bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Expression Changes Associated with Acute Mountain Sickness After Rapid Altitude Exposure at The South Pole
Authors: Byrd M. T. , Wheatley‐Guy Courtney M. , Grill Diane E. , Shea Meredith G. , Herman Nicole M. , Johnson Bruce D.
Published: May 2022

Many individuals travel to high altitude each year. Acclimatization to altitude occurs over the course of several days as the body adequately adapts to function in an environment with reduced partial pressure of oxygen. However, with a faster accent to attitude, comes an increased potential for maladaptations to occur, leading to the development of acute mountain sickness (AMS). Each year scientists and support workers are transported by plane from McMurdo Station in Antarctica (sea level) to the Amundsen‐Scott South Pole Station (2835m). This uniform and rapid deployment to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression, which may help to illustrate the body’s ability to acclimatize to these environmental conditions.

Purpose

To detect pathway specific gene expression changes associated with the development of AMS and help identify potential targets that may aid in the prediction of risk in the development of AMS symptoms due to rapid exposure to high altitude.

Methods

Venous blood samples were collected from 53 (height 176.65±8.91cm, weight: 80.49±14.36kg) healthy subjects (38 (27 males; 11 females) that did not develop AMS and 15 (10 males; 5 females) that did develop AMS, as defined by the Lake Louis symptom questionnaire, collected at 2 different time points, with the first being at sea level and the second being after 48hr of altitude exposure). Microarray analysis was performed on the peripheral blood mononuclear cells (PBMCs) from the collected samples, and a logistic regression was performed to determine probe set association with AMS.

Results

There was a total of 178 significant (p<0.05) probe sets, with 156 of the probe sets being associated with AMS and 22 of the probe sets being associated with no AMS. The probe sets were entered into the Reactome Pathway Database (reactome.org). Pathways identified in association with AMS involved immune system pathways (interleukin signaling) and cellular responses to stimuli pathways (oxidative stress induced senescence signaling), and gene transcription pathways (mitochondrial biogenesis signaling, differentiation of hematopoietic stem cells). Interestingly, pathways identified in association with no development of AMS involved cell cycling pathways (mitotic signaling), chromatin organization pathways (chromatin modifying enzymes), metabolism pathways (utilization of ketone bodies) and the circadian clock pathway (circadian gene expression signaling).

Conclusion

These findings indicate potential maladaptive responses within the immune system and mitochondrial function, may play a key role in why some individuals develop AMS symptoms. An individual’s ability to sufficiently utilize different metabolic fuel sources and adapt their circadian rhythm may also prevent the development of AMS symptoms.
Statins stimulate voltage‐gated Ca2+ channels in arterial smooth muscle cells to induce rapid vasoconstriction in resistance‐size cerebral arteries
Authors: Zerin Farzana , Menon Sreelakshmi , Hasan Raquibul
Published: May 2022

Statins are cholesterol‐lowering drugs with a range of beneficial vascular effects. Vascular effects are believed to result from long‐term statin therapy and reduction of cholesterol and mevalonate biosynthesis, and the inhibition of mevalonate‐dependent Rho/ROCK signaling. However, no studies have investigated the direct effects of acute statin application on fresh isolated resistance cerebral arteries using therapeutic concentrations of statins. Here, we examined acute vascular effects of rosuvastatin and simvastatin on Sprague Dawley rat cerebral arteries and underlying molecular mechanisms. We used pressurized arterial myography, simultaneous measurement of vessel Ca2+ fluorescence and diameter, pharmacological modulation, RNAi, Western blotting and biotinylation. Intravascular pressure in the mounted cerebral artery segments was gradually increased to 60 mmHg to stimulate the development of myogenic tone. At 60 mmHg, cerebral arteries developed ~36% myogenic tone, after which a cumulative dose response (0.01‐1000 nM) to rosuvastatin was performed and diameter changes recorded. Our data shows that, at 0.1, 1 and 10 nM concentrations, rosuvastatin induced a potent vasoconstriction – an observation never reported previously with any statin. Intriguingly, rosuvastatin, at 100 nM and 1000 nM concentrations (supratherapeutic), abolished myogenic tone and caused robust vasodilation. Rosuvastatin‐induced vasoconstriction reached a plateau at 1 nM concentration, which was used as reference for mechanistic studies. Within 2‐3 minutes of application, rosuvastatin and simvastatin constricted cerebral arteries by ~26 μm and ~24 μm, respectively. Endothelium denudation (intact ~23 μm vs denuded ~25 μm), or mevalonate supplementation did not alter statin‐induced vasoconstriction (control ~28 μm vs mevalonate ~29 μm), suggesting endothelium‐ and HMG‐CoA reductase‐independent mechanism. In contrast, removal of extracellular Ca2+with EGTA (~6 μm) or the application of nimodipine (~5 μm), a selective blocker of smooth muscle cell voltage‐gated Ca2+ channel, CaV1.2, or siRNA knockdown of CaV1.2 each abolished cerebral artery vasoconstriction and reduced [Ca2+]i, indicating that the Ca2+ entry through CaV1.2 plays a critical role here. Application of thapsigargin, a blocker of SR/ER membrane Ca2+‐ATPase pump, SERCA, but not ryanodine, a blocker of ryanodine receptor‐mediated Ca2+ release, fully reversed statin‐induced cerebral artery constriction and reduced [Ca2+]i. Arterial biotinylation data showed that short‐term statin exposure did not alter the surface expression, distribution and function of CaV1.2 channels.

