadecan‐19‐yl 20‐hydroxyeicosa‐6(Z),15(Z)‐dienoate (20‐SOLA) and N‐disodium succinate‐20‐hydroxyeicosa‐6(Z),15(Z)‐diencarboxamide (AAA) displayed potent half‐maximal inhibitory concentrations (IC50s) of 8.059 e‐10 and 5.356 e‐10 M, respectively. The 19(R)‐HETE analogue sodium (19(R)‐hydroxyeicosa‐5(Z),14(Z)‐dienoyl)glycinate (19(R)‐HEDGE) also demonstrated a strong yet less potent IC50 response of 6.715 e‐9 M. Further studies are necessary to better understand the structure‐function relationships between 20HR agonists, partial agonists, and receptor blockers. These data would allow for the development of novel 20HRBs for the treatment of various pathologies associated with elevations in 20‐HETE including hypertension, cancer, diabetes, and obesity.
Pharmacodynamics and Pharmacokinetics of the Non‐Fentanyl Synthetic Opioid, Isotonitazene, in Male Rats
Authors: Baumann Michael H. , Glatfelter Grant C. , Walther Donna , Walton Sara E. , Logan Barry K. , Krotulski Alex J.
Published: May 2022

Illicitly manufactured fentanyl is fueling the current overdose epidemic, and non‐fentanyl mu‐opioid receptor (MOR) agonists are emerging in street drug markets worldwide. The etonitazene analog, isotonitazene, is one example of a non‐fentanyl MOR agonist linked to overdose deaths. Little is known about the biological effects of isotonitazene in humans or animal models. To this end, we examined the pharmacodynamics, pharmacokinetics, and metabolism of isotonitazene in rats. Male rats were fitted with surgically implanted intravenous (i.v.) catheters and subcutaneous (s.c.) temperature transponders under ketamine/xylazine anesthesia. One week later, rats received s.c. isotonitazene (3, 10, or 30 μg/kg) or its vehicle, and blood samples (0.3 mL) were collected via catheters at 15, 30, 60, 120, 240 min post‐injection. Plasma was assayed for isotonitazene and its metabolites by liquid chromatography tandem mass spectrometry. Pharmacodynamic effects ‐ including hot plate latency, catalepsy score, and body temperature ‐ were assessed at each blood withdrawal. Isotonitazene produced dose‐dependent increases in hot plate latency (ED50=4.2 μg/kg) and catalepsy (ED50=8.7 μg/kg), while 30 μg/kg produced marked hypothermia. Isotonitazene concentrations in plasma rose linearly with increasing dose, Cmax (0.5 to 6.6 ng/mL) was achieved within 15 min, and drug half‐life ranged from 40 to 60 min. Isotonitazene metabolites were detectable but below the level of quantification. Analgesia, catalepsy, and hypothermia were correlated with mean isotonitazene concentrations. Radioligand binding assays revealed that N‐desethyl isotonitazene displays higher affinity at MOR (Ki=2.2 nM) than the parent compound (Ki=15.8 nM). Our findings reveal that isotonitazene is a MOR agonist that is ~1000‐fold more potent than morphine (ED50=4.2 mg/kg) as an analgesic agent. Plasma concentrations of isotonitazene are in the low ng/mL range, whereas metabolites are found in even lesser amounts. Although the N‐desethyl metabolite of isotonitazene displays high affinity at MOR, extremely low levels are formed in vivo. The ultra‐high potency of isotonitazene presents challenges for forensic detection and likely poses grave risk to users who are inadvertently exposed to the drug.
Heart Rate Fragmentation in Rats with Streptozotocin‐induced Diabetes
Authors: Silva Thais M. , Silva Carlos Alberto A. , Galdino Gabriela A. M. , Catai Aparecida Maria , Salgado Helio Cesar , Silva Luiz Eduardo V. , Fazan Rubens
Published: May 2022

