bluuebunny/crossref_metadata_2025 huggingface dataset:
Elucidating the role of adenine nucleotide transporter in mitochondrial swelling: an experimental and computational approaches
Authors: Chapa‐Dubocq Xavier R. , Garcia‐Baez Jorge F. , Bazil Jason N. , Javadov Sabzali R.
Published: May 2022

Mitochondria produce over 90% of cellular ATP and actively participate in maintaining ion homeostasis, redox status, lipid metabolism, and cell growth. Changes in the matrix volume of mitochondria affect their functional and structural integrity. Modest volume increases associated with modulation of the inner mitochondrial membrane can activate electron transfer chain and oxidative phosphorylation, whereas excessive swelling impairs structural organization of the membrane and initiate mitochondria‐mediated cell death mechanisms. Therefore, clarifying the precise mechanisms of excessive mitochondrial swelling is important for regulation of mitochondria‐mediated cell death pathways in response to energy and oxidative stresses. Opening of non‐selective mitochondrial permeability transition pores (mPTP) is the primary cause of excessive matrix swelling. The molecular identity remains unknown and recent studies suggest the existence of two or more types of mPTP that can be composed of different protein(s). The adenine nucleotide translocator (ANT) and FOF1‐ATP synthase were proposed to be potential mPTP core components that can act together or independently each other. Here, we elucidated the role of ANT in mPTP opening by applying both experimental and computational approaches. mPTP opening was evaluated in cardiac mitochondria that were exposed to moderate and high Ca2+ concentrations in the absence and presence of respiratory substrates and ADP. We developed a detailed model of the ANT transport mechanism including the matrix (ANTM), cytosolic (ANTC), and pore (ANTP) states of the transporter that was able to simulate our experimental data. In addition, we corroborated and simulated our ANT model based on previous ANT kinetics data. The model was successful not only in simulating ANT pore state transition, but also explained the potential role of ANT in mPTP opening in cardiac mitochondria.
The Loss of PPARα in Adipocytes Induces Lipogenesis via the PASK‐SREBP1 Signaling Axis
Authors: Kipp Zachary A. , Xu Mei , Yiannikouris Frederique , Morris Andrew , Stec Donald , Wahli Walter , Stec David , Hinds Terry
Published: May 2022

PPARα is a nuclear receptor and a key player in regulating adiposity by activating b‐oxidation while inhibiting de novo lipogenesis within the liver. Its role in extrahepatic tissues is currently unknown. Therefore, we established the first adipose‐specific PPARα knockout (PparaFatKO) mice to determine the signaling position of PPARα in adipose tissue expansion and adipocyte hypertrophy that occurs during the development of obesity. To assess the function of PPARα in adiposity, female and male mice were placed on a high‐fat diet (HFD) or normal chow for 30 weeks. Only the male PparaFatKO animals had significantly more adiposity in the inguinal white adipose tissue (iWAT) (p=0.0091) and brown adipose tissue (BAT) (p=0.0031) with HFD compared to littermates. No changes in adiposity were observed in female mice compared to littermate controls. Lipidomics analysis of iWAT via liquid chromatography–mass spectrometry (LC‐MS) showed that PparaFatKO males had an increase in cholesterol esters. Interrogation of the signaling mechanisms with the loss of PPARα in adipocytes showed that the transcription factor sterol regulatory element‐binding protein‐1 (SREBP‐1) had more of the mature form present in the KO males and its target genes were higher, which might be due to the significantly (p=0.009) elevated expression of the Per‐Arnt‐Sim Kinase (PASK), a kinase that activates SREBP‐1. The hyperactivity of the PASK‐SREBP‐1 axis significantly (p<0.05) increased the lipogenesis proteins fatty acid synthase (FAS) and stearoyl‐Coenzyme A desaturase 1 (SCD1) and raised the expression of genes for cholesterol metabolism (Scarb1, Abcg1, and Abca1). Hence, we have uncovered a new signaling paradigm, the PASK‐SREBP‐1 axis in adipocytes, that drives lipogenesis, which PPARα inhibits. Our results demonstrate the first adipose‐specific actions for PPARα in protecting against lipogenesis and cholesterol ester accumulation that leads to adipocyte hypertrophy in obesity.
YM‐254890 Promotes the Subcellular Redistribution of Gαq
Authors: Randolph Clinita , Dwyer Morgan , Aumiller Jenna , Dixon Alethia , Inoue Asuka , Osei‐Owusu Patrick , Wedegaertner Philip
Published: May 2022

Uveal melanoma (UM), though a rare form of cancer, is the most common primary intraocular cancer in adults. Approximately 50% of patients succumb to metastatic UM for which there are no effective treatments resulting in a survival period of 2‐8 months. Interestingly, most patients with UM harbor a mutually exclusive mutation in Gαq or Gα11. However, no drugs are clinically approved to target these proteins to treat UM. YM‐254890 (YM) and FR‐900359 (FR) have gained much attention due to their ability to inhibit constitutively active Gαq by preventing the release of GDP and thereby inhibiting GDP to GTP exchange. However, their mechanism of action and potential to prevent uveal melanoma progression is not fully understood. While Gα subunits are known to signal at the plasma membrane (PM), some studies also show that these proteins traffic into the cytoplasm or to intracellular organelles particularly upon activation. The objective of this study is to understand the changes in localization of Gαq wild type (WT) and constitutively active GαqQ209L that occur by inhibition with YM. By immunofluorescence microscopy we show that Gαq WT and GαqQ209L display a subcellular redistribution from the PM to the cytoplasm upon YM treatment. We hypothesize that this relocalization is important for YM’s ability to inhibit GαqQ209L. By adding PM‐targeting motifs (myristylation, palmitoylation, or polybasic sites) to constitutively active Gαq we show that the PM‐restricted mutants do not redistribute into the cytoplasm upon YM treatment and, surprisingly, retain their ability to signal upon YM treatment. Interestingly, our signaling assays show that G protein‐coupled receptor (GPCR) activated PM‐restricted Gαq WT remains sensitive to inhibition by YM. Using pull down techniques to determine if YM promotes an inactive conformation of the PM‐restricted GαqQ209L mutants we show that both GαqQ209L and PM‐restricted GαqQ209L mutants display increased binding to Gβγ and decreased binding to RGS2 upon YM treatment, suggesting that YM promotes an inactive conformation in these PM‐restricted mutants in the same manner as with Gαq WT and GαqQ209L. Our studies suggest that an additional way in which YM inhibits GαqQ209L is by promoting the subcellular redistribution of constitutively active Gαq from the PM to the cytoplasm.
Transcriptional regulation of Rab21 GTPase in leukemia
Authors: Dovat Sinisa , Ding Yali , Song Chunhua , Bogush Daniel , Bastihali Tukaramrao Diwakar , Sharma Arati , Desai Dhimant
Published: May 2022

