bluuebunny/crossref_metadata_2025 huggingface dataset:
Endoplasmic reticulum‐associated, aggregation‐prone protein substrates inform protein disaggregation in mammalian cell systems
Authors: Daskivich Grant J. , Brodsky Jeffrey L.
Published: May 2022

Neurodegenerative diseases afflict over 5 million Americans and are caused primarily by proteins and protein aggregates that disrupt proteostasis; the process which maintains protein function and quality control in the cell. Healthy cells can process small aggregates through the action of molecular chaperone assemblies and protein degradation pathways. However, highly stable aggregates irreversibly disrupt proteostasis and trigger disease onset. In contrast to human cells, the chaperone Hsp104 can resolve highly stable aggregates in yeast. Problematically, humans lack Hsp104. Therefore, we hypothesize that metazoan cells have developed alternative machinery to resolve stable protein aggregates. To address this hypothesis, we developed multiple endoplasmic reticulum (ER) localized substrates that have aggregation‐prone cytosolic motifs. Substrates were comprised of either yeast or mammalian protein‐derived membrane anchors and aggregation‐prone and amyloid‐like motifs. Because of their structure, we hypothesized that these substrates were dependent on ER associated degradation (ERAD) and became insoluble under stress conditions. We evaluated these substrates in HEK293H cells with cycloheximide chase and detergent fractionation assays and discovered each substrate largely depended on the proteasome for degradation while only some were insoluble at elevated temperatures. We then used biotin proximity labeling to identify potential protein chaperones associated with our substrates.
Role of the SWIRM Domain in Regulating the HAT Module of the SAGA Complex
Authors: Haile Sara T. , Wolberger Cynthia
Published: May 2022

The SAGA (Spt‐Ada‐Gcn5 acetyltransferase) co‐activator complex regulates most yeast genes and both acetylates and deubiquitinates chromatin. SAGA contains the acetyltransferase, Gcn5, which is found in the four‐protein subcomplex known as the HAT module. On its own, Gcn5 is unable to acetylate nucleosomal and chromatin substrates. To acetylate chromatin, Gcn5 depends on two of the other HAT subunits, Ada2 and Ada3, and its activity is further enhanced by the presence of the last subunit, Sgf29. While recent cryo‐EM structures have resolved much of the SAGA complex, the structure of the HAT module could not be resolved due to poorly defined density. HAT module subunit, Ada2, contains a SWIRM domain, which is found in other chromatin modifying enzymes but whose function varies based on context. Here we show that the yeast S. cerevisiae SWIRM domain is required for incorporation of the HAT module into the SAGA complex. This study investigates how loss of the SWIRM domain affects the HAT module’s function in yeast and its ability to acetylate and bind nucleosomes. The insights from this work will aid in understanding why the SWIRM chromatin reader domain is important for histone acetylation, a vital step in making chromatin accessible to transcription.
Phosphoglycerate Mutase 5 Expression Modulates Hepatocellular Steatosis
Authors: Johnston Andrea , Moeller Cambri , Liu Chin‐Chi
Published: May 2022

Phosphoglycerate Mutase 5 (PGAM5) is a serine/threonine phosphatase that plays a role in oxidant injury sensing, mitochondrial biogenesis, mitophagy, and multiple cell death pathways.

Pgam5 null mice are protected from high‐fat diet induced obesity. This metabolic phenotype was attributed to upregulation of adaptive thermogenesis in brown adipose tissue, but hepatocyte specific lipid metabolism has not been evaluated. We hypothesize that hepatic PGAM5 expression level modulates hepatic steatosis. Using primary cultures of Pgam5 knockout hepatocytes, we have shown that palmitate induced steatosis is attenuated and that treatment with the long‐chain fatty acid impairs oxygen consumption rate specifically reducing the maximal respiratory capacity. Further, over‐expression of PGAM5 in human hepatoma cells exacerbates palmitate induced steatosis. Cumulatively, these results suggest that PGAM5 regulates hepatic lipogenesis and mitochondrial long‐chain fatty acid beta‐oxidation.
Pulmonary Arterial Smooth Muscle Cell Monocarboxylate Transporter 1/4 Contributes to Extracellular Acidosis following Chronic Hypoxia‐induced Pulmonary Hypertension
Authors: Tuineau Megan N. , Herbert Lindsay M. , Resta Thomas C. , Jernigan Nikki L.
Published: May 2022