Heart rate fragmentation (HRF) is a recently proposed approach to evaluate sino‐atrial instability characterized by the presence of numerous inflections points in a series of successive values of RR intervals (RRi). HRF has been shown to be increased in aging and coronary diseases and seems to be linked to the risk of life‐threatening cardiovascular events and death. We recently demonstrated that cardiac autonomic modulation in involved in HRF in rats. Moreover, HRF is increased in a rat model of heart failure. Diabetes mellitus (DM) markedly alters HRV indices and might affect HRF as well. Therefore, we hypothesize that HRF is altered in streptozotocin‐ (STZ) induced diabetic rats. Male Sprague‐Dawley rats were injected with STZ (50 mg/kg), or vehicle (citrate buffer) into penile vein. Development of diabetes was confirmed 72h after STZ by the presence of hyperglycemia (> 350 mg/dL). Electrocardiographic (ECG) recordings were performed in conscious rats, 7 days (acute) or 4 weeks (chronic) after STZ or vehicle administration. Series of RR intervals were generated and processed by the customized software PyBios as follows: RRi values were symbolized as “‐1”, “0,” or “1” when the differences between successive RRi were negative, null, or positive, respectively. Transitions between symbols “‐1” and “1” were labeled as “hard” (H) inflection points, while those between “‐1” (or “1”) and “0” were labeled as “soft” (S) inflection points. The percentage of inflection points (PIP) was quantified, as well as sequences of 4 consecutive symbols, so‐called “words”, computing the occurrence of words with zero (W0), one (W1), two (W2), or three (W3) inflections points. The occurrence of words with only hard (WH) or only soft transitions (WS) were also quantified. Both groups of diabetic rats were bradycardic as compared to their control counterparts. Data of HRF are shown in the table below. Occurrences (%, mean ± SEM) of PIP, words with 0 to 3 inflection points, and words with only soft or hard transitions of symbols. As hypothesized, diabetes affects HRF, increasing fragmented indices (PIP, W3 and WH) and decreasing fluent ones (W1 and WS). Moreover, our results showed that changes in HRF due to diabetes in this model is time‐dependent. In addition to traditional methods of analyzing heart rate variability, HRF is a new method that can contribute to the diagnosis and prognosis of cardiovascular diseases in diabetic patients. Further studies are necessary to understand better the mechanisms involved in our findings.
Cyclic di‐GMP Signaling in Paracoccus denitrificans
Authors: Alatishe Aishat , Serrano Fred , Islam Mohammad S. , Yukl Erik
Published: May 2022

Cyclic di‐GMP (c‐di‐GMP) signaling has been discovered to regulate many functions such as biofilm formation, motility, and virulence in gram negative bacteria. Intracellular concentrations of this secondary messenger are controlled by activities of diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes, which synthesize and degrade it, respectively. In general, elevated levels of c‐di‐GMP are associated with increased biofilm formation. However, a hyperbiofilm phenotype in a DGC mutant of Paracoccus denitrificans suggests that c‐di‐GMP downregulates biofilm formation in this organism. Further, a gene for the heme‐based sensor protein H‐NOX, which has been implicated in biofilm formation in other organisms, is found adjacent to one of these DGC genes. Consistent with previous observation, we show that the Δhnox mutant of P. denitrificans is deficient in biofilm formation. We further use mass spectrometry to determine levels of intracellular c‐di‐GMP and proteomics to demonstrate altered expression and phosphorylation for several proteins, indicated signaling pathways perturbed by the mutation. In order to understand the mechanism of H‐NOX action, it and its cognate DGC were expressed and purified from E. coli. P. denitrificans H‐NOX lacks the proximal His residue found in other H‐NOX homologues to coordinate the heme cofactor. Indeed, this H‐NOX purifies with no heme bound, making it uncertain how it could act as a sensor of environmental conditions. We have performed activity assays for the DGC in the presence and absence of H‐NOX to test its function as a DGC inhibitor and are currently working to determine structures of these proteins by X‐ray crystallography or NMR. Taken together, this work advances our knowledge of the diverse mechanisms by which c‐di‐GMP regulates biofilm formation in bacteria.
Synthesis of the transmembrane domain of the accessory protein ORF7a of SARS‐CoV‐2 using solid phase peptide synthesis and analyzing the oligomerization state
Authors: Hobart Tyler G. , Reichart Timothy M.
Published: May 2022