Rab GTPase family proteins have critical roles in membrane trafficking. Rab21 is a member of the Ras oncogene family, whose dysregulation has been associated with drug resistance in cancer. Regulation of Rab21 expression is still unknown. Here we present data that RAB21 expression is regulated by Casein Kinase II (CK2) and by the Ikaros protein in B‐cell acute lymphoblastic leukemia (B‐ALL). Ikaros is a DNA‐binding protein, encoded by the Ikzf1 gene, which acts as a tumor suppressor in leukemia. Analysis of global DNA binding showed increased occupancy of Ikaros at promoter of the Rab21 gene in several B‐ALL cell lines and primary B‐ALL cells. The role of Ikaros in transcriptional regulation of RAB21 expression in B‐ALL was determined by gain‐of‐function and loss‐of‐function experiments. Overexpression of Ikaros in B‐ALL via retroviral transduction resulted in increased Ikaros binding to the RAB21 promoter and decreased RAB21 transcription. In contrast, shRNA‐mediated knockdown of Ikaros inhibited Ikaros binding to the RAB21 promoter and resulted in an increased RAB21 expression. These data suggest that Ikaros directly repress RAB21 transcription. Our previous studies showed that CK2 directly phosphorylates Ikaros and reduces its DNA‐binding affinity. We tested whether CK2 affects Ikaros’ ability to regulate RAB21 expression in B‐ALL. Treatment of B‐ALL cells with a specific CK2 inhibitors, CX‐4945, resulted in increased Ikaros binding at the RAB21 promoter and reduced RAB21 expression. The use of luciferase reporter assay showed that both wildtype Ikaros and Ikaros phosphoresistant mutants strongly repress Rab21 transcription, while phopshomimetic Ikaros mutants at CK2 phosphorylation sites lost the ability to repress RAB21 transcription. Together, these data demonstrate that Ikaros and CK2 regulate transcription and overall expression of RAB21 in B‐cell ALL. Results uncovered an interplay between CK2 and Rab GTPase signaling pathways and suggest a mechanism by which CK2 regulates Rab21 activity in leukemia.
Effects of Omega‐6 Fatty Acids on Interleukin‐6, Phospholipase A2, and Prostaglandin‐Endoperoxide Synthase 2 Gene Expression in Lipopolysaccharide‐Challenged Murine Macrophages and Adipocytes
Authors: Swartzmiller Cory , Hallinan Ryan , Caldari‐Torres Cristina
Published: May 2022

Obese adipose tissue (AT) has been described as an organ involved in meta‐inflammation‐a chronic low‐grade inflammation linked to the predisposition to chronic diseases. Both adipocytes and macrophages are found in AT, with obese AT containing a higher percentage of macrophages compared to lean AT. Adipocytes and macrophages can produce pro‐inflammatory molecules (adipokines), with research showing that the macrophage is the primary contributor to inflammatory adipokine release by obese AT. Our lab has described differential effects of omega‐6 (n‐6) fatty acids (FAs) on the production of adipokines by 3T3‐L1 murine adipocytes and RAW 264.7 murine macrophages. The objective of this study was to compare the effects of n‐6 FAs on gene expression of inflammation‐related molecules in 3T3‐L1 and RAW264.7. Gene expression of interleukin‐6 (IL‐6), prostaglandin‐endoperoxide synthase‐2 (PTGS2), and phospholipase A2 (PLA2) were quantified in both of these cell lines after incubation with n‐6 FAs and a lipopolysaccharide (LPS) challenge. PGTS2 and PLA2 are enzymes involved in the production of inflammatory eicosanoids, while the IL‐6 gene codes for the IL‐6 protein. Macrophages were pre‐incubated with 100 μM of linoleic acid (LA; n‐6) or arachidonic acid (ARA; n‐6) for 24 h, followed by a 6 h, 0.01 μg/ml LPS challenge. Fibroblasts (3T3‐L1) were differentiated into adipocytes with the addition of 100 μM of one of the n‐6 FAs throughout the differentiation period, followed by a 6 h, 1 μg/ml LPS challenge once they were fully differentiated. After LPS challenge, RNA was extracted for quantitative PCR analysis. Fatty acid treatments had no effect on expression of the target genes (IL‐6, PTGS2, PLA2) in 3T3‐L1 cells (two‐way ANOVA). PTGS2 expression was downregulated in LA (0.24 ± 0.13 2ΔΔCt) and ARA (0.16 ± 0.13 2ΔΔCt)‐treated macrophages challenged with LPS compared to control (1.07 ± 0.13 2ΔΔCt) and LPS (1.70 ± 0.13 2ΔΔCt)‐challenged macrophages (two‐way ANOVA; p<0.0001). There were no significant LPS or LPS+n‐6 FA effects on gene expression of IL‐6 and PLA2in macrophages (two‐way ANOVA). Time‐response experimentation is necessary to determine the kinetic model of gene expression of these target genes in both cell lines.
Targeting ligand surface density controls both complement activation and targeting efficiency of immunoliposomes
Authors: Nong Jia , Glassman Patrick M. , Wang Zhicheng , Marcos‐Contreras Oscar A. , Brenner Jacob S. , Muzykantov Vladimir M.
Published: May 2022