Pulmonary hypertension (pHTN) is defined by mean pulmonary arterial pressure at rest exceeding 20 mmHg with mortality often resulting from right ventricular failure that is secondary to increased afterload. During pHTN, increased reliance on aerobic glycolysis and inhibition of mitochondrial oxidative respiration is observed in pulmonary arterial smooth muscle cells (PASMCs). This metabolic shift is associated with hyperproliferation and resistance to apoptosis contributing to the pathogenesis of pHTN. Along with these phenotypic changes, cells undergoing aerobic respiration exhibit increased glucose uptake and subsequent lactate and H+ efflux, contributing to extracellular acidosis. Acid‐sensing ion channel 1 (ASIC1) is a voltage‐independent, proton‐gated cation channel that contributes to the development of chronic hypoxia (CH)‐induced pHTN. While the function of ASIC1 is well characterized in pHTN, it has not been determined whether the metabolic shift observed during pHTN contributes to the activation of ASIC1. Therefore, we hypothesized that enhanced glucose uptake and subsequent acidification of the extracellular microenvironment in PASMCs lead to the activation of ASIC1 in CH‐induced pHTN. As an initial step in testing this hypothesis, rats were housed in a hypobaric chamber (barometric pressure: ~380 mmHg) to induce pHTN and compared to age‐matched normoxic controls. After 4 weeks, PASMCs were collected and transiently cultured under normoxia for 3‐4 days. As expected, PASMCs from CH rats showed a reduced oxygen consumption rate (p<0.0001) and greater extracellular acidification (p<0.0001) compared to those from control animals, indicating increased glycolytic activity. Utilizing the fluorescent pH indicators SNARF‐5/AM, we found that PASMCs from CH rats exhibit intracellular alkalization (p<0.0001 vs. control) and extracellular acidosis (p<0.001 vs. control). We further examined the H+ transporter(s) involved in mediating extracellular acidosis by inhibiting carbonic anhydrase IX (CA‐IX), the Na+/H+ exchanger (NHE1), the vacuolar‐type H+‐type ATPase (V‐ATPase), or monocarboxylate transporters 1 and ‐4 (MCT1 and MCT4). The dual MCT1/4 inhibitor, syrosingopine, prevented intracellular alkalization and extracellular acidosis following CH (Figure 1A), which corresponded with an increase in MCT4, but not MCT1, expression (Figure 1B). These data suggest MCT plays an important role in regulating PASMC pH homeostasis. Future studies will assess the role of MCT to activate ASIC1 and contribute to pHTN.
Nitazenes are potent mu‐opioid receptor agonists with profound respiratory depression
Authors: Palkovic Barbara , Malcom Nicholas J. , McCorvy John D. , Langer Thomas M. , Callison Jennifer J. , Stuth Eckehard A. , Zuperku Edward J. , Stucke Astrid G.
Published: May 2022

Background

Nitazene opioids (2‐benzylbenzimidazole opioids) were first identified in multiple overdose fatalities a few years ago. It has been shown that nitazene drugs are highly active at the mu‐opioid receptor, with potency and efficacy exceeding fentanyl. Because of this, isotonitazene was added to Schedule I of the controlled substance list in August 2020 by the Drug Enforcement Agency. Further studies have shown the high potency of the N‐desethyl‐isotonitazene metabolite, which was similar in potency of isotonitazene itself. The objective of this study was to compare the pharmacokinetics with regards to respiratory depression between N‐desethyl‐isotonitazene and fentanyl in vivo rabbit model.

Methods

The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3‐4kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet. Phrenic neural activity, respiratory rate, arterial blood pressure, and airway carbon dioxide concentration were recorded on a computerized chart recorder (Powerlab/16SP; ADInstrumentes, Australia). We determined the magnitude and time course of respiratory rate depression from 1mcg/kg N‐desethyl‐isotonitazene and fentanyl and subsequently the dose required to achieve apnea. Data were collected using LabChart data and exported to SigmaPlot 11 (Systat Software, USA) for data processing, data plotting and statistical analysis. Using the phrenic neurogram, we determined the respiratory rate and peak phrenic activity. We plotted the average respiratory rate for each minute normalized to control frequency, removed artefacts, and determined the best fitting curve for each plot. Plots were averaged for each dose and displayed as mean± standard error.

Results

Fentanyl (1mcg/kg) depressed respiratory rate by 25±5% from baseline (n=3) while N‐desethyl‐isotonitazene caused a 38±2% depression (n=6). Recovery to baseline was 150±15 minutes for N‐desethyl‐isotonitazene and 46±8 minutes for fentanyl. N‐desethyl‐isotonitazene caused apnea at 4mcg/kg (n=6), and complete recovery to baseline respiratory rate was 201±33 min minutes. Fentanyl caused apnea at 8mcg/kg (n=4), and 64±7 minutes were needed to recovery.

Conclusions

Nitazenes are potent synthetic opioids. At equal doses, they cause more profound and longer lasting respiratory rate depression than fentanyl.
Is P‐glycoprotein functionally active at the Blood‐CSF Barrier?
Authors: Sun Austin D. , Wang Joanne
Published: May 2022

The brain is protected from circulating metabolites and xenobiotics by two major barriers: the blood‐brain barrier (BBB) and the blood‐cerebrospinal fluid (CSF) barrier (BCSFB). The drug efflux transporter P‐glycoprotein (P‐gp) is known to be expressed luminally at the BBB where it limits the entry of circulating drugs and toxins into the brain. The BCSFB, formed by the choroid plexus epithelial cells, similarly expresses membrane transporters to mediate solute efflux or influx into the CSF. There are conflicting findings on P‐gp activity at the BCSFB, and limited studies of P‐gp localization posit the transporter is expressed apically or subapically, implying a paradoxical function to mediate a blood‐to‐CSF transport of xenobiotics and endobiotics. The objective of this study is to clarify the localization and function of P‐gp at the BCSFB utilizing a quantitative fluorescence microscopy approach. Calcein‐AM, commonly used to evaluate P‐gp‐mediated efflux, is a permeable, non‐fluorescent P‐gp substrate that is converted by intracellular esterases into calcein, a fluorescent and impermeable compound. Time‐dependent uptake of 250 nM calcein‐AM was performed in lateral ventricle choroid plexus isolated from adult FVB mice, and confocal imaging was utilized to observe the accumulation of calcein formed in the choroid plexus epithelial cells. Image segmentation was performed using ImageJ to individually quantify choroid plexus cell and blood capillary accumulation of calcein. Coincubation with 2 µM elacridar, a potent P‐gp inhibitor, increased accumulation of calcein in the choroid plexus cells by 5.7‐fold and increased blood capillary accumulation by 11.2‐fold, indicating inhibition of apical efflux transport. Our data suggests that that P‐gp is indeed functionally active at the apical, CSF‐facing membrane of the BCSFB. As CSF drug concentrations are frequently used as a surrogate measure of CNS drug exposure, elucidating the functional role of P‐gp at the BCSFB is critical for understanding the interplay of BBB and BCSFB transporters and their impact on CNS pharmacokinetics and pharmacodynamics.
VX‐745 Inhibits Smooth Muscle Cell p38 Mitogen‐Activated Protein Kinase Alpha (MAPKα) and Heat‐Shock Protein 27 (Hsp27) Phosphorylation to Induce Vasodilation in Resistance Mesenteric Arteries
Authors: Menon Sreelakshmi , Zerin Farzana , Pandey Ajay K. , Hasan Raquibul
Published: May 2022