This research project consists of synthesizing and characterizing the accessory protein ORF7a of SARS‐CoV‐2. ORF7a plays a role in viral assembly and inhibition of this protein would prevent viruses from assembling and spreading within a host’s cells. The goal of this project is to determine the oligomerization state of the protein through a fluorescence assay, which would determine the protein’s structure and how it folds. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Synthesis of the protein was done using a solid phase peptide synthesis technique. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Fluorescence assay was performed using a UV‐vis spectrometer. This presentation will highlight current progress with the oligomerization state with the goal of devising an effective inhibitor for ORF7 activity in SARS‐CoV‐2 particle assembly.
Targeting Keratin 17 in Pancreatic Cancer: A Novel Rewired Pathway of Nucleotide Metabolism that Drives Chemoresistance
Authors: Pan Chun‐Hao , Tseng Robert , Donnelly Katie , Leiton Cindy V. , Hogg Simon J. , Marchenko Natalia , Singh Pankaj K. , Shroyer Kenneth R. , Escobar‐Hoyos Luisa F.
Published: May 2022

Pancreatic ductal adenocarcinoma (PDAC) is characterized by two molecular subtypes, of which the basal‐like subtype is associated with the worst survival and is highly resistant to the first‐line chemotherapy. We previously reported that keratin 17 (K17), a signature gene in basal‐like subtype, is a novel negative‐prognostic and predictive biomarker, whose overexpression results in resistance to Gemcitabine (Gem) and 5‐fluorouracil, the major chemotherapeutic agents in standard‐of‐care treatments, and leads to shortened patient survival. Here, we set out to uncover the mechanisms of chemoresistance and explore targeted therapies for K17‐expressing PDAC. We hypothesized that K17 reprograms cancer metabolism and leads to therapeutic resistance. We manipulated the expression of K17 in multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells and orthotopic xenografts, for drug‐testing, metabolomic and mechanistic studies. To uncover the mechanisms associated with K17‐induced chemoresistance, we performed unbiased metabolomic studies in isogenic PDAC cell lines and found that compared to control cells, K17 increases intracellular levels of deoxycytidine (dC) by four‐fold that promote Gem (dC analogue) resistance. Interestingly, K17‐expressing cells are more sensitive to a compound that targets de novo pyrimidine biosynthesis. Based on previous findings that K17 enters nucleus to regulate gene expression, weexplored whether K17 triggers metabolic reprogramming at the transcriptional level and found that enzymes involved in pyrimidine biosynthesis are positively correlated with K17 expression in PDAC cells. Given that it is still poorly understood how K17 regulates gene expression, we performed domain‐prediction analyses. We discovered and validated a novel chromatin remodeling domain in K17 that is required for metabolic reprogramming. Importantly, mice bearing tumors with the deletion of the chromatin remodeling domain in K17 survived significantly longer than those with tumors expressing wild type K17. We are now performing ATAC‐seq, ChIP‐Seq and RNA‐Seq to understand how this domain alters pyrimidine biosynthesis. In summary, we identified a novel pathway of chemoresistance that could result in developing a biomarker‐based personalized therapy.
Mathematical and Experimental Analysis of Body Temperature Fluctuations During Weight Change
Authors: Concepción‐Díaz Lemuel , Perez‐Salas Rosanie A. , Martinez‐Boschetti Javier , Rolon‐Rey Raphel , Esteras‐Robles Sebastian , Malpica‐Diaz Kimberly , Monagas‐Vazquez Shalyver , Pereira‐Rodriguez Fernando , Arencibia‐Albite Francisco
Published: May 2022