Coupling monoclonal antibodies (mAb) and their derivatives to the surface of nanoparticles is a widely tested strategy to improve the specificity and magnitude of targeting to specific organs and cells. However, immediately after intravascular injections, nanoparticles are opsonized by many proteins, including complement protein C3. Opsonization promotes two undesirable processes that limit the utility of nanoparticles: 1) rapid elimination by phagocytes and 2) an anaphylactoid syndrome known as complement activation‐related pseudoallergy (CARPA), which manifests as capillary leak, hemolysis, hypotension, and can be catastrophic to critically ill patients. These processes can be further enhanced by the Fc region of coupled mAbs, which enhances both recognition by phagocytes via FcγRs and complement activation, making antibody‐targeted nanoparticle a “double‐edged sword”. In order to maximize the potential of targeted nanoparticles, CARPA must be minimized, while drug delivery is maintained. In this study, we investigated the effect of antibody coating density on complement activation, side effect, and targeting efficacy of targeted nanoparticles. Incubation of immunoliposomes with a range of mAb coating density with mouse serum revealed that C3a release (a marker of complement activation) was correlated with coating density (Fig A). Following intravenous injection, increasing mAb coating density on liposomes promoted hemoconcentration (Fig B), increased numbers of white blood cells in circulation (Fig C), and increased liposome clearance by the liver (Fig C). Liposomes that were coated with a mAb recognizing vascular cell adhesion molecule were injected into mice to study their blood and tissue pharmacokinetics. Liposomes with a lower coating density had delayed clearance from the blood and, as a result, improved delivery to the target tissue (brain) (Figure D). Future studies will elucidate the role of Fc receptors on liposome biodistribution, as well as the safety and efficacy of drug‐loaded immunoliposomes coated with different densities of targeting ligands.
Measurement of Cerebrovascular Hemodynamics in Awake Freely Behaving Mice
Authors: Vigderman Abigail , Longden Thomas
Published: May 2022

Hemodynamic changes are frequently used as a readout for neuronal activity in functional imaging studies, and the mechanisms by which neurons communicate activity‐dependent energy needs to the surrounding vasculature to evoke an increase in blood flow (functional hyperemia) are collectively termed neurovascular coupling (NVC). Various techniques are utilized to measure the hemodynamic changes resulting from NVC but none provide precise, cellular resolution data in awake and freely behaving animals. Here, we repurpose novel in‐vivo imaging technology to measure brain blood flow at the single capillary level using miniature head mounted microscopes (miniscopes) and compare this to gold‐standard multiphoton imaging of blood flow. We combine the use of miniscopes for data collection with automated analyses for rapid, accurate and impartial assessment of blood flow through complex vascular networks in deep brain structures. By adapting miniscopes to study hemodynamics in detail, we aim to generate high‐resolution data of NVC and functional hyperemia that provides a window into the dynamics of these mechanisms in awake, freely behaving animals.
Stretch‐induced Myocardial Injury and Inflammation After Transient Vasopressin‐Mediated Left Ventricular Pressure Overload in Swine
Authors: Rolland Tyler J. , Shihab Ronzon R. , Nsumbu Dorcas , Tajlil Arezou , Canty John M. , Weil Brian R.
Published: May 2022

Objective

Using the α1‐adrenergic agonist phenylephrine (PE), we have previously demonstrated that an acute episode of transient pressure overload (TPO) elicits stretch‐induced myocardial injury and reversible left ventricular (LV) systolic dysfunction. Although this was accompanied by leukocyte mobilization, a rise in inflammatory cytokine levels, and increased pro‐inflammatory gene expression in the heart, the extent to which this resulted from direct activation of α1‐adrenergic receptors on immune cells vs. an inflammatory response to mechanical stretch‐induced myocardial injury is unclear. Accordingly, the present study was designed to determine if an inflammatory response to myocardial injury is evident when TPO is produced by vasopressin (VP), a vasoconstrictor without direct α1‐adrenergic agonist properties.

Methods

Hemodynamic and echocardiographic parameters were assessed in propofol‐anesthetized swine before, during, and after a 30‐minute infusion of VP (VP‐TPO; 0.04 U/kg/min, n=7) to increase LV end‐diastolic pressure (EDP) to ~30mmHg. Serial jugular venous blood sampling was performed to quantify circulating cardiac troponin I (cTnI), leukocytes, and inflammatory cytokines (TNF‐α and IL‐6) at baseline and 3‐hours after cessation of VP‐TPO. Leukocyte and cytokine levels were compared with those observed after PE‐mediated TPO in a separate group of animals (PE‐TPO; n=6).

Results

VP elicited a significant rise in mean arterial pressure (91±5 to 165±11 mmHg) and LV EDP (10±1 to 29±2 mmHg; both p<0.05 vs. baseline). This was accompanied by a significant rise in LV end‐diastolic volume (73±5 to 110±7 mL) and end‐systolic volume (20±3 to 53±6 mL), as well as a reduction in LV ejection fraction (EF; 72±3 to 52±3 %; all p<0.05 vs. baseline). One hour after cessation of VP‐TPO, LV EF remained depressed vs. baseline (60±3 %; p<0.05) and circulating cTnI levels began to rise, reaching a concentration that was ~10‐fold higher than baseline concentrations at the 3‐hour post‐TPO timepoint (18±5 to 118±21 ng/L; p<0.05). These changes were accompanied by a significant rise in circulating neutrophils, monocytes, TNF‐α, and IL‐6 that was similar to that observed after PE‐TPO (Figure).