VX‐745, a selective inhibitor of the alpha isoform of the mitogen‐activated serine/threonine protein kinase p38 MAPK, is under clinical investigation for its efficacy in Alzheimer’s disease (AD) and dementia with Lewy Bodies (DLB). Although enhanced vascular tone and hypertension contribute to cognitive impairment in AD and DLB, no studies have examined if acute VX‐745 application could relax resistance arteries that control vascular resistance and blood pressure. Here, we investigated if VX‐745 could induce vasodilation in resistance‐size rat mesenteric arteries and the underlying mechanisms. Freshly isolated fourth order rat mesenteric arteries were cannulated and maintained in a temperature‐controlled perfusion chamber. Arterial segments were subjected to a stepwise increase in intraluminal pressure to 80 mmHg. Development of sustained myogenic tone at 80 mmHg generated a baseline diameter after which a cumulative concentration‐responses (100 pM to 10 μM) to VX‐745 were performed and diameter changes recorded. Our pressure myography data showed that VX‐745 produced a dose‐dependent vasodilation in mesenteric arteries. On application of 10 μM VX‐745 the mesenteric arteries dilated by ~μm after 2 to 3 minutes of drug application. Vasodilation induced by VX‐745 remained unchanged upon endothelium denudation (intact ~41 μm vs denuded ~38 μm), suggesting an endothelium‐independent mechanism. Endothelium denudation or pharmacological inhibition of endothelium‐derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) had no effect on such vasodilation. Our Western blotting data revealed that acute VX‐745 treatment significantly reduced the phosphorylation of p38 MAPKα by 33.74±0.3.7% compared to that in control arteries and its downstream target heat‐shock protein 27 (Hsp27) involved in cytoskeletal reorganization and smooth muscle contraction by 37.75±1.19% compared to control arteries. Likewise, non‐selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation by 32.25±0.1.8% and 31.57±3.47% respectively compared to control arteries and induced vasodilation in mesenteric arteries. VX‐745‐evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell large‐conductance Ca2+‐activated K+ channels (BKCa), ATP‐sensitive K+ channels (KATP), and voltage‐gated K+ channels (KV). Altogether, our data for the first time demonstrates that, in resistance mesenteric arteries, VX‐745‐mediated selective inhibition of p38 MAPKα reduces the phosphorylation of the actin‐associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which VX‐745 is being investigated.
Formation of the Major Circulating Metabolite of Cannabidiol by Non‐P450, Cytosolic Enzymes
Authors: Beers Jessica L. , Authement Aurora , Isoherranen Nina , Jackson Klarissa D.
Published: May 2022

Cannabidiol (CBD), a natural product derived from cannabis, was recently FDA‐approved to treat epilepsy in children. CBD is extensively metabolized by hepatic cytochrome P450 (P450) enzymes to form the pharmacologically active metabolite 7‐hydroxy‐cannabidiol (7‐OH‐CBD). This metabolite is further converted to the major circulating metabolite, 7‐carboxy‐cannabidiol (7‐COOH‐CBD), prior to hepatobiliary excretion. CBD is mainly eliminated in the form of 7‐COOH‐CBD and related hydroxylated derivatives, and the plasma area under the curve of 7‐COOH‐CBD is up to 40 times higher than that of the parent compound. While prior work has shown that 7‐OH‐CBD is mainly formed by CYP2C9 and CYP2C19, the enzymes involved in 7‐COOH‐CBD formation have not been clearly established. Preliminary data from in vitro experiments with hepatocytes and hepatic subcellular fractions suggests that cytosolic enzymes may contribute to formation of 7‐COOH‐CBD. The objective of this study was to define the roles of P450 versus non‐P450 enzymes in 7‐COOH‐CBD generation. We hypothesized that 7‐OH‐CBD may be metabolized through a multi‐step reaction involving an aldehyde intermediate prior to conversion to 7‐COOH‐CBD. Reaction phenotyping experiments were performed with 7‐OH‐CBD and human liver microsomes, cytosol, and S9 fraction in the presence of selective enzyme inhibitors and cofactors to determine their effects on 7‐COOH‐CBD formation. Results show that 7‐COOH‐CBD formation is largely NAD+‐dependent, and the cytosolic enzyme aldehyde dehydrogenase can generate 7‐COOH‐CBD. In addition, reactions performed in the presence of the trapping agent methoxylamine show decreased 7‐COOH‐CBD formation in human liver S9 fraction compared to those lacking this agent, suggesting that a reactive aldehyde intermediate may be formed from 7‐OH‐CBD. These data fill a critical research gap in our understanding of cannabinoid metabolism and help identify potential safety risks and drug‐drug interactions for patients and consumers taking CBD.
H2S Therapy Improves Diastolic Dysfunction and Exercise Capacity in Two‐Hit Murine Model of Heart Failure with Preserved Ejection Fraction
Authors: Doiron Jake E. , Lapenna Kyle , Li Zhen , Xia Huijing , Goodchild Traci , Xian Ming , Kang Jianming , Lefer David
Published: May 2022