Human body temperature fluctuates in proportion to the balance between metabolic heat production and heat dissipation and needs to remain nearly constant as a requirement for normal physiology. The biology of temperature homeostasis is well‐understood, and a low resting body temperature has been linked to increased longevity in humans and other organisms. Substantial weight loss in rodents, as result of underfeeding, leads, for instance, to a significant drop in mortality that occurs in parallel to a reduction in rectal temperature. The effects of weight gain or obesity on resting temperature are, however, not understood; and for the most part are controversial, e.g., obesity increases mortality, yet some lines of research argue that a low body temperature underlies its pathophysiology. The present work uses mathematical modeling and analytical methods to investigate the effects of body weight fluctuations on temperature. The latter methodology leads to a simple equation that estimates the whole‐body heat transfer coefficient (hc). Computational simulations, on the other hand, predict two possible scenarios: 1. Resting temperature is an increasing function of body mass if hc is unaltered by weight change; 2. If body temperature is insensitive to weight change, then hc will change in the same direction as body weight. Experimental measurements validate the first case and show that obesity leads, in men and women, to a significant increment in resting temperature relative to normal body mass index subjects. This analysis also shows that hc is a decreasing power function of the temperature gradient, i.e., the difference between the body temperature and the external temperature. Our data indicates, overall, that another mechanism by which obesity increases human mortality is by producing a persistent increment in body temperature. To our knowledge, we are the first to identify such adverse obesity effect.
Isolation and characterization of the novel Mycobacterium smegmatis phage Dawnguard
Authors: Konieczka Tobias A. , Wolyniak Michael J.
Published: May 2022

Bacteriophages are the most ubiquitous organisms found within the biosphere, composed of an estimated 1031 particles worldwide. The evolutionary “arms‐race” between bacteriophages and bacteria have set the stage the natural selection process that has underpinned biological complexity. Because of the sheer volume of phage particles, it is practically impossible to identify every unique phage available; however, the combined power of laboratory techniques and computer prediction software has made the identification of a few of these phages possible. Under the guidance of the Howard Hughes Medical Institute (HHMI) Science Education Alliance‐Phage Hunters Advancing Genomics and Evolutionary Science (SEA‐PHAGES) isolation guide, solid and liquid samples from three locations across the Hampden‐Sydney College campus in central Virginia were tested for the presence of M. smegmatis phage. Although we were unable to isolate novel phages from these areas, we were successful in performing bioinformatic characterization of a phage previously isolated but not studied in detail. Using the computer programs DNA Master and Phage Evidence Collection and Annotation Network (PECAAN), we discovered a significant error in previous annotations done on the M. smegmatis phage Samcrothat we were able to correct prior to submission of the phage's gene annotation to GenBank for publication. The phage, renamed Dawnguard, possesses several genes of interest, including a glycosyltransferase, immunity repressors, and a resolvase, that may explain its unique adaptations to effective reproduction in M. smegmatis.
Regional binding of [3H]‐MK801 to NMDA receptors in young and aged rat brain membranes
Authors: Aljohani Yousef , Dezfuli Ghazaul , Kellar Kenneth J.
Published: May 2022

Norepinephrine (NE) and dopamine (DA) are essential neuromodulators in the central nervous system. They regulate several crucial physiological processes, including arousal/attention, focus, neuroendocrine functions, and the sleep‐wake cycle; they also play complex roles in regulating cognition and affect; therefore, dysfunctions in catecholaminergic neurotransmission are associated with various neurological consequences. Previously, we confirmed that in brain slices from aged Fischer 344 rats, there is an age‐related decrease in glutamate‐stimulated NE release in the cerebral cortex and hippocampus (Gonzales et al., 1991 & Dezfuli et al., 2019) and a regionally specific decline in glutamate‐stimulated DA release, with a deficit in the striatum but not in the olfactory tubercle (Aljohani et al., 2021a). Importantly, our studies demonstrated that NMDA receptors mediate glutamate‐stimulated NE and DA release. We also have shown that the alpha‐2 adrenergic receptors agonist clonidine decreases this glutamate‐stimulated NE release in a concentration‐dependent manner; while in contrast, blockade of these receptors with phentolamine, an alpha‐1 and alpha‐2 adrenergic receptor blocker, or with idazoxan, a selective alpha‐2 receptor blocker, markedly potentiates glutamate‐stimulated NE release in cortical brain slices (Aljohani et al., 2021b). Since aging has heterogeneous effects on NMDA receptors in different brain areas, we have now begun to examine [3H]‐MK801 binding to NMDA receptors to compare changes in receptor density and responsiveness during aging in the cortex, hippocampus, striatum, and olfactory tubercle, as a possible mechanistic explanation for the changes in NE and DA release. The data from cortical membrane preparations suggest that young and old rats have similar NMDA receptor densities but may differ in their response to stimulation with glutamate/glycine.
Maternal angiotensin II‐induced hypertension impairs salt sensitivity and estrogen regulation of salt/water intakes in female adult offspring
Authors: Xue Baojian , Beltz Terry G. , Guo Fang , Johnson Alan K.
Published: May 2022