Conclusions

VP‐TPO elicits myocardial injury, leukocyte mobilization, and an acute rise in circulating pro‐inflammatory cytokines, thereby recapitulating several features of PE‐TPO in the absence of direct α1‐adrenergic stimulation. These findings suggest that stretch‐induced myocardial injury provokes an inflammatory response that may play an important role in promoting the development of cardiac fibrosis and diastolic dysfunction after exposure to repetitive episodes of TPO.
Development of the Göttingen Minipig Model: GB and GF Toxicity
Authors: Yue Xiaomin , Stevenson Robert , Burgan Bradley , Langston Jeffrey , MYERS Todd M.
Published: May 2022

The dwindling availability of nonhuman primates (NHP) and increased ethical concerns over their use as experimental animals has driven a search for alternative animal models that can predict the effects of organophosphorus nerve agent (OPNA) in humans and to develop better medical countermeasures (MCM). Göttingen minipigs are physiologically and anatomically similar to humans, and are being considered as a potential alternative species for OPNA and MCM research. In the current study, fifteen sexually mature male Göttingen minipigs were utilized to determine the intramuscular (IM) 24‐hour median lethal dose (LD50) for the nerve agent sarin (GB) and cyclosarin (GF) using an up‐and‐down dose selection method. The initial doses for GB and GF were selected by approximating established lethality data for VX in Göttingen minipigs across IM and subcutaneous (SC) routes of administration. All animals exhibited typical signs of nerve agent poisoning immediately after being exposed to GB/GF. Three unique doses were required to estimate a GB LD50 of 40.00 µg/kg (95% confidence interval 33.25 µg/kg ‐ 49.76 µg/kg) and a GF LD50 of 40.57 µg/kg (95% confidence interval 33.03 µg/kg ‐ 49.73 µg/kg) with maximum likelihood estimation. The progression of toxic signs and the LD50 estimates obtained support the further development and use of Göttingen minipigs as a model to study MCMs and OPNAs.
Long Term Pulmonary Effects of E‐cigarettes on in‐utero Exposed Mice
Authors: Alghothani Omar , Aslaner David M. , Gorr Matthew W. , Miller Roy A. , Wold Loren E.
Published: May 2022

Introduction

The in‐utero environment is delicate, as it fosters the proper health and growth of the fetus. Negative environmental effects during gestation increase the chances of long‐term developmental consequences. As e‐cigarettes gain popularity in pregnant mothers, the aim of this study is to observe the chronic pulmonary function of adult mice exposed to e‐cigarette vapor during gestation.

Methods

Pregnant mice underwent exposure for 4 hours a day, 5 days a week beginning from the first day of pairing for breeding and ending at birth. Mice were separated into three exposure groups each receiving one 70 mL e‐cig vapor puff per minute of (1) E‐cig vapor with nicotine Nic(+) (2% Nic 50:50 propylene glycol:vegetable glycerin), (2) E‐cig exposure without nicotine Nic(‐) (propylene glycol: vegetable glycerin), or (3) continuous filtered air exposure (FA)HEPA filter. Resulting pups from each group were examined at adulthood (4‐5 months). Data including baseline lung mechanic scan and a dose response to different concentrations of methacholine, a drug used to induce airway hyperreactivity, were obtained via the FlexiVent FX2 (SCIREQ Inc.)

Results

Male and female offspring of mice exposed to Nic(‐) and Nic(+) witnessed a significant decrease in overall lung function as compared to the FA group. Baseline compliance (Crs) was significantly decreased in the Nic(‐) female group and Nic(‐) and Nic(+) male groups (P < 0.05 via ANOVA). Female and male mice in the Nic(‐) had greater stiffness (Ers) than the corresponding FA group (P < 0.05). Upon addition of methacholine, female mice in the Nic(‐) and male mice in the Nic(+) had an increased amount of lung resistance (Rrs)upon inhalation (P < 0.05). Female mice in the Nic(‐) had decreased forced expiratory volume (FEV0.1) in comparison with the FA group (P < 0.05).

Conclusion

Preliminary data supports that in‐utero exposed mice to e‐cigarette vapor with or without nicotine causes long term alterations in lung function. Future directions aim to understand the mechanisms of change through observing changes in histology, altered gene expression, protein analysis, and epigenetic changes through DNA methylation. Results of this work demonstrate that vaping may not be a safe alternative to smoking during pregnancy as the health of the offspring have detrimental pulmonary effects in adulthood.
Dent Disease in Drosophila: An Unexpected Model to Study the Relationship Between a Cl‐ Transporter and Ca2+
Authors: Reynolds Carmen , Dow Julian , Romero Michael
Published: May 2022

Mutations in the 2Cl‐/H+ transporter CLC‐5 cause Dent Disease Type 1 (DD1) and lead to progressive renal failure by age 20‐40. A major characteristic of DD1 is the renal mishandling of Ca2+that increases urinary Ca2+ excretion, calcium oxalate (CaOx) kidney stones, and kidney calcification. Interestingly, none of the DD1 symptoms include or relate to Cl‐ homeostasis despite impaired transport function. Thus, how does a Cl‐ transporter cause Ca2+ dysregulation?

Our laboratory has identified Drosophila Clc‐c to be the homolog of CLC‐5 with conserved amino acids at DD1 mutation sites. We hypothesize that Clc‐c shares functional similarities in Cl‐transport and Ca2+ homeostasis. To test our hypothesis, we used electrophysiology to measure ion transport, and then assessed CaOx crystal formation in adult fly renal tubules (Malpighian tubules, MTs) of Drosophila.