Altogether, our data demonstrate that statins at therapeutic concentrations directly stimulate smooth muscle cell CaV1.2 and Ca2+ influx to induce rapid vasoconstriction in resistance cerebral arteries.
Targeting Neuropilin‐2 in Lymphatic Malformation
Authors: Gao Yao , Balasubbramanian Dakshnapriya , Greene Arin , Chen Hong , Bielenberg Diane
Published: May 2022

Lymphatic malformations (LM) are sporadic, non‐familial congenital lesions consisting of dilated, abnormal lymphatic vessels that can cause severe lymphedema, disfigurement, organ dysfunction, and in some cases morbidity. These vascular anomalies grow slowly but progress over time and recur after surgery; no effective drugs or preventions currently exist for LM. Genome sequencing identified that approximately 80% of LM patients have somatic activating mutations in the PIK3CA gene. Lymphatic endothelial cells (LEC) expressing mutant PIK3CAH1047Rinduce both macrocystic and microcystic LM in mouse models. Our goal is to understand the cellular and molecular processes that regulate LM growth in order to develop novel therapeutic strategies for patients with LM. Neuropilin‐2 (NRP2) is an essential co‐receptor for VEGFC/VEGFR3 signaling through the PI3K/AKT pathway which is crucial for LEC viability and motility, yet NRP2 also mediates inhibitory signals from its Semaphorin‐3F (SEMA3F) ligand to attenuate activation of AKT and induce cytostasis. Our results using immunohistochemistry to detect NRP2 protein in paraffin‐embedded human samples indicate that NRP2 is highly upregulated in LM lesions compared to normal lymphatic vessels. LM‐LEC primary cell lines, derived separately from several patients, show increased NRP2 levels and hyper‐activated AKT compared to primary LEC derived from healthy human dermis. Additionally, LEC isolated from the skin of transgenic mice (prox1‐creERT2;Nrp2 f/f) specifically lacking Nrp2 in lymphatic vessels following tamoxifen induction (LEC‐Nrp2‐KO) show suppressed proliferation and migration following VEGF‐C stimulation compared to normal LEC isolated from wildtype mice. Importantly, SEMA3F exogenous protein, in a dose‐dependent manner, inhibits the proliferation of normal LEC and LM‐LEC and attenuates the phosphorylation of AKT. Taken together, our study suggests that NRP2 may be an important target for therapy in LM patients. Future studies using SEMA3F protein or specific antibodies targeting the VEGF‐binding domain of NRP2 will be tested in preclinical trials.
Role of the Proximal Region of CYP1A2 in the heteromeric P450 complex with POR and Cytochrome‐b5
Authors: Saha Aratrika , Reed J. R. , Connick J. P. , Backes Wayne L.
Published: May 2022

Previous studies have shown that CYP1A2 is capable of forming a CYP1A2‐CYP1A2 homomeric complex that involves the proximal region of the enzyme. Using molecular cloning, specific residues on the proximal face of CYP1A2 (residues L91‐K106: P1 region, residues P131‐S155: P2 region and residues T434‐K447: P3 region) were swapped with homologous residues from CYP2B4 (CYP2B4: T92‐S107 for P1, E132‐E163 for P2 and D438‐R448 for P3). CYP2B4 was chosen as we have evidence that shows that CYP2B4 does not form a homomeric complex in vitro. These constructs were then expressed in HEK‐293T cells and bioluminescence resonance energy transfer (BRET) was used to measure complex formation and disruption. The P1 region of the proximal face was shown to be involved in the homomeric interaction. Interestingly, when we examined the activity of the P1 chimera, lowered activity was observed which suggested that the CYP1A2‐POR (NADPH‐cytochrome P450 reductase) interaction had been affected. POR interacts with P450s on the proximal face. The activity data suggested that the P1 mutation had affected the ability of the chimera to associate with POR. Consequently, BRET competition studies established that the P1 region was involved in the CYP1A2‐POR complex. Subsequent studies with other residues from the proximal face (regions P2 and P3) suggested that these residues were not involved in either the CYP1A2‐CYP1A2 homomeric complex or the CYP1A2‐POR heteromeric complex. We have extended this analysis to examine the effects of these proximal CYP1A2 modifications on its ability to interact with cytochrome b5. BRET competition studies were performed with wild‐type CYP1A2 and wild‑type b5 (tagged with GFP and Rluc respectively) co‐transfected at a high GFP: Rluc ratio with increasing amounts of unlabeled CYP1A2, P1, P2 and P3chimera as the competitor. Preliminary data suggests that the unlabeled P1, P2 and P3chimera were unable to disrupt the wild‐type CYP1A2‐b5 complex as well as the unlabeled CYP1A2, thereby suggesting that the proximal face of CYP1A2 may be involved in the CYP1A2‐ b5 complex. Studies are underway to establish the role of the proximal face in the CYP1A2‐b5 complex and to elucidate the interactions between CYP1A2, POR and cytochrome b5.
Targeting HDAC6 in the Dorsal Root Ganglia Attenuates Peripheral Nerve Injury‐induced Hypersensitivity
Authors: Giosan Ilinca M. , Zimering Jeffery , Serafini Randal A. , Pryce Kerri D. , Zachariou Venetia
Published: May 2022