Background

HFpEF is a complex multi‐organ disease expected to represent >60% of all clinical heart failure cases by 2030. To date, the majority of therapeutic approaches to treat HFpEF have largely failed in either preclinical or clinical trials. Given the recent reports of diminished H2S bioavailability in HFpEF combined with the robust cardioprotective actions of H2S in cardiovascular disease, we sought out to test the effects of H2S therapy in a well‐established pre‐clinical HFpEF murine model.

Methods

HFpEF was induced in nine‐week‐old C57/BL6N mice (n=10 per group) by high fat diet and L‐NAME (0.5g/L/day) via drinking water for 5 weeks. Mice were treated with either vehicle or the H2S donor, JK‐1 (100 mg/kg/day B.I.D., I.P.) for a period of 5 weeks. Echocardiography and exercise capacity were performed at study weeks 0, 5, and 10 for monitoring of cardiac function and progression of exercise intolerance. At week 10, endothelium‐dependent and independent aortic vascular reactivity and left ventricular invasive hemodynamic studies were performed.

Results

Treatment with JK‐1 significantly improved cardiac function as measured by decreases in both left ventricular end diastolic pressure (LVEDP) and E/e’ after 5 weeks of treatment. Similarly, endothelium‐dependent vascular relaxation with acetylcholine exhibited significant improvement. Treadmill exercise performance expressed as total work showed improvement for the JK‐1 group, demonstrating changes in exercise capacity are irrespective of body weight.

Conclusion

These results suggest that the H2S donation therapy exerts beneficial effects in the setting of severe HFpEF. Studies are currently ongoing to determine the mechanisms by which H2S therapy improves both cardiac and vascular function in this devastating cardiovascular disease.
HMGN2 regulates transcription factor activity through chromatin modifications and protein interactions, developmentally modulated by microRNA‐23
Authors: Amendt Brad A. , Eliason Steven , Su Dan
Published: May 2022

The chromatin‐associated high mobility group protein N2 (HMGN2) regulates transcription factor activity through both chromatin and protein interactions. Several homeodomain and HMG‐box domain transcription factors regulate development and differentiation and are modulated by protein interactions and epigenetic factors. We demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull‐down and co‐immunoprecipitation assays show HMGN2 directly interacts with Lef‐1 through its HMG‐box domain. Furthermore, electrophoretic mobility shift assays demonstrate that HMGN2 inhibits Lef‐1 DNA binding activity. Thus, HMGN2 inhibits the transcriptional activities of Lef‐1, Dlx2, FoxJ1 as well as Pitx2. Pitx2 and Lef‐1 activate the Hmgn2 promoter and HMGN2 expression inhibits their transcriptional activation. Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter demonstrating Hmgn2 association with open chromatin. Hmgn2 expression is developmentally regulated however, the post‐transcriptional mechanisms that regulate Hmgn2 expression remain unclear. We demonstrate that miR‐23a and miR‐23b directly target Hmgn2, promoting transcriptional activation of several gene promoters including the amelogenin promoter, regulated by Pitx2. Hmgn2 expression decreases correlating with increased miR‐23 expression in craniofacial tissues as the murine embryo develops. Analyses of Hmgn2 LacZ expression demonstrate that Hmgn2 expression is gradually decreased during embryonic development. Ablation of Hmgn2 in mice results in increased amelogenin expression due to increased Pitx2, Dlx2, Lef‐1 and FoxJ1 transcriptional activity. We demonstrate both post‐transcriptional regulation of Hmgn2 by miR‐23a/b and post‐translational gene expression by Hmgn2 protein interactions. HMGN2 appears to regulate tooth development through its interaction with multiple transcription factors.
Mitochondrial Transfer Between Mesenchymal Stem Cells and Cardiac H9c2 Cells
Authors: Kortenhoeven Miranda , Boomsma Robert
Published: May 2022

Mesenchymal stem cells (MSC) have been shown to improve heart function after myocardial infarction, but the exact mechanisms are not completely understood. Studies have shown that stem cells are able to transfer mitochondria through cytosol extensions to change the abilities and programming of surrounding cells. The purpose of this study was to determine whether mitochondrial transfer takes place between MSCs and cardiac H9c2 cells, and the effect of hypoxic conditions on this process. Mouse bone marrow MSC were cultured in Mesencult + 10% mouse supplement (Stem Cell Technologies) + 10% FBS and H9c2 cells were cultured in DMEM + 10% FBS. MSC mitochondria were stained using MitoTracker Red CMX Ros (Invitrogen), while H9C2 cells were stained using CellTracker Green CMFDA (Invitrogen) according to the manufacturer’s instructions. Following staining, the cells were co‐cultured for 24 hours in Fluorobrite DMEM (Gibco) + 10% FBS in 4‐well glass culture slides. After washing with PBS and mounting with ProLong Live Antifade Reagent (Invitrogen), cells were observed using an Olympus BH2 fluorescent microscope. Our results showed close interactions between MSC and H9c2 cells with mitochondria in long filamentous extensions that made contact with H9c2. There was some evidence that mitochondria were transferred from MSC to H9c2 cells. Experiments are underway to determine whether mitochondria transfer from H9c2 cells to MSC, and the effect of hypoxia. These results continue to suggest that mitochondrial transfer may be one mechanism used by MSC to improve heart function after myocardial infarction.
Disassembly of fascin bundled actin filaments via their Mical associated oxidation
Authors: Rajan Sudeepa , Yoon Jimok , Yang Taehong , Terman Jonathan R. , Reisler Emil
Published: May 2022