Voltage clamp experiments with Xenopus oocytes show that Clc‐c is electrogenic with the same outward‐rectifying current in Cl‐ solutions as observed with human CLC‐5. Next, knockdown flies (Clc‐c‐KD) were generated by crossing Uro‐Gal4 (MT driver) with UAS‐Clc‐c‐RNAi flies, resulting in 50% Clc‐c mRNA expression in the MTs. Crystals in adult MT (7 days after eclosure) were present in 100% of Clc‐c‐KD compared to 71% of control flies. The average number of crystals in adult tubule pairs was significantly greater in Clc‐c‐KD flies (15±4 crystals per pair) than control flies (5±2 crystals, P= 0.02). When fed a diet with 2mM NaOx ClcC‐KD for 4 days, Clc‐c‐KD flies had a significantly greater number of crystals in each tubule (24±5 crystals) than controls (4±2 crystals, P=0.005). However, the KD flies did not produce more crystals than control flies when fed a supraphysiological concentration of sodium oxalate (20 mM NaOx diet) for 1 or 4 days and in ex vivo assays of dissected MTs submerged in solution containing 10 mM NaOx for 1 hour.

In conclusion, Drosophila Clc‐c has both identical sequences at DD1 mutation sites and similar biophysical functions to human CLC‐5. Furthermore, Clc‐c‐KD increases incidence of CaOx crystals which corresponds to the increased risks for kidney stones in DD1 patients. With this fly‐DD1 model, we will be able to assess the relationship of Clc‐c Cl‐ transport to renal Ca deposits. Future experiments will evaluate renal pathophysiology of DD1 mutations and their relationship to Ca2+ transport in Clc‐c transgenic flies.
Role of HSP90 Family Molecular Chaperone in Regulating Chloroplast Protein Homeostasis and Plant Development in Arabidopsis
Authors: Zhao Rongmin , Mu Bona , Jiang Tim , Tseng Wei‐Tse , Nair Adheip M.
Published: May 2022

Chloroplasts carry out photosynthesis in plants and provide most food sources for living organisms. Either divided or differentiated from proplastids and etioplasts, the biogenesis of thylakoid membranes is the hallmark of chloroplast maturation and functionality, and this process requires a large set of photosynthetic proteins that must be synthesized on cytosolic ribosomes and then imported into the chloroplast stroma. Thylakoid lumen proteins travel further through the stroma and then cross the thylakoid membrane through one of the ancestral translocases. Like the cytosolic environment and the other major organelles, a cohort of molecular chaperones and proteases reside within the chloroplast to ensure proper protein homeostasis. We have identified and confirmed the interaction between the essential plastid HSP90 family heat shock protein HSP90C and a thylakoid lumen located photosystem II subunit PsbO1. To understand the biological significance of the interaction, we adopted transgenic approaches, specific HSP90 inhibitors, biochemical and in organello protein transport assays to study how the HSP90 family protein facilitates the representative lumen proteins transport across the thylakoid membrane. Specifically, we showed that the expression of the molecular chaperone is developmentally regulated, and its expression is highly correlated with some photosynthetic proteins while not with the others. Additionally, we demonstrated that HSP90C physically interacts with the thylakoid SEC translocon subunit, and that its chaperone activity is required for efficient PsbO1 transport. We also investigated the signature sequences of different HSP90 subfamily proteins and provided evidence that the chloroplast located HSP90C contains features specialized for some photosynthetic proteins interaction, and thus the chloroplast maturation and functionality.
Mitochondrial Fission and Fusion Dynamics are Regulated by Multiple Pathways in Renal Proximal Tubule Cells Treated with High Glucose
Authors: Cleveland Kristan H. , Schnellmann Rick G.
Published: May 2022

In type 2 diabetes, hyperglycemia leads to proximal tubular dysfunction, which is accompanied by altered mitochondrial homeostasis. We previously demonstrated that in renal proximal tubule cells (RPTC) grown in high glucose, as well as in diabetic db/db mice, proteins involved in mitochondrial dynamics were altered. Phosphorylation of the mitochondrial fission protein Drp1 increased and expression of the mitochondrial fusion protein Mfn1 decreased. One potential activation pathway of Drp1 is the RhoA/ROCK1/Drp1 signaling cascade in the presence of high glucose, leading to increased mitochondrial fission. Conversely, Mfn1 can be activated by MEK/ERK signaling. However, these pathways have not been investigated in the proximal tubule. Therefore, we determined the signaling pathways responsible for altered Drp1 phosphorylation and Mfn1 expression in RPTC. Primary cultures of RPTC were grown in high glucose (17mM), mannitol (17mM) or no glucose for 96 hr and were co‐treated with either RhoA (CCG‐1423), ROCK1 (Y‐27632) or MEK 1/2 (GSK 1120212) inhibitors 24 hr prior to harvesting. Cells were subjected to GTPase pulldown assays to measure Drp1, RhoA and Mfn1 activity and maximal mitochondrial respiration was measured using Seahorse XF96 analyzer. RPTC treated with glucose for 96 hr exhibited an increase in both RhoA and pDrp1 and an increase in GTP‐bound RhoA and Drp1. Co‐treatment with CCG‐1423 or Y‐27632 prevented glucose‐induced increases in RhoA and Drp1, respectively. Inhibition of RhoA and ROCK1 restored maximal mitochondrial respiration. Co‐treatment with GSK 1120212 prevented glucose‐induced decreases in Mfn1. Together, these results demonstrate that treatment of RPTC with high glucose increases RhoA and Drp1 activity and maximal respiration through the RhoA/ROCK1/Drp1 signaling pathway and is responsible for increased mitochondrial fission and respiration. In contrast, the MEK/ERK signaling cascade prevents the decrease in Mfn1 observed in the presence of high glucose. These data indicate that mitochondrial dynamics in high glucose in RPTC, are regulated by two independent signaling pathways.
Sex‐Differences in Acute Cannabinoid Response and Tolerance as a Function of Mouse Strain
Authors: Lulek Courtney F. , Morgan Daniel J. , Henderson‐Redmond Angela N.
Published: May 2022