Histone deacetylase 6 (HDAC6) is a Class IIb histone deacetylase, which is primarily localized to the cytoplasm and plays an important role in cell structure and dynamism, transcriptional repression, exocytosis and endocytocis. Preclinical evidence has suggested that HDAC6 inhibitors alleviate signs of chemotherapy‐induced peripheral neuropathy (CIPN), such as mechanical allodynia. However, no group to our knowledge has investigated the mechanism of action of HDAC6 inhibitors in a severe nerve injury model, which has a different pathophysiology than milder models such as CIPN. In this study we use genetically modified mice and the tibial spared nerve injury (SNI) model to determine the impact of dorsal root ganglion (DRG) specific HDAC6 knockout in the induction and maintenance of sensory hypersensitivity. Downregulation of HDAC6 in the DRG was achieved by injection of the left sciatic nerve of adult male HDAC6fl\fl mice with AAV8‐CMV‐Cre‐EGFP or AAV8‐CMV‐EGFP vectors. DRG‐knockdown of HDAC6 prevented the development of mechanical allodynia after SNI. Using in situ hybridization (RNAScope), we also demonstrate that HDAC6 is upregulated at three weeks after the induction of nerve injury in L3‐6 DRG neurons. Furthermore, treatment with the peripherally acting HDAC6 inhibitor ACY1215 (Regenacy Pharmaceuticals, Waltham, MA) after SNI leads to full recovery from mechanical allodynia. We are continuing this work by performing bulk RNA sequencing on L3‐6 DRGs from mice who received 21 days of treatment with ACY1215 or vehicle starting at three weeks after SNI or Sham operation, in order to understand the transcriptomic events and upstream pathways associated with recovery from prolonged neuropathic states.

Our findings highlight a promising therapeutic role of HDAC6 inhibitors for the prevention or recovery from sensory hypersensitivity behaviors associated with prolonged peripheral nerve injury. Our future work aims to define the mechanisms underlying HDAC6 action in the DRG and identify new pathways associated with recovery from sensory hypersensitivity to influence novel treatment strategies for pain.
In Vitro FRET‐Based Helicase Activity Assay as a Drug Screening Tool
Authors: Sheehan Hugo , Fernander Gaddiel , Nyman Karl , Borysov Sergiy I.
Published: May 2022

The Cdc45‐Mcm2‐7‐GINS (CMG) helicase is a protein complex that unwinds double‐stranded DNA into single strands during DNA replication at the S‐phase. Previous research has pointed to the CMG helicase as a potential target for cancer treatment. Indeed, the CMG helicase activity is an essential and vulnerable component of the cell cycle machinery of cancerous cells. However, the development of anti‐cancer helicase targeting molecules requires a robust and quantifiable helicase assay.

Here we report a novel CMG helicase activity assay that utilizes FAM‐BlackFQ dual‐labeled DNA oligonucleotides as a substrate. The underlying principle of the Fluorescence Resonance Energy Transfer (FRET) allows for a quick, reliable, real‐time measurement of helicase activity in vitro. As a source of active CMG helicases, we prepared a nucleoplasmic extract (NPE) derived from the cell‐free system of Xenopus egg extracts, a canonical system widely used to study vertebrate DNA replication in vitro. This NPE is a rich source of the Xenopus CMG helicase, which is highly analogous to its human counterpart.

In this report, we present optimization of our novel assay. Remediation of potential contaminations with endogenous DNases is addressed. We utilized our helicase assay to study inhibition of the CMG helicase by therapeutically relevant peptides. We also extended its utility by screening natural compounds of the NCI/DTP library N5.
Peroxiredoxin 6 suppresses erastin‐induced ferroptosis in lung endothelial cells
Authors: Vazquez‐Medina Jose P. , Torres‐Velarde Julia M. , Allen Kaitlin N. , Salvador‐Pascual Andrea , Ensminger David C.
Published: May 2022

Ferroptosis is a newly described form of regulated cell death driven by an iron‐dependent accumulation of phospholipid hydroperoxides. Glutathione peroxidase 4 (GPx4) suppresses ferroptosis through its ability to reduce phospholipid hydroperoxides. Peroxiredoxin 6 (Prdx6) expresses phospholipid hydroperoxide glutathione peroxidase activity, but its role in the regulation of ferroptosis remains unexplored. We compared Prdx6 and GPx4 mRNA levels in murine lungs. Prdx6 expression was five times higher than GPx4 expression. We then visualized the spatial localization of lung Prdx6. Prdx6 was expressed in both pulmonary epithelial (epCAM+) and endothelial (Ve‐cadherin+) cells. We studied the role of Prdx6 on lung ferroptosis induced by erastin, an inhibitor of the cystine‐glutamate antiporter, which is critical to glutathione production. Erastin (1‐5μM, 24h) increased lipid peroxidation and cell death in mouse pulmonary microvascular endothelial cells (MPMVECs) in primary culture; this effect was higher in Prdx6‐KO MPMVECs than in WT‐MPMVECs. Co‐treatment with the ferroptosis suppressor ferrostatin‐1 prevented erastin‐induced lipid peroxidation and cell death in both WT and Prdx6‐KO MPMVECs. Similarly, knockdown (KD) of Prdx6 in human pulmonary microvascular endothelial cells (HPMVECs) increased erastin‐induced cell death, suggesting that Prdx6 limits ferroptosis in the pulmonary endothelium. We then conducted extracellular flux assays and RNA‐seq analyses in Prdx6‐deficient cells to further explore how the loss of Prdx6 sensitizes cells to ferroptosis. Cellular respiration and mitochondrial function were blunted in Prdx6‐deficient cells compared to corresponding controls. Top differentially expressed genes in Prdx6‐KD HPMVECs include known regulators of iron metabolism and ferroptosis such as hmox1, cybrd1, and sfxn1. Upregulated Reactome pathways enriched in Prdx6‐KD HPMVECs include mitochondrial and amino acid metabolism and p53‐mediated DNA damage response, while downregulated pathways include biological oxidations, transport of bile salts and organic acids, metal ions and amine compounds, cytochrome P450, and phase 1 metabolism. These results show that loss of Prdx6 sensitizes lung endothelial cells to ferroptosis by promoting lipid peroxidation and altering transcriptional signatures associated with cell cycle progression and mitochondrial metabolism.
Dynamic gating of substrate binding in β‐amylase2 from Arabidopsis thaliana
Authors: Berndsen Christopher , Monroe Jonathan
Published: May 2022