Actin filaments are the major component of cellular substructures involved in cell motility and cell sensing, depending on the cues from the microenvironment. Actin’s diverse functions are enabled by its transitions from a monomeric (G‐actin) to filamentous (F‐actin) form, which in turn can be converted into higher order structures, like bundles, with the help of various actin‐crosslinking proteins. The mechanism of actin bundles formation has been previously reported, but how they disassemble is understood less well. Using real‐time solution‐based assays and purified proteins we try to unravel the specific mechanism by which the MICAL family of monooxygenase induces the disassembly of bundled actin. These experiments were further supported by our in vivo studies using a model cellular system. We showed that the rate of disassembly is enhanced upon addition of cofilin, a well‐known F‐actin severing protein. Overall, our results shed new light on the mechanism of actin bundle disassembly and its direct impact on cellular remodeling.
DNA methylation levels of CEBPβ gene differentially affected by saturated and polyunsaturated fatty acids in adipogenesis of 3T3‐L1 cells
Authors: Li Yi , Archdeacon Kyle , Brown Dionne
Published: May 2022

In obesity and type 2 diabetes, adipose and muscle tissues are significantly affected. It has been demonstrated that many of the changes are associated with gene expression. Cultured cell models were used to study the involvement of epigenetic modifications in mediating gene expression affected by hyperlipidemia. Cultured L6 muscle cells and differentiating 3T3‐L1 adipocytes were treated with saturated or polyunsaturated fatty acids to mimic hyperlipidemia conditions. After the treatments, methylation sequencing was conducted to identify the changes of the methylation levels of the genomic DNA samples from the cells. And restriction digestion followed PCR were used to validate the methylation sequencing results. In general, methylation levels of most of the genes were increased while methylation levels of some of the genes were decreased and small portion of the genes changed slightly among the genes with altered methylation levels in differentiating 3T3‐L1 cells treated with a saturated or a polyunsaturated fatty acid. We have demonstrated that both saturated type and polyunsaturated type fatty acids enhanced adipogenesis of differentiating 3T3‐L1 cells. While both saturated and polyunsaturated fatty acids decreased methylation levels of PPARγ gene, only polyunsaturated fatty acids increased methylation levels of C/EBPβ gene. These results are consistent with the knowledge that expression of both PPARγ and C/EBPβ gene are increased in adipogenesis and intake of saturated fat is associated with obesity.
Increased Oxidation From Glucose Limiting Conditions or SOD Deletions Result in Activation of No‐Go Decay and Subsequent P‐body Assembly
Authors: Segal Scott , Cianflone Emily , Jorgensen Anna , McManus Gretchen , Rolin Kyle
Published: May 2022

The budding yeast, Saccharomyces cerevisiaepreferentially metabolizes glucose anaerobically when it is abundant. However, as glucose becomes depleted, yeast will enter diauxic shift. Under this condition, growth will slow, and glucose becomes metabolized aerobically. Upregulation of aerobic glucose metabolism ultimately leads to the increase of intracellular reactive oxygen species (ROS). The increase of ROS can lead to oxidation of nucleic acids through formation of 8‐oxoguanine bases (8‐oxoG). In mRNA the presence of 8‐oxoG can elicit ribosome stalls, resulting in translational shutdown and the activation of the no‐go mRNA quality control pathway. No‐go decay is mediated by the proteins Hbs1p and Dom34p, which act to remove stalled ribosomes, and will promote cleavage of the mRNA at the stall site. The remaining 5’ fragment is subject to 3’‐5’ decay by the cytoplasmic exosome, while the 3’ fragment is degraded 5’‐3’ by exonuclease Xrn1p in P‐bodies, which are sites of mRNA degradation.

Upon growing yeast into diauxic shift, an increase in P‐body assembly was observed. P‐bodies contain mRNA decay enzymes and are sites where nontranslating mRNA will localize to be degraded. This observed increase in P‐body assembly appears to be due to an upregulation of no‐go Decay, as P‐body assembly is reduced in strains lacking either Dom34p or Hbs1p. Upon exposure to the antioxidant quercetin during diauxic shift, P‐body assembly is abrogated showing that generation of ROS is responsible for the activation of no‐go decay and subsequent P‐body assembly. The same result is phenocopied by exposing yeast to glucose limiting conditions (0.5% glucose). As expected with the increased oxidation from glucose limiting conditions, global translation is also reduced. Interestingly, strains lacking either Sod1p or Sod2p, also show a reduction in global translation, along with an upregulation of no‐go decay and P‐body assembly. These data suggest that physiological stresses that lead to an increase in ROS, which can damage mRNA leading to activation of No‐Go Decay and subsequent P‐body assembly.
Structural characterization of the cone‐rod homeobox protein binding to DNA
Authors: Buchholz Christine N. , Sholes Abigail E. , Enke Raymond A. , Berndsen Christopher
Published: May 2022