Cannabinoids have been increasingly used to alleviate chronic pain; however, tolerance to the antinociceptive effects of cannabinoids, including delta‐9‐tetrahydrocannabinol (Δ9‐THC), may limit their therapeutic utility. Likewise, with more women than men now using medical cannabis for pain relief, it is imperative that we understand how sex may influence cannabinoid‐mediated antinociception and subsequent tolerance. While studies in rats have consistently found female rats to be more sensitive to the acute antinociceptive effects of cannabinoids compared to male rats, work in our lab consistently finds the opposite finding that male mice are more sensitive to the acute antinociceptive effects of both Δ9‐THC and CP55,940 compared to female littermates. Studies in our lab have consistently utilized mice on a C57BL6/J (B6) background. Therefore, the purpose of the present study is to examine whether our observed sex‐differences in Δ9‐THC‐induced antinociception and tolerance are consistent across multiple mouse strains or are strain‐dependent. Male and female B6 and DBA mice were first assessed for differences in acute Δ9‐THC‐induced antinociception using the tail‐flick assay across a range of doses of (0‐100 mg/kg). After a significant washout period, these mice were subsequently assessed for sex‐differences in antinociceptive tolerance development to 30 mg/kg Δ9‐THC following once‐daily treatment for seven consecutive days. Consistent with our previous findings, male B6 mice were more sensitive to the acute antinociceptive effects of Δ9‐THC than female B6 mice. Male and female DBA, however, mice did not differ in their antinociceptive response to Δ9‐THC, suggesting that sex‐differences in cannabinoid‐induced antinociception in mice is likely strain‐specific. These studies highlight the therapeutic potential of Δ9‐THC in pain management and underscore the importance of considering sex when evaluating their clinical utility.
Force and Shortening Velocity are Reduced in 24‐month Fischer 344 Rats
Authors: Davis Leah A. , Forero Luis , Hernandez‐Vizcarrondo Genesis , Han Young‐Soo , Mantilla Carlos B. , Sieck Gary C.
Published: May 2022

The diaphragm muscle (DIAm) shortens and produces force to generate negative intra‐thoracic pressure for airflow into the lungs and positive intra‐abdominal pressure for airway clearance and expulsive behaviors. Breathing is a low force repetitive behavior that requires recruitment of fatigue resistant type I and IIa DIAm fibers. In contrast, short duration airway clearance behaviors are infrequent and associated with greater DIAm force that require the additional recruitment of more fatigable type IIx/IIb fibers. Aging is characterized by selective atrophy of type IIx/IIb DIAm fibers and decreased specific force, i.e., sarcopenia. Older individuals are able to breathe without difficulty but have trouble with some higher force airway clearance behaviors of the DIAm such as coughing and sneezing. In 6‐ and 24‐month Fischer 344 rats (n=6 per group), DIAm strips were dissected from the mid‐costal region, mounted in a tissue chamber filled with Rees‐Simpson solution, and stimulated directly to measure maximum isometric specific force (Po) and maximum shortening velocity (Vmax) using a Cambridge dual‐mode length‐force servo controller (model 300B). In addition, power fatigue was assessed during repetitive isovelocity (30%Vmax) contractions evoked by 75 Hz in 400‐ms‐duration stimulus trains repeated for a 2‐min period. Additional muscle segments were rapidly frozen at optimal sarcomere length in melting isopentane, and transverse muscle fiber sections were cut at 10 µm thickness for determination of muscle fiber type (based on myosin heavy chain immunoreactivity) and fiber cross‐sectional areas. The relationship between force (load) and shortening velocity was determined using the Hill equation. Aging significantly reduced maximum isometric force, maximum shortening velocity, and maximum power output of the DIAm in older rats. The reduction in power during repetitive isovelocity shortening was reduced in older rats, but the residual power output after 2 min stimulation was comparable between young and old rats. These changes in mechanical and fatigue properties were consistent with the selective atrophy of type IIx/IIb DIAm fibers and reflected the sustained output of type I and IIa DIAm fibers, which are recruited for essential breathing behaviors.
Investigating the Effects of IRF6 on Focal Adhesions in Keratinocytes
Authors: Lowenberg Sarah , Antiguas Angelo , Dunnwald Martine
Published: May 2022

Focal adhesions are one of the two main types of adhesions between cells and the extra‐cellular matrix (ECM). Focal adhesions are dynamic complexes anchored to the actin cytoskeleton through intracellular adaptor proteins, and to the ECM through heterodimers of α and β integrins, forming a transmembrane link between the intra‐ and extra‐cellular space. β‐1 integrin is the primary β subunit found in keratinocytes, and its corresponding heterodimers show affinity for a wide range of ECM proteins including collagen, fibronectin, and laminin. Collagen is the main structural protein in cutaneous ECM (the dermis), while fibronectin is a glycoprotein found in the ECM that binds integrins and plays a crucial role in wound healing. Interferon regulatory factor 6 (IRF6) is a transcription factor expressed by epithelial cells that regulates keratinocyte differentiation, cell‐cell adhesions, and migration. It is well known that focal adhesions are required for cell migration, however the role of IRF6 in regulating focal adhesions is unknown. Therefore, we hypothesize that IRF6 regulates focal adhesion formation. To determine if IRF6 levels affect attachment to the ECM, we seeded wildtype and IRF6 knockdown keratinocytes on collagen IV and plastic and quantified the number of cells attached to the plate after thirty and sixty minutes. No differences were found between wild‐type and IRF6 knockdown keratinocytes on plastic. However, less IRF6 knockdown keratinocytes were attached on collagen IV compared to wildtypes. To explore how IRF6 affects focal adhesions, we plated IRF6 knockdown and wild‐type keratinocytes on different types of ECM that were representative of both normal and wounded conditions of the dermis. Immunofluorescent staining for β‐1 integrin and phalloidin was performed, followed by quantitative analysis of focal adhesion length and intensity. Initial observations indicate that IRF6 knockdown keratinocytes consistently have longer focal adhesions compared to the wildtype. Variations of β‐1 fluorescent intensity was also observed in wild‐type cells compared to IRF6 knockdown keratinocytes. These preliminary results suggest a role for IRF6 in regulating focal adhesions, providing a potential mechanism for the underlying defect observed in the migration of IRF6 deficient keratinocytes.
Deletion of Ch. IV Telomeres via Genetic Engineering of a Circular Chromosome in the Budding Yeast S. cerevisiae
Authors: Mefford Melissa A. , Hoover Blake
Published: May 2022