β‐Amylases (BAMs) in plants are hydrolases involved in starch degradation, which is important for providing sugar when current photosynthate is unavailable. Plant genomes contain multiple BAM genes some of which have unknown functions. BAM2 in Arabidopsis thaliana is an unusual b‐amylase in that it is tetrameric with sigmoidal kinetics. Other catalytically active BAMs are monomeric with hyperbolic kinetics. A deep groove formed between BAM2 dimers forms a secondary binding site (SBS) for starch and this binding is necessary for activity. To investigate the nature of this catalytic mechanism we aligned BAM2 orthologs with other BAMs and noticed several regions that are uniquely conserved. One of these regions, a surface loop between the active site and the SBS, which we refer to as the HED loop, is three‐amino acids longer in BAM2 than in other BAMs. The loop is proximal to the conserved substrate binding sites suggesting a role for these amino acids in substrate binding. When this loop is shortened, the KM for starch is about 2‐fold lower with a sigmoidal kinetics curve. Using molecular docking, we compared the affinity of WT and mutated BAM2 models for maltose and maltodextrin substrates and found that the mutant shows higher affinities. A second unique site is a Ser residue adjacent to a catalytic Glu that when mutated to Gly, the residue conserved in typical BAMs, results in a 7.5‐fold lower KM for starch. Simulation of the dynamics of BAM2 with the Ser to Gly substitution shows increased tendency for the HED loop to be away from the active site. These data suggest the presence of an active site gate in BAM2 which controls substrate binding and is unique to BAM2.
Targeting Angiogenesis Receptors in Retinoblastoma: New Treatment Opportunities
Authors: Chan Ashley
Published: May 2022

Retinoblastoma (Rb) is a cancer that affects the retina of the eye, and can be fatal if the malignancy is left untreated. According to previous investigations, current treatments including chemotherapy, focal therapy, radiation therapy, and enucleation, can cause damage to normal cells. In an effort to offer an alternate treatment, we investigated three pharmacologic agents that target receptor proteins involved in the angiogenic pathway. Previous studies have demonstrated that the proteins prostaglandin F receptor, histone acetyltransferase, cyclooxygenase‐1, and kinase‐C are involved in the angiogenic pathway and have a strong association with cancer progression. In order to determine if these proteins can be targeted, we executed several protein‐ligand induced fit docking simulations using curcumin, OBE022, and AL‐8810. Our simulations demonstrate that these pharmacologic agents can bind efficiently and therefore may assist in the inhibition of angiogenesis.
Prenatal Hypoxia is Associated with Coronary Artery Endothelial Dysfunction in Male and Female Offspring
Authors: Hula Nataliia , Pasha Mazhar , Quon Anita , Kirschenman Raven , Cooke Christy‐Lynn M. , Spaans Floor , Davidge Sandra T.
Published: May 2022

Introduction

Fetal hypoxia is a common consequence of complicated pregnancies and is linked to the development of cardiovascular (CV) dysfunction in the offspring. We showed an impaired cardiac function in adult prenatal hypoxia offspring, however, the mechanisms are not known. Coronary artery endothelial dysfunction has been shown to contribute to the development of cardiac dysfunction, however, whether endothelial function is impaired in coronary arteries of prenatal hypoxia offspring is unknown. We hypothesize that prenatal hypoxia leads to impaired coronary artery endothelial function in adult male and female offspring.

Methods

Pregnant Sprague‐Dawley rats were exposed to normoxia (21% O2) or hypoxia (11% O2; p‐Hyp) on gestational days 15‐21 (term=22 days). Male and female offspring were aged to 9‐9.5 months. Left anterior descending coronary arteries were isolated (n=5‐7/group), and endothelium‐dependent vasodilation to methylcholine (MCh) and endothelium‐independent vasodilation to the nitric oxide (NO) donor sodium nitroprusside (SNP) were assessed by wire myography. To assess the contribution of endothelial vasodilation pathways, MCh‐curves were performed in the presence/absence of inhibitors of nitric oxide synthase (L‐NAME), prostaglandin H synthase ([PGHS]; meclofenamate), or small‐ and intermediate‐conductance Ca2+‐activated potassium channels (apamin and TRAM‐34, respectively). Data were summarized as Emax (maximum vasodilation response), pEC50 (concentration required to produce 50% of Emax) or Area Under the Curve (AUC). Data were compared by two‐way ANOVA (Sidak's post hoc test); p<0.05 was significant.

Results

Mch‐induced vasodilation (Emax) was decreased in p‐Hyp males (p=0.006) and females (p=0.032) compared to normoxia offspring. L‐NAME completely inhibited vasodilation (AUC) in normoxia and p‐Hyp males (p<0.0001). In the female offspring, incubation with L‐NAME completely inhibited vasodilation in the normoxia group (p<0.0001), but to a lesser extent (Emax ~15%) in the p‐Hyp offspring (p<0.0001). In contrast, meclofenamate did not alter MCh‐induced vasodilation (AUC) in males, while in females, meclofenamate increased vasodilation to MCh in the p‐Hyp group only (p=0.04). Apamin and TRAM‐34 did not alter responses to MCh in any of the groups. SNP sensitivity (pEC50) was decreased in p‐Hyp females compared to normoxia controls (p=0.012), without changes in males.