Roses are red, some eyes are blue, you wouldn’t see this without CRX too. The Cone‐Rod Homeobox protein or CRX is an essential transcription factor for the development of cells in the eye called photoreceptors. Photoreceptor cells (PRs) convert light into signals that the brain can understand. CRX plays a role in the differentiation of these photoreceptors into rods and cones, which detect achromatic and colored light, respectively. While there are details known about the molecular function of CRX, there is little known about the structural information of the protein. It is known that CRX contains a DNA binding domain (DBD) and two activation domains (ADs). Changes to the amino acid sequence in any of these regions are linked to a number of diseases associated with congenital blindness. We would like to better understand the structure and function of the AD and DBD to describe the regulation of photoreceptor development by CRX. We used Small‐Angle X‐Ray Scattering (SAXS) and DNA binding assays on the DBD showing that the DBD is globular and can bind to DNA in the absence of the activation domain. We crosslinked the DBD peptide to DNA with glutaraldehyde in an attempt to get a bound structure of the peptide‐DNA complex. The peptide‐DNA complex will show more accurately how the DBD binds to DNA and the stoichiometry of the complex. We are working to confirm preliminary findings suggesting a 2:1 DBD:DNA complex. These experiments will help us to understand the role that CRX plays in regulating transcriptional networks in PR neurons.
Endothelial connexin hemichannels as a novel endothelial regulatory mechanism of vasodilation
Authors: Lillo Mauricio A. , Burboa Pia C. , Alves Natascha G. , Figueroa Xavier F. , Contreras Jorge E. , Duran Walter N.
Published: May 2022

The vascular endothelium regulates vasomotor tone in arterioles through intercellular conduction of electrical signals by gap junctions. Endothelial vasodilation is mediated by nitric oxide (NO) and by a hyperpolarizing current initiated in the endothelium. Both signals are transmitted to smooth muscle cells evoking relaxation. Endothelial cells express connexin (Cx) 37,40,43. Cx hemichannels allow the exchange of ions and molecules between intracellular and extracellular compartments that may be associated with endothelial electrical activity. Whether or not Cx hemichannels regulate vasomotor tone is unknown. We evaluated the role of Cx‐hemichannels in the vasodilation process by measuring dye uptake in primary culture of endothelial cells and in isolated mouse mesenteric arterioles. We detected that both acetylcholine (ACh) and bradykinin (BK) increase dye permeability, which was inhibited by connexin mimetic peptides that block Cx43 hemichannels (Gap19). We determined that NO mediates Cx43 hemichannels activity inasmuch as blocking NOS inhibits hemichannel activity. Using biotin switch assay, we observed that ACh induced S‐nitrosylation only in Cx43 at cysteine 271. ACh and BK open endothelial Cx43 hemichannels activity and promote endothelial Ca2+entry from the extracellular space and lead to endothelium hyperpolarization signaling in isolated mesenteric arteries. Blocking Cx43‐hemichannels reduces calcium entry and hyperpolarization. To further assess the physiological role of Cx43 endothelial hemichannels activity, we cannulated isolated mesenteric arterioles and studied ACh‐induced arteriolar relaxation in the presence and absence of connexin mimetic peptides that block Cx43 hemichannels (Gap19) and Cx40 (40Gap 27, a connexin mimetic peptide). Gap19 attenuates ACh‐induced relaxation, but 40Gap27 fails to reduce relaxation. These results indicate that endothelial Cx43 hemichannels participate in arteriolar dilatation. In addition, we replaced cysteine 271 by serine to create a Cx43C271S mouse that failed to cause S‐nitrosylation of Cx43. We detected that this knockin mouse displayed hypertension compared to wild type mice, indicating that Cx43 hemichannels and S‐nitrosylated Cx43 play a role in vascular resistance. Our results suggest that Cx43 hemichannels are a potential therapeutic target in hypertension and vascular disease.
Biomolecular Assessment and Assay of Kisspeptin, Oxytocin and Melatonin in Female Depressed Patients on Antidepressant Therapy: A Comparative Study
Authors: Oseyemwen Ndudi K. , Iniaghe Loretta O. , Bafor Enitome E.
Published: May 2022

Depression, a common psychiatric disorder with a prevalence of 4.4%, a leading cause of disability and the fourth highest contributor to the global burden of diseases remains underdiagnosed. Biomarkers have been suggested to enable a better understanding of the variable responses to therapy observed in depression. Kisspeptin, oxytocin and melatonin have been implicated in the etiology of anxiety and neuropsychiatric disorders such as seasonal affective depression. This study aimed to compare the differences in the serum levels of oxytocin, melatonin, kisspeptin and their messenger RNAs between antidepressant naïve females (control group) and those who had received at least a four‐week course of antidepressant therapy (test group). The mean serum levels of oxytocin, melatonin and kisspeptin among the test subjects was were 3.18 ±.77 pg/mL, 41.8 ± 2.53 pg/mL, and 291.56 ±106.11 nmol/L, respectively; and 3.14 ±.8.1 pg/mL, 42.06 ±3.18 pg/mL, and 268.89 ± 98.92 nmol/L, respectively for the control group. The total mRNA with the test group was an average of 17.5 ± 18.64), while for the control group it was 11.5 ± 18.41). There were slight correlations between the clinical symptoms of the patients and their mRNA levels but no significant changes in the total mRNA levels of oxytocin and kisspeptin in individuals on antidepressant therapy. There was also no significant relationships in the mRNAs and the serum levels of oxytocin, melatonin and kisspeptin between the test and control groups. Results from this study suggest that kisspeptin, oxytocin and melatonin may not be suitable biomarker for depression but provides a platform for further research.
PMSF and SFN Reduce Alpha‐synuclein Aggregation in a Yeast Model of Parkinson’s Disease
Authors: Kozub Noah J. , Luden Devyn E. , Furey Daniel P. , Haak Victoria M. , Sexton Zachary , Austriaco Nicanor
Published: May 2022