Chromosome structure varies from a single circular chromosome in prokaryotes to multiple linear chromosomes in eukaryotes. Linear chromosomes differ in that they possess specialized ends, called telomeres, that play important roles in protecting the ends from degradation. Furthermore, telomeres cannot be fully copied during DNA replication without the enzyme complex telomerase. Since telomerase is not expressed in all cells, telomeres shorten with aging. On the other hand, overexpression of telomerase is observed in >85% of human cancers. Given that telomeres create this end‐replication problem, our lab is interested in ultimately understanding why linear chromosomes evolved in the first place. To address this broad question, we are taking a novel genetic approach to systematically convert each of the linear chromosomes into circularized versions using the single‐celled eukaryote Saccharomyces cerevisiae. Our approach involves building two DNA cassettes with selectable marker elements that are integrated into the left and right arms of each chromosome in a haploid yeast strain. The cassettes are designed so that recombination between the two regions will reconstitute a functional marker gene, allowing genetic selection of circularized chromosomes. To date, we have isolated a circularized version of Ch. IV, the longest of the 16 yeast chromosomes. We have verified recombination between the cassette via PCR across the newly formed junction. The circularized Ch. IV yeast form colonies slightly smaller in size and exhibit a ~10% decrease in doubling time relative to wild‐type. Together, these data demonstrate that our approach can generate viable yeast with individual chromosomes circularized. We are currently analyzing cellular morphology using light microscopy, testing the ability of the circularized yeast to undergo mating and sporulation, and analyzing impacts on gene expression. A longer‐term goal of the lab is to use this approach to attempt to circularize the other 15 chromosomes and explore how linear chromosomes evolved.
Central hemodynamic response during submaximal and exhaustive exercise in humans with high affinity hemoglobin and compensatory polycythemia
Authors: Webb Kevin L. , Regimbal Riley J. , Morkeberg Olaf H. , Senefeld Jonathon W. , Baker Sarah E. , Joyner Michael J. , Wiggins Chad C.
Published: May 2022

Humans with high affinity hemoglobin (HAH) develop compensatory polycythemia, an elevated hematocrit, which increases blood viscosity and may compromise cardiac output during exercise. In addition, humans with HAH have shown alterations in muscle blood flow during exercise in normoxia and hypoxia. As such, we aimed to investigate the influence of HAH and compensatory polycythemia on the central hemodynamic response during both normoxic and hypoxic exercise.

METHODS

Seven humans with HAH (4 females, 36 ± 13 years) and 11 healthy controls with normal oxygen binding affinity (4 females, 39 ± 10 years) performed cycling exercise at two intensities, 1) at a power output corresponding to 15% below normoxic gas exchange threshold (GET) for four minutes and 2) at a power output requiring 85% of normoxic maximal aerobic capacity until exhaustion. This protocol was performed during both normoxia and hypoxia (21% O2 and 15% O2, respectively). Hemodynamic measurements were taken after three minutes of cycling at 15% below normoxic GET, three minutes at 85% V̇O2max, and at exhaustion. Cardiac output (Q̇) was estimated using an open‐circuit acetylene wash‐in technique. Stroke volume (SV) was calculated by dividing Q̇ by heart rate (HR). Baseline characteristics were compared between groups with student’s two‐tailed unpaired t‐tests. Hemodynamics were analyzed using a two‐way repeated measures analysis of variance to examine the effects of group and exercise intensity.

RESULTS

As expected, hematocrit was ~15% greater in the HAH group than the control group (HAH: 58 ± 7 % vsCTL: 43 ± 3 %, P<0.001). Both groups elicited an increase in Q̇ and HR as exercise intensity increased during both normoxia (P<0.001 for both) and hypoxia (P<0.001 and P=0.002, respectively). There was no significant difference in Q̇ between groups during normoxia (P=0.85) or hypoxia (P=0.69). In addition, neither HR nor SV were different between groups during exercise in normoxia (P=0.13 and P=0.39, respectively) or hypoxia (P=0.89 and P=0.73, respectively). There was no group by intensity interaction for Q̇, HR, or SV during normoxia (all, P>0.42) or hypoxia (all, P>0.63).

CONCLUSION

Our data suggest that cardiac output is not compromised during normoxic or hypoxic exercise in humans with HAH and compensatory polycythemia. Therefore, compensatory polycythemia may improve exercise capacity in humans with HAH by raising oxygen carrying capacity without limiting cardiac output.
Mechanistic study of nucleotide‐sugar binding and catalysis in SECRET AGENT O‐GlcNAc Transferase
Authors: Nelson Dana , Philbrick Kaylee , Dallmier Kate , Chalfant Kaitlyn , Olson Brian , Joiner Cassandra
Published: May 2022