Conclusions

Our data suggest that prenatal hypoxia leads to coronary artery endothelial dysfunction in male and female offspring. In prenatal hypoxia males, NO contribution to endothelium‐mediated vasodilation was reduced. In females, prenatal hypoxia increased PGHS‐dependent vasoconstriction and decreased smooth muscle sensitivity to NO. Understanding the mechanistic pathways involved in programming of CV disease may allow for the development of future prenatal and postnatal therapeutic interventions.
MicroRNA‐195 regulates blood pressure by inhibiting NKCC2A
Authors: Hao Shoujin , Zhao Hong , Hao David , Ferreri Nicholas R.
Published: May 2022

We previously showed that inhibition of renal tumor necrosis factor‐alpha (TNF) production induced by high salt intake increases Na+‐K+‐2Cl−cotransporter isoform A (NKCC2A) mRNA, protein, and phospho (p)NKCC2 expression. MicroRNA (miRNA) contributes to blood pressure regulation, however no studies have addressed the regulation of NKCC2 by miRNA. miR‐195 is one of the most abundant miRNAs expressed in the kidney and qRT‐PCR experiments showed that injection of TNF (5ng/g bw) directly into the kidney of male mice up‐regulates miR‐195 in the renal outer medulla (OM) by approximately 3.5‐fold (p<0.05). Subsequently, a potential miR‐195 binding site in the 3′‐UTR of NKCC2A was identified using the prediction tool TargetScan v5.1, and luciferase reporter gene assays confirmed that NKCC2A mRNA is directly targeted and repressed by miR‐195. Primary cultures of medullary thick ascending limb (mTAL) cells and freshly isolated mTAL tubules express NKCC2 isoforms A and F. However, only miR‐195 and NKCC2A, but not NKCC2F mRNA, were up‐regulated in mTAL cells exposed for 2 h to a change in osmolality from 300 to 500 mOsmol/kgH₂O, produced with NaCl. Transient transfection of mTAL cells with an shRNA vector targeting TNF prevented increases in miR‐195 induced by high NaCl concentration. Moreover, transfection of a miR‐195 mimic into mTAL cells suppressed NKCC2A mRNA, but not NKCC2F mRNA, by approximately 83% (p<0.05) in cells exposed to high NaCl. In contrast, transfection with anti‐miR‐195 increased NKCC2A mRNA without altering NKCC2F mRNA abundance. Laser‐scanning cytometry detected a 1.5 fold (p<0.05) increase in pNKCC2 protein expression in mTAL cells transfected with anti‐miR‐195, and a miR‐195 mimic prevented the increase of pNKCC2 induced by silencing TNF in cells exposed to high NaCl. Next, we determined whether miRNA‐195 regulates HS‐dependent changes in blood pressure by inhibiting NKCC2A. Both TNF and miR‐195 expression increased in OM from mice given 1% NaCl in the drinking water (HS) for 7 days. Intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6‐TNF‐ex4) inhibited the expression of miR‐195 (p<0.05), while increasing NKCC2A mRNA expression in mice ingesting HS. However, intrarenal injection of murine recombinant TNF significantly upregulated the expression of miR‐195 and suppressed the increases of NKCC2A mRNA in mice ingesting HS. Intrarenal administration of miR‐195 prevented the increase of NKCC2A mRNA abundance in the OM from mice injected with the TNF silencing lentivirus (U6‐TNF‐ex4) compared with the control lentivirus. Moreover, the HS‐induced increase in blood pressure detected by radiotelemetry after renal silencing of TNF was markedly attenuated in mice given an intrarenal injection of miR‐195. Collectively, the study identifies miR‐195 as a salt‐sensitive and TNF inducible miRNA that inhibits HS‐mediated increases in blood pressure by inhibiting NKCC2A.
Frequency, variation, and structure of the median artery in a cadaver sample from New Mexico
Authors: Broderick Steven P. , Hill Ethan C.
Published: May 2022

The median artery is presently understood to be an embryonic structure, which typically begins to regress around the 8th week of intrauterine life. However, this artery can sometimes persist well into adulthood. It can exist as one of two types, forearm or palmar, which is determined by the site of termination. The purpose of this study was to examine the prevalence of the persistent median artery in a sample size of 41 cadavers from the University of New Mexico School of Medicine Anatomy Lab. Additionally, site of origin and termination were recorded. Out of 82 forearms from 41 cadavers, the persistent median artery was observed in eight (9.75%). Of these, five (62.5%) were determined to be the forearm type and three (37.5%) the palmar type. The artery was present bilaterally in only one case (12.5%). The most common site of origin was the common interosseus artery (5; 62.5%), followed by the ulnar artery (2; 25%), and the anterior interosseus artery (1; 12.5%). The persistent median artery was present in close to 10% of this population, which is a lower prevalence than observed in other similar studies. Presence of the persistent median artery is relevant to health care providers and worth considering due to role it may play in conditions such as carpal tunnel syndrome, as it would reduce the available space inside this pathway.
Dissecting the Mechanism of Allosteric Regulation of SIRT1
Authors: Leong Emily , Dosanjh Reena , Huynh Johnson , Hur Yujin , Cabreros Christiane , Lee Yue Tong , Wang Ningkun
Published: May 2022

