bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining Vascular Responses to Passive Movement in Premenopausal Females: Comparisons Across Sex and Menstrual