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) transferase (OGT) is an essential protein that is highly conserved across all eukaryotes. Misregulation of OGT’s activity has been implicated in cancers and neurodegenerative diseases. OGT is unique in that it transfers a GlcNAc sugar from UDP‐GlcNAc to over 1,000 nuclear and cytoplasmic proteins in almost every cellular process. The mechanisms of nucleotide‐sugar binding and sugar transfer have been well characterized in human OGT. Secret Agent (SEC), an OGT structural homolog in Arabidopsis thaliana, also uses UDP‐GlcNAc as its sugar donor, but the mechanisms of nucleotide‐sugar binding and catalysis are not well characterized. We investigated how the SEC homolog selects UDP‐GlcNAc, looking particularly at the residues that are hypothesized to interact with the GlcNAc portion of UDP‐GlcNAc in OGT and drive deprotonation of protein substrates. However, it is unknown if the function of these residues is conserved in SEC. Active site residues in SEC corresponding to known essential residues in OGT were mutated using site‐directed mutagenesis and expressed and purified from E. coli. Their catalytic activities were analyzed using glycosyltransferase activity assays and compared to the wildtype enzyme. These mutations led to lower relative activity, similar to OGT studies, suggesting that the binding and orientation mechanism of UDP‐GlcNAc required for sugar transfer is conserved between SEC and OGT.
Profiling the Cytochrome P450 2J2 Active Site
Authors: Motl Nicole , Smith Richard D. , Carlson Heather A. , Scott Emily E.
Published: May 2022

Human cytochrome P450 2J2 (CYP2J2) is a membrane bound, heme‐containing monooxygenase primary expressed in the heart. This enzyme oxidizes the double bonds in polyunsaturated fatty acids such as arachidonic acid and linoleic acid to epoxides, generating epoxyeicosatrienoic acids (EETs). Various EET products are implicated in cardioprotective effects, but also the promotion of cancer tumor growth and metastasis. Additionally, CYP2J2 can act on a range of pharmaceuticals. Thus, CYP2J2 is of clinical interest, but there is no structural information to understand how the CYP2J2 active site accommodates this wide variety of ligands.

In this study, the CYP2J2 active site was defined by its ability to bind an array of >75 substrates, inhibitors, and xenobiotics, including a broad panel of azole compounds. The binding modes and affinities were determined by monitoring the shifts of the CYP2J2 heme Soret absorbance, which is indicative of heme environment perturbation. From these experiments the following spectral shift responses were observed: displacement of the heme‐coordinated water (type‐I binding mode), compound coordination to the heme iron (type‐II), a red‐shifted type‐I binding mode, compound coordination to heme coordinated water (reverse type‐I binding mode), or no spectral shift. As the latter result can be consistent with no binding or binding in the active site in a way that does not perturb the heme center, such compounds were investigated via a luminescence‐based inhibition assay to verify their interaction with CYP2J2. As imidazole‐containing ligands provide a fixed, known reference point for their interaction with the CYP2J2 heme iron, cheminformatics approaches were applied to these compounds to help provide insight into the chemical makeup of the CYP2J2 active site. The physical properties of these ligands were analyzed to identify key ligand features that drive CYP2J2 ligand preference. In the absence of protein structure information, this strategy provides a route to defining the CYP2J2 ligand binding site, potentially supporting the development of selective inhibitors that can help define the role of CYP2J2 modulation in vivo and potentially facilitate therapeutic strategies.
Roles of GABAergic and Glutamatergic Prebötzinger Complex Circuits in Opioid‐Induced Respiratory Depression
Authors: Schumacker Kayla S. , Scarpellini Carolina , Montandon Gaspard
Published: May 2022

Introduction

. Opioids are widely prescribed analgesic medication but are highly addictive and can be lethal with overdose. Respiratory depression is the primary cause of death by opioid overdose, and it limits the ability to use opioid analgesics safely and effectively. Respiratory depression by opioids is mediated by the inhibition of neural circuits in the brainstem, such as the preBötzinger Complex (preBötC), a small region of the medulla involved in respiratory rhythm generation. The generation of respiratory rhythm requires the activation of excitatory and inhibitory neurons in the preBötC. Glutamate is a main excitatory neurotransmitter, and preBötC glutamatergic neurons are involved in generating respiratory rhythm. γ‐aminobutyric acid (GABA) is a main inhibitory neurotransmitter, and GABAergic neurons are found in the preBötC. Interestingly, GABAergic neurons often co‐express µ‐opioid receptors, and disinhibition of GABAergic neurons by opioid drugs is involved in opioid addiction and euphoria. Here, we aim to determine the roles of excitatory glutamatergic and inhibitory GABAergic neurons in opioid‐induced respiratory depression.

Methods

To control glutamatergic and GABAergic neurons, we expressed opsin channels in cells expressing the vesicular glutamate transporter 2 (vGLUT2) and the vesicular GABA transporter (vGAT), respectively. Using a cre‐lox recombination approach, we injected into the preBötC cre‐dependent adeno‐associated viruses to locally express the inhibitory archaerhodopsin or the excitatory channelrhodopsin in vGLUT2 or vGAT‐expressing cells. Following a 4‐week incubation period, we applied 554 nm or 470 nm lights through optical fibres focused onto the preBötC while recording diaphragm and genioglossal muscle activities in anesthetized mice. Following systemic injection of fentanyl (5µg/kg), we inhibited or activated glutamatergic or GABAergic cells, respectively.

Results

Our preliminary data indicate that inhibition of vGLUT2 cells severely depressed respiratory rate, mimicking the respiratory depression observed with the opioid fentanyl. We are currently performing optical inhibition of vGAT preBötC cells in anesthetized mice and determining their role in respiratory depression by fentanyl.

Discussion

We will identify the role of GABAergic and glutamatergic preBötC cells in coordinating respiratory rhythm in preBötC cells and whether GABAergic cells contribute to respiratory depression by opioid drugs. Understanding the role of the populations of cells involved in respiratory depression by opioid drugs is critical to identify new molecular targets to reduce respiratory depression and provide safe and reliable pain killers without the risks associated with opioid pain killers.