The enzyme SIRT1 is a lysine deacetylase that plays a part in various pathways in the body, including insulin secretion and neurodegeneration, rendering it as a potential drug target for common diseases such as Type II diabetes and Alzheimer's disease. The activity of SIRT1 can be modulated allosterically by Sirtuin Activating Compounds (STACs), such as resveratrol, which bind to the STAC Binding Domain (SBD) of SIRT1 in the N‐terminus. End‐point activity assays have shown that resveratrol can have different effects on SIRT1 activity depending on the acetylated peptide substrate used, including an increase, decrease, or no change in deacetylation activity. The goal of our project is to dissect the activity of SIRT1 with and without resveratrol against different acetylated peptide substrates. Rather than only examining the overall catalytic efficiency, our goal is to assess the effect of resveratrol on the catalytic rate (kcat) and substrate recognition (KM) of SIRT1 against different acetylated peptide substrates. Upon addition of resveratrol, a large change in the KM of SIRT1 with a select peptide would imply that resveratrol had an effect on the substrate recognition of the enzyme, whereas a large change in the kcat may indicate that resveratrol caused an alteration in the catalytic rate of the enzyme for that specific peptide. We hypothesize that resveratrol allosterically alters SIRT1 activity by changing the conformation of the SBD region relative to the catalytic core, which would likely result in a large change in the KM, such as a two‐fold change in KM or higher, indicating a change in the efficiency of SIRT1 substrate recognition. To test this hypothesis, enzyme‐coupled assays involving SIRT1 acting on the peptide substrates Ac‐p300, Ac‐H4, and Ac‐p53 with and without resveratrol have been performed. Previous end‐point assay studies have shown that resveratrol decreases SIRT1 deacetylation activity against Ac‐p300, but increases activity against Ac‐H4, and does not affect SIRT1 activity against Ac‐p53. The Michaelis‐Menten parameters for SIRT1 activity with and without resveratrol were compared, allowing observation of the effects of resveratrol on SIRT1’s KMand kcat values. Understanding how resveratrol affects SIRT1 deacetylation activity will provide further insight into the development of drugs that can alter SIRT1 activity in a specific way to mitigate the effects of harmful diseases in humans.
Expression and Purification of Human Circadian Protein hRORγ for Structural and Functional Studies
Authors: Salazar Wendy V.
Published: May 2022

The circadian rhythm is an internal biological clock composed of different autonomous oscillators that regulate many physiological activities such as the sleep wake cycle. Long term disruption of circadian rhythm can lead to sleep disorders, neurodegenerative disease, metabolic syndrome, and high risks of cancer. The mechanism of circadian rhythm is driven by interlocked time‐delayed feedback loops consisting of positive and negative transcriptional regulators. For instance, Retinoic Acid Receptor‐Related Orphan Receptors (RORs) are nuclear receptors to improve circadian rhythm stability and functionality. A naturally occurring flavonoid Nobiletin (NOB) was reported to enhance circadian amplitude due to its high affinity to human ROR gamma (hRORγ). The interaction between NOB and hRORγ has not been studied in detail which hinders the future application of NOB as a promising candidate of circadian interfering medicine to ameliorate related diseases. The goal of this project is to solve the atomic three‐dimensional structure of hRORγ/Nobiletin complex to understand their interaction. The hRORγ gene was cloned and expressed using E. coli BL21 cells. Expression protocols were optimized to produce large amounts of recombinant hRORγ which have been purified using affinity chromatography and size exclusion chromatography. Circular dichroism has been used to verify that the purified recombinant hRORγ is properly folded. High concentration homogenous recombinant hRORγ will be co‐crystalized with Nobiletin. The atomic structure of the complex will be determined using X‐ray crystallography. The resultant structural information will shed light on Nobiletin’s enhancement mechanism on circadian rhythm and build the foundation for future drug development.
Efficient Incorporation and Template‐Dependent Polymerase Inhibition are Major Determinants for the Broad‐Spectrum Antiviral Activity of Remdesivir
Authors: Gordon Calvin J. , Lee Hery W. , Tchesnokov Egor P. , Perry Jason K. , Feng Joy Y. , Bilello John P. , Porter Danielle P. , Gotte Matthias
Published: May 2022

Remdesivir (RDV) is a direct‐acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 (COVID‐19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). RDV exhibits broad‐spectrum antiviral activity against positive‐sense RNA viruses, e.g. SARS‐CoV‐2 and hepatitis C virus (HCV) and non‐segmented negative‐sense RNA viruses, e.g. Nipah virus (NiV), while segmented negative‐sense RNA viruses such as influenza (Flu) virus or Crimean‐Congo hemorrhagic fever virus (CCHFV) are not sensitive to the drug. The reasons for this apparent pattern are unknown. Here, we expressed and purified representative RNA‐dependent RNA polymerases (RdRp) and studied three biochemical parameters that have been associated with the inhibitory effects of RDV‐triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV‐TP, (ii) the effect of the incorporated RDV‐monophosphate (MP) on primer extension, and (iii) the effect of RDV‐MP in the template during incorporation of the complementary UTP. The results of this study revealed a strong correlation between antiviral effects and efficient incorporation of RDV‐TP. Inhibition in primer extension reactions is heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template‐dependent inhibition of UTP incorporation opposite the embedded RDV‐MP is seen with all polymerases. Molecular modeling suggests a steric conflict between the 1’‐cyano group of RDV‐MP and conserved residues of RdRp motif F. We conclude that future efforts in the development of nucleotide analogues with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1’‐modification.

C.J.G, H.W.L, and E.P.T contributed equally to this work
Enhanced Metabolism and Altered Paracrine Signaling in Glioblastoma Following Cytomegalovirus Infection
Authors: Harrison Mark A. , Hochreiner Emily M. , Benjamin Brooke P. , Zwezdaryk Kevin J.
Published: May 2022

Glioblastoma (GBM) is an aggressive central nervous system neoplasia with an extremely poor prognosis. Despite intense research, 5‐year survival rates have not increased for the past 20 years, pointing to the need for alternative therapeutic strategies. Accumulating evidence has described a role for cytomegalovirus (CMV) in GBM, yet the mechanism of action is still unclear. We hypothesized that CMV may be metabolically reprogramming GBM cells and that these changes may alter the tumor microenvironment (TME) resulting in enhanced tumor progression. To test this hypothesis we infected GBM cells with CMV and employed the Seahorse Bioanalyzer and flow cytometry to examine their metabolic function. We witnessed increased glycolysis and oxidative phosphorylation relative to control cells. These changes were also accompanied by increases in lactate and reactive oxygen species (ROS) production. Since both lactate and ROS are capable of producing significant tumor promoting effects within the TME, we examined the impact that CMV‐infected cells had on their neighbors by employing an indirect co‐culture model. We found that, independent of viral transmission, the secretome of infected GBM cells was able to alter the levels of key metabolic proteins and epigenetic marks of their neighbors. These findings suggest the potential for major alterations within the TME following CMV infection or reactivation. They point to an oncomodulatory role for CMV in the context of GBM and may underlie the enhanced disease progression seen in CMV positive patients. Further research into these findings may yield new therapeutic targets which could be exploited to treat GBM more effectively.
Circulating MicroRNAs in Canine Congestive Heart Failure – Why Exosomes?
Authors: Ewing Carl A. , Ewing Coralis W. , Bohan Amy
Published: May 2022

By influencing protein translation, microRNAs (miRNAs) have emerged as powerful regulators of a wide range of biological processes. miRNAs can be found freely circulating in blood as well as in circulating exosomes. It has been speculated that evaluating the quantity of miRNAs contained in exosomes may serve as a better diagnostic and prognostic tool than those found freely circulating.

A good test case for this relationship is to look at miRNA dysregulation effecting Canine Congestive Heart Failure, resulting from Myxomatous Mitral Valve Disease (MMVD), in these two contexts. In order to do this, we are assessing levels of miR‐1, miR‐126, and miR‐133a, in nine small breed, older dogs with this disorder, compared to healthy dogs, using Realtime qPCR analysis.

We hypothesize that our results will confirm the assertion that exosomes reveal a more reliable indication of MMVD than by quantifying freely circulating miRNAs.
Effect of Viscosity on Fouling: Investigation into Pre‐Treatment of Saliva Samples for a Point‐of‐care SARS‐CoV‐2 Device
Authors: Hyman Jenna M. , Stennett Elana M.
Published: May 2022

Since the emergence of COVID‐19, the focus has been on curing, vaccinating against, and detecting the virus that spread into virtually every community. With that effort came the need for not just increased testing but also cheap and fast testing. One possible solution is a point‐of‐care device. Similar to a pregnancy test, a device in which users could deposit a saliva sample and receive a measurable result could be accessible to millions all over the world. However, building such a device requires a lot of forethought, including any pre‐treatment of the sample. The goal of pre‐treatment, in this case, is to lessen the variability and complexity of the saliva for testing. Egg white and glycerol solutions were prepared to mimic saliva and determine if viscosity played a role in flux or possible membrane fouling using a custom flux setup designed in the lab for a method to ready samples for a rapid SARS‐CoV‐2 test. By comparing flux rate of fluorescein‐labeled bovine serum albumin (BSA) (0.1 g/L) in phosphate‐buffered saline (PBS) (pH 7.4, 15mM), buffer/glycerol solutions, and egg white solutions through a membrane, the effect of viscosity on fouling of black polycarbonate track‐etched (BPCTE) membranes was observed. Fluorescence imaging of BPCTE membranes allowed for further observation of fouling. Higher viscosity solutions showed a decrease in the measured flux, and no fouling occurred for any solutions. However, egg whites and imaging indicated alternate implications of pre‐treatment for saliva‐based rapid tests.
Investigating the Interaction Interface between MEK and ERK Kinase
Authors: Lin Sabrina , Naagaard Mats D. , Pham Tammy , Fera Daniela
Published: May 2022

The mitogen‐activated protein kinase (MAPK) pathway is pertinent to various cellular processes such as cell proliferation, cell division, and cell death. Dysregulation of the MAPK pathway can therefore lead to numerous illnesses. Most notably, the dysregulation of proteins in this pathway is a significant cause of melanomas and other cancers. Due to this oncogenic potential, various drugs and inhibitors exist to regulate MAPK overexpression. As drug resistance and paradoxical activation are perpetual challenges, additional regulatory targets are needed to effectively suppress the effects of MAPK oncogenic mutations, calling for further investigation into downstream interactions such as that between MEK and ERK kinase. By identifying and characterizing the binding interface between MEK and ERK, we can identify additional drug targets that can modulate the MAPK pathway.

To help map the MEK‐ERK interface, we first performed computational docking experiments to find possible interaction regions between MEK and ERK. In several trials, we found that certain MEK alpha helices interacted with ERK. We then proceeded to identify the corresponding binding partners on ERK. We introduced mutations in the most promising regions on ERK and expressed the mutant proteins using a bacterial expression system. Using biolayer interferometry and co‐elution assays, we compared the binding affinities of our mutants to that of the corresponding wild‐type.

Our studies showed that introducing the K231E and K114E mutations in ERK altered the binding kinetics between MEK and ERK. Our co‐elution studies showed that a stable complex does not occur, suggesting that the interaction between MEK and ERK is either weak or transient and may need another molecule to stabilize the complex. Cross‐linking experiments are in progress, and so far, we identified 1% glutaraldehyde incubated for 5 minutes as sufficient for covalent linkage of MEK and ERK. These findings will help towards obtaining a 3D MEK‐ERK complex structure to better understand signaling in this pathway.
The Influence of Androgen Receptors on Muscle Repair and Hypertrophy
Authors: Hatt Aidan , Fortino Stephen , Wageh Mai , Kamal Michael , Brown Alexander , Joanisse Sophie , Parise Gianni
Published: May 2022

The mechanisms underlying inter‐individual variation in adaptation to exercise have yet to be fully elucidated. Recently, high responders to resistance training (RT) have demonstrated greater levels of androgen receptor (AR) content, and therefore AR may be a factor underlying the variation in the adaptive response to resistance exercise. Moreover, there has been inconclusive findings surrounding the impact of AR content in females as well as following damage. In the current study, twenty‐six healthy young men (n=13) and women (n=13) underwent an acute bout of damaging exercise consisting of 300 eccentric kicks, followed by 10 weeks of full‐body RT to investigate changes in AR content following acute muscle damage and following chronic resistance exercise. This was assessed in the context of the satellite cell response and during muscle repair and hypertrophy. Skeletal muscle biopsies from the vastus lateralis were taken at baseline, 48 hours post damage and 48 hours following the last bout of RT. RT‐qPCR and western blots were performed to quantify AR gene expression and protein content, respectively. An immunofluorescence staining protocol was optimized for the visualization of AR protein abundance. Results were considered in the context of the SC response and muscle hypertrophy data from the same cohort. AR content increased from baseline (30457 +/‐ 4399 a.u.) to post‐damage (66448 +/‐ 8083 a.u.) by 218% (p=0.005) and post RT (60889 +/‐ 6636 a.u.) by 199% (p=0.003). Those with the greatest increase in SC content post‐damage also had the greatest increase in AR content (p=0.01). Individuals with the greatest increase in leg‐lean mass following RT also had the greatest increase in AR content (p=0.05). Preliminary data from western blot analysis revealed that AR protein content post‐damage in males was correlated with activated SC number (MyoD+ cells) (p=0.01). We then aimed to investigate sex differences in the context of AR content since there is a paucity of work in females. Males (61550 +/‐ 10961 a.u.) had a 198% greater change in AR content than females (31104 +/‐ 7499 a.u) from baseline to post‐damage (p=0.03). Males also had a greater change in AR content (3.186 +/‐ 0.5443) than females (2.003 +/‐ 0.2625) post‐RT, relative to baseline. In males, those with greater relative gains in muscle fibre CSA (high responders), post‐RT, had a 373% greater change in AR content (64936 +/‐ 11783 a.u.) from baseline to post‐RT than low responders (17388 +/‐ 15438) (p = 0.03). Interestingly in females, those with greater relative gain in CSA post‐RT (1.681 +/‐ 0.4120) had a trend towards lower AR content post‐RT compared to low responders (2.502 +/‐ 0.1946) (p = 0.06). This study is the first to immunofluorescently stain AR content for visualization and quantification in skeletal muscle. Collectively, these findings suggest that AR content influences SC activity and exhibits sex differences following damage and chronic resistance exercise.
Synthesis and New Cytotoxicity Screening Approach of Novel Dirhodium Complexes
Authors: Saulino Emily N. , Dunham Stephen , Thévenin Anastasia
Published: May 2022

Transition metal–based chemotherapeutics have been used to treat a variety of cancers since the 1960s with cisplatin being the most prominent of these drugs used in a clinical setting. Many cancers become chemoresistant to cisplatin, so there is currently a need to synthesize and explore the anticancer abilities of other transition metal complexes. Rhodium, one of the six platinum–group metals, demonstrates cytotoxic effects in cancer cells in the form of dirhodium tetraacetate (Rh2A4). However, Rh2A4 is known to be less cytotoxic than cisplatin in HeLa cervical cancer cells. This research aims to modify Rh2A4 to increase its cytotoxicity to that of cisplatin. One of the acetate ligands of Rh2A4 was selectively replaced with 6‐maleimidohexanoic acid (M) to form a dirhodium triacetate maleimide (Rh2A3M) complex. Maleimides are known to readily react with alkylamines (R‐NH2) under basic conditions to produce amine conjugates. Alkylamines were chosen because of this reactivity and because their presence in a variety of metabolic and physiological processes may aid in the uptake of the dirhodum complex in cancer cells. The Rh2A3M complex was successfully reacted with four alkylamines: benzylamine, cyclohexylamine, ethanolamine, and glucosamine. This reaction was done in a 1:10 molar ratio in a phosphate buffer of pH 7.25 at room temperature for 72 hours to form subsequent Rh2A3M‐NH‐R complexes. The crude reaction mixture was then purified by preparative HPLC and characterized via 1H 1D NMR. A new screening method was developed using MTT reagent to compare the cellular viability of HeLa cervical cancer cells treated with the four crude reaction mixtures and the four HPLC purified Rh2A3M‐NH‐R complexes. Each pure complex had a similar percent cellular viability to its corresponding crude reaction mixture. This procedure will allow for future screens of many Rh2A3M + R‐NH2 crude reaction mixtures to indicate cytotoxicity of Rh2A3M‐NH‐R products without the need for the purification and characterization steps. Out of the four pure complexes, Rh2A3M‐cyclohexylamine was found to be the most cytotoxic at ~20 μM, suggesting it is comparable in cytotoxicity to cisplatin. Current work is focused on the determination of the IC50 value for this complex to definitively compare its cytotoxicity to cisplatin.