Blood pressure (BP) is associated with body fluid homeostasis and the intake and excretion of water and salt. Female sex hormones play an important role in regulating salt and water intake. Recent evidence indicates that prenatal insults can have a strong influence on BP salt sensitivity in offspring. In this study, we investigated if maternal angiotensin II‐induced hypertension (MHT) alters BP salt sensitivity in female offspring exposed post‐weaning to a high salt diet (HSD, 4% salt, 12 weeks), and if estrogen modulates the HSD‐induced changes in BP and salt/water intake. Post‐weaning HSD feeding elicited a significant increase in BP in intact female offspring of normotensive (NT) dams when compared with normal salt diet (NSD) fed female offspring (MAP, 114.5±0.7 to 124.1±1.6 mmHg). However, HSD feeding did not alter BP in intact female offspring of MHT dams (MAP, 114.7±1.7 to 110.6±1.2 mmHg), indicating that MHT blunted salt sensitivity in the offspring. Ovariectomy (OVX) diminished the HSD‐induced BP increase in offspring of NT dams (111.2±1.6 mmHg) but surprisingly significantly increased BP in HSD fed offspring of MHT dams (118.5±1.2 mmHg). There were no differences in HSD and water intakes and body fluid composition (measured by nuclear magnetic resonance spectroscopy) between intact offspring of NT and MHT dams. OVX in offspring of NT dams resulted in significant increases in the intake of HSD and water, and in body fluid composition. The offspring of MHT dams had only a slight increase in HSD intake (i.e., less than that of offspring of NT dams), and they had no changes in water intake or body fluid composition. This suggests that estrogen effects on salt and water intakes were compromised by MHT. The results indicate that MHT induced by angiotensin II during pregnancy may downregulate salt sensitivity and compromise the regulatory action of estrogen to salt and water intakes, thereby altering the BP response to HSD. It is notable that these results seem to be contrary to current dogma that prenatal insults sensitize the development of hypertension and that estrogen plays protective role. These observations need further investigation.
Differences in Cellular Metabolism and Metabolic Regulation between Non‐diabetic and Diabetic Human Coronary Artery Endothelial Cells
Authors: Xu Cynthia M. , Karbasiafshar Catherine , Braga Karla M. S. , Teixiera Rayane B. , Sellke Frank W. , Abid M. R.
Published: May 2022

Objective

To determine the differences in cellular metabolism and metabolic regulation in non‐diabetic and diabetic human coronary artery endothelial cells (HCAEC and DM‐HCAEC) to further the understanding of the effects of metabolic syndrome on endothelial dysfunction in the setting of cardiovascular disease.

Methods

Both HCAEC (n = 5) and DM‐HCAEC (n = 5) were cultured to passage 6. The cells were then starved for 18 hours. After treatment, a Reactive Oxygen Species (ROS) Assay was performed, and cell lysates were made for proteomics analysis and western blotting.

Results

The ROS assay demonstrated that the total ROS was significantly higher in DM‐HCAEC (p<0.0001) than in HCAEC. Proteomics analysis and western blotting showed that DM‐HCAECs had significantly altered cellular transport, RNA processing, protein biogenesis, and antioxidant pathways. Proteomics analysis demonstrated that there were about 1,500 proteins significantly upregulated or downregulated in DM‐HCAEC compared to HCAEC. Analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed a significant increase in the “vesicle‐mediated transport” pathway and significant decrease in the “mRNA metabolic process” pathway in DM‐HCAEC with false discovery rates (FDR) of 1.40 x 10‐58 and 4.90 x 10‐92, respectively. Western blot analysis showed a significant changes in phosphorylated AMPK/total AMPK, phosphorylated mTOR (serine 2481), total mTOR, and several enzymes responsible for the regulation or elimination of ROS (Figure 1). The increased levels of AMPK activation and dysregulation of mTOR may be due to the excessive ROS in DM‐HCAEC, which are consistent with the ROS assay findings and significant changes in expression of enzymes that control ROS levels. Further research is needed to determine the mechanisms of downstream effects.