Parkinson’s Disease, PD, is the second most common neurodegenerative disease in humans. PD is marked by Lewy body formation in the brain, which disturbs the dopamine transfer system across neurons. Previous studies have shown that the protein, α‐synuclein, is a major contributor in the formation of Lewy bodies, which are a pathological hallmark of PD. In this study, we modeled α‐synuclein aggregation in the budding yeast, Saccharomyces cerevisiae and treated the cells with phenylmethylsulfonyl fluoride (PMSF) in one trial, and sulforaphane (SFN) in another. Our data suggest that a 4mM concentration of PMSF and a 200μg/ml concentration of SFN significantly reduces α‐synuclein aggregation. We are currently working to uncover the mechanism of action of these drugs.
Prenylated Stilbenoids as Potential Adjuvants for Paclitaxel in the Treatment of Triple‐Negative Breast Cancer
Authors: Mohammadhosseinpour Sepideh , Ho Linh‐Chi , Weaver Alexx , Medina‐ Bolivar Fabricio
Published: May 2022

Breast cancer is the most prevalent type of cancer among women worldwide. Triple‐negative breast cancer (TNBC) is unresponsive to typical hormonal treatments causing it to be one of the deadliest forms of breast cancer. Investigating alternative therapies to increase survival rates for this disease is essential. This study aims to examine the role of prenylated stilbenoids as an adjuvant for paclitaxel, a chemotherapeutic drug with severe side effects. To produce prenylated stilbenoids, hairy root cultures of peanut were co‐treated with elicitors and further purified via semi‐preparative high performance liquid chromatography. The cytotoxicity of prenylated stilbenoids was studied in TNBC cell lines MDA‐MB‐231 and MDA‐MB‐436 and the non‐cancerous cell line MCF‐10A. At low micromolar concentrations, the prenylated stilbenoid arachidin‐1 showed no significant cytotoxicity in the non‐cancerous cell line, whereas it was cytotoxic in the TNC cell lines. High cytotoxicity levels correlated with increased levels of the apoptosis markers caspase‐3 and caspase‐7. Arachidin‐1 also decreased the IC50 of paclitaxel by approximately 4‐fold and blocked cell division in S and G2‐M phases in the TNBC cells. Furthermore, paclitaxel combined with arachidin‐1 induced apoptosis through the intrinsic pathway by activation of caspase‐9. This highlights the significance of continuing research with prenylated stilbenoids in TNBC. Current studies focus on elucidating the signaling pathways affected by these compounds in TNBC cells to advance our understanding of the anticancer mechanisms of these natural products.
Gut It Out: An Anatomy Card Game and Study Tool for Medical Students
Authors: Stiver Mikaela L. , Ding Linda , Lalande Alexia , Lau Cat , Lin Winnie , Fejtek Monica , Jeon Sean , Leavitt Sarah , Krebs Claudia
Published: May 2022

Gamification in medical education involves integrating game principles—including mechanics, design, or experiences—into a learning context. Game‐based approaches in higher education have been shown to improve student engagement, motivation, and performance when applied effectively. Many medical students report feeling overwhelmed by the complexity and content‐heavy nature of anatomy, making it an ideal subject for gamification. In this project, we developed and designed an educational anatomy card game intended for medical students.

Students in the inaugural cohort of the Certificate in Biomedical Visualization and Communication (BMVC) at the University of British Columbia teamed up with the Hackspace for Immersive Virtual Experiences (HIVE) and faculty advisors for their capstone project. The team collected survey responses from current medical students to determine desirable game features and dynamics, followed by several rounds of prototyping and piloting. The students also enlisted the help of a subject matter expert in game design.

The final card game, entitled “Gut it Out”, is appropriate for players possessing any level of anatomy knowledge, with optional mechanics designed for medical students. Gameplay involves players competing to build fully innervated and vascularized organs. Organ cards specify the number of blood vessel cards (arteries and veins) and nerve cards (somatic, sympathetic, and parasympathetic) required for completion. Additional clinical cards can be played to gain advantages or sabotage opponents. An optional gameplay component challenges players to name each blood vessel and nerve associated with the organs they have completed in exchange for double points. Aesthetics feature a high‐contrast colour palette and bold, simple organ illustrations. The team developed multiple versions of the rules that are currently being play tested by medical students and anatomists. Future directions include incorporating an interactive augmented reality component to enrich the educational value of the organ cards and conducting a mixed methods study to assess the efficacy of the game as a study tool.

“Gut it Out” applies gamification to anatomy education to create a fun and educational card game featuring accessible, fast‐paced gameplay. This game was designed to complement and expand the current ecosystem of anatomy resources hosted by the HIVE.
Age‐dependent behavioral and neuropathological assessment of analysis of AppNL‐G‐F/NL‐G‐Fknock‐in (KI) mice
Authors: Wang Dongsheng , Head Brian , Wang Shanshan
Published: May 2022

Mitochondria play a crucial role in Alzheimer's disease (AD) onset and progression. Traditional transgenic AD mouse models share the limitation that the proteolytic processing of overexpressed mutant APP results in overproduction of amyloid‐beta (Aß) and other APP peptide fragments resulting in artificial, non‐clinically relevant phenotypes. Here we performed an in‐depth and time‐resolved behavioral and metabolic characterization of a clinically relevant AD mouse model engineered to express normal physiological levels of APP harboring humanized Swedish (K670N/M671L), Beyreuther/Iberian (I716F), and Arctic (E693G) mutations, (AppNL‐G‐F/NL‐G‐F), termed APP knock‐in (APPKI) mice. At 7 months of age, APPKI mice exhibited only learning deficit confirmed by both the water maze test and fear conditioning test. At the same age, we also observed mitochondrial dysfunction consisting of decreased ATP production, higher membrane potential with subsequent greater production of reactive oxygen species (ROS). Furthermore, mitochondria were larger in volume, accompanied by lower molecular markers of mitochondrial dynamics, mitofusin‐2 (Mfn2), at 6‐8 m. In contrast, the oxygen consumption rate (OCR) from 6 m APPKI hippocampal homogenates was similar to that of WT mice. At 12 months of age, APPKI mice exhibited learning and memory deficit, together with significantly decreased mitochondrial oxygen consumption rate detected by high‐resolution respirometry. Histopathology revealed minor amyloidosis accompanied by microgliosis (Iba1) as early as 3 months, which became more apparent at 6 and 12 m. These data indicate a wide range of brain mitochondrial dysfunction and associated cognitive deficits in APPKImice. Moreover, the identified behavioral and bioenergetic alterations in this physiologically relevant AD transgenic mouse model provide a valuable tool to optimize the temporal component for therapeutic interventions to treat AD.
Identification of a Novel Amino Acid Motif Responsible for Peptide Aggregation in vitro.
Authors: Torres Daniel J. , Nyman Karl , Glinka Tomasz , Borysov Sergiy I.
Published: May 2022

Peptides and their derivatives comprise a substantial sector of drug development initiatives due to their specific biological activities and broad chemical versatility. Among the challenges of peptide drug development is a reduction in peptide solubility that leads to peptide aggregation. This dramatically reduces a peptide’s biological availability and prevents its further testing in vivo.

Here we report a physicochemical analysis of peptides that inhibit the CMG helicase, the main replicative helicase within the cell. We demonstrate that the inhibitory peptides had a pronounced propensity to form aggregates in vitro by utilizing light microscopy and a fluorescence‐based peptide aggregation assay. By applying a peptide chase approach and abovementioned testing assays, we identified a short amino acid motif of X‐Leu‐Met‐Leu‐X that was responsible for peptide aggregation. Amino acid substitutions at these key residues were studied to confirm their contribution towards aggregation. Interestingly, substitution of either Leucine residues with Serine residues dramatically increased peptide solubility.
Plant Phytochrome B Assembles an Asymmetric Dimer with Unique Signaling Potential
Authors: Li Hua , Burgie Sethe , Gannam Zachary T. , Vierstra Richard , Li Huilin
Published: May 2022

Phytochromes (Phy) sense red and far‐red light and reversibly interconvert between inactive Pr and active Pfr conformers. Phys typically consist of a photo sensory module (PSM) formed by sequential N‐terminal PAS, GAF, and PHY domains, followed by a C‐terminal histidine kinase‐related domain (HKRD). There are four plant isoforms Phy A through D, and they all contain two additional PAS domains (PAS1 and PAS2) inserted between PSM and HKRD. Despite extensive biochemical and structural studies, there has been a lack of a full‐length structure, hindering our understanding of plant Phy signaling. Here we report a 3.3‐Å cryo‐EM structure of Arabidopsis PhyB dimer. Instead of the widely anticipated parallel association of the two monomers resembling the prokaryotic relatives, Plant PhyB adopts a topologically complex dimeric architecture with the two PSMs in a head‐to‐tail antiparallel form while the two HKRDs in a head‐to‐head parallel form. The PAS2 mediates the antiparallel dimerization of PSM that leads to a parallelogram‐shaped platform. And a preceding ‘modulator’ loop assembles tightly with the PSM of its own protomer. We show that these inter‐ and intra‐protomer connections contribute to stabilizing of dimer and accelerate thermal reversion of Pfr back to Pr. Lean of HKRDs towards the platform results in profound asymmetry of PhyB that might enable distinct signaling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate‐sensitive surface for interaction with their downstream signaling partners.
Surgical Implications in Multiple Variants in the Axillary Artery
Authors: Ramos Alicea Daniel A. , Marcano Anaya Jordan , Pagan John M. , Ramirez Norman , I. Quiñones Rodriguez Jailenne
Published: May 2022

The third segment of the axillary artery (TSAA) contributes crucial irrigation to muscles in mostly all compartments of the upper limb. Numerous studies have reported atypical branching patterns of the TSAA, which creates obstacles in operative assessments involving structures irrigated by this segment of the artery. In our current study, we observed three atypical branching patterns in the TSAA in which the subscapular artery gave rise to the posterior humeral circumflex artery and a second subscapular artery. In addition, a variant was found in the origin of the thoracodorsal artery; two collateral horizontal arteries irrigating the deep medial surface of the latissimus dorsi. Anatomical variants, not only in the axillary artery but in the whole body, have a remarkable importance in surgery as a whole and are a cause of the continuous adjustment of surgical standards and patient management. In this case report, we aim to evaluate these variants from a clinical perspective for the proper management of axillary traumas and pathologies.