Conclusions

AMPK and mTOR are key regulators of energy homeostasis, cellular growth and metabolism, and are altered in diabetes in the setting of increased oxidative stress. These findings will guide further experiments to assess the effect of metabolic syndrome and diabetes on therapeutics targeted at improving the outcomes of both acute and chronic myocardial ischemia. This also underlines the need for further development of therapies or the use of current therapies to address the metabolic disturbances found in diabetes to improve clinical outcomes in cardiovascular disease.
Liver Proteomic Profile in Mice on a High‐Fat Diet: Modulation with Anti‐Tumor Intervention
Authors: Harber Isabelle , Zedan Dania , McClintock Shannon , Varani James , Aslam Muhammad N.
Published: May 2022

ntroduction

Nonalcoholic fatty liver disease (NAFLD) is becoming a primary cause of liver damage in Western society. Nonalcoholic steatohepatitis (NASH) is an advanced stage of NAFLD and may lead to liver cancer. Our previous long‐term murine studies have shown the beneficial role of Aquamin, a red marine algae‐derived (calcium, magnesium, additional trace element‐rich) supplement, in reducing liver injury and decreasing liver tumor incidence. The main objective of the study was to determine how manifestations and effects of NASH can be mitigated using Aquamin. We hypothesized that mice on a high‐fat Western diet (HFWD) with minerals would exhibit less liver injury than mice on a HFWD without minerals.

Methods

Two cohorts of MS‐NASH mice (8‐week‐old) were placed on a HFWD with and without minerals for 16 weeks. An additional cohort of MS‐NASH mice on the same HFWD was treated with obeticholic acid (OCA). C57BL/6 mice on a regular chow were included as a control. During the in‐life phase of the study, weight changes were assessed weekly. At termination, livers were histologically assessed for steatosis and fibrosis by using hematoxylin and eosin, and picrosirius red staining. Liver tissue samples were also subjected to tandem mass tag (TMT) mass‐spectroscopic proteomics for protein expression profile in individual mice.

Results

Mice on HFWD gained more weight than mice on control diet, but there was no overall change in weight for mice on HFWD, irrespective of interventions. On histological assessment, there was no difference in steatosis between the two high‐fat groups, but mice on Aquamin showed reduced picrosirius red staining or collagen deposition as compared to placebo (on HFWD) mice. Regarding proteomic profile, there was a clear distinction among control and HFWD groups. Placebo mice were used for comparison. Aquamin intervention altered 91 proteins while OCA altered 84 proteins in liver samples with a 2‐fold change. Of these, 57 proteins were common between Aquamin and OCA. Most of the proteins were upregulated. Aquamin upregulated 39 unique pathways (with a p‐value <0.05) as assessed by the Reactome database. Some of the pathways significantly impacted by Aquamin were formation of cornified envelope; keratinization; gap junction assembly; type I hemidesmosome assembly; apoptosis‐related; plasma lipoprotein assembly, remodeling and clearance; and hedgehog “off” state pathways. Proteins downregulated by OCA are mainly involved in the synthesis of bile acids and bile salts.

Conclusion

The addition of dietary minerals may play a protective role in interfering with downstream advancement from steatosis to NASH. Our studies provide mechanistic insight into how mineral supplementation may contribute to the reduction in liver injury and tumor formation (one of the most devastating consequences) of fatty liver disease in the context of HFWD‐induced steatosis.
Aberrant glycogen accumulation alters gene expression and promotes lung tumor progression in vivo
Authors: Lamb Jessica F. , Conroy Lindsey R. , James Alexis N. , Buoncristiani Michael D. , Relich Eric R. , Brainson Christine F. , Sun Ramon C.
Published: May 2022

Lung cancer is one of the most prevalent cancers worldwide, accounting for nearly two million new cases each year. Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer and is the leading cause of cancer related mortalities worldwide. LUAD has an extremely poor five‐year survival rate, which is likely due to cases being diagnosed at advanced stages and thus being more difficult to treat the metastatic disease. Identifying the molecular processes that contribute to disease progression is a critical goal in lung cancer research that could lead to predictive biomarkers and novel therapies. Glycogen is the primary form of carbohydrate storage in mammalian cells, and its degradation product, Glucose 6‐Phosphate (G6P) feeds directly into glycolysis, making glycogen intimately connected to central carbon metabolism. Recently, aberrant glycogen accumulation in lung tumors has been reported to promote lung cancer progression, driven by largely unknown mechanisms. We recently generated a transgenic KRASG12D/p53‐/‐LUAD mouse model that also lacks the glycogen phosphatase laforin (LKO). This model develops increased glycogen accumulation in the lungs and displays accelerated tumor growth compared to the KRASG12D/p53‐/‐control (WT), as measured by Ki67 staining. To identify potential biological processes perturbed by LUAD‐glycogen, we performed RNA‐sequencing on lung tumors resected from WT and LKO mice. We identified over 300 genes that are differentially expressed between WT and LKO lung tumors. Specifically, gene set enrichment analysis (GSEA) revealed genes that were up‐regulated in LKO lung tumors were involved in extracellular matrix remodeling, cell‐to‐cell communication, and cell proliferation. All of these processes are implicated in tumor growth, which supports our initial observation that LKO mice have increased tumor burden and disease progression. Our findings ultimately highlight several potential mechanisms by which LUAD‐glycogen promotes LUAD tumor progression, which can be further investigated to identify novel biomarkers and therapeutic targets.
ErbB3 Promotes Intestinal Barrier Function and Expression of the Tight Junctional Protein Pmp22
Authors: Hsieh Jonathan J. , Almohazey Dana , Frey Mark R.
Published: May 2022

Background

ErbB3 is a member of the ErbB/EGFR family of receptor tyrosine kinases, and is the constitutively‐expressed neuregulin (NRG) receptor in the intestinal epithelium. Pathways downstream of ErbB3 have been implicated in regulating tight junctions in other systems, but a role for ErbB3 in barrier function in the gut has not been described. Here we tested the hypothesis that ErbB3 is required for maintenance of normal barrier function in the intestine.

Methods

We generated Villin‐Cre;ErbB3flox/flox mice (ErbB3‐IEKO) with ErbB3 deletion in the intestinal epithelium. All comparisons are to ErbB3flox/flox littermate controls. Mice were given a FITC‐dextran 4 kDa gavage to assess intestinal permeability. Ileal samples were collected for RT‐qPCR, immunohistochemistry, and bulk RNA‐seq analysis. Enteroids were generated and treated with the ErbB3 ligand NRG‐1β. Caco‐2BBe cells were grown on Transwells, treated with NRG‐1β, and assessed for TEER and FITC‐dextran (4kDa) flux.

Results

ErbB3‐IEKO mice displayed increased intestinal barrier permeability to 4kDa FITC‐dextran compared to controls (p < 0.01). RNA‐seq analysis showed a decrease in the expression of the putative tight junction protein Pmp22in ErbB3‐IEKO mice, while other tight junctional components were unchanged. RT‐qPCR analysis of Ileal samples showed an 80% Pmp22downregulation with ErbB3 loss (p < 0.01) compared to controls, with similar results in ErbB3‐deficient enteroids. Immunohistochemical analysis confirmed near complete loss of PMP22 in ErbB3‐IEKO mice. In vitro, treatment with NRG‐1β induced Pmp22expression in control enteroids (p < 0.001) but not in ErbB3‐deficient enteroids. Furthermore, treatment of Caco‐2BBe monolayers with NRG‐1β increased TEER and reduced FITC‐dextran flux, demonstrating active regulation of permeability.

Conclusions

We demonstrate that loss of ErbB3 in the intestinal epithelium leads to reduced expression of the tight junction protein PMP22 and impaired barrier function in mice. This may represent a mechanism for regulation of the intestinal barrier through ErbB3 and PMP22. Furthermore, increased TEER in Caco‐2BBe monolayers following NRG‐1β treatment suggests induction of PMP22 may represent a means to enhance barrier function. Since disrupted intestinal barrier function contributes to the pathophysiology of chronic inflammatory conditions such as inflammatory bowel disease, these results may point to potential future therapeutic interventions targeting the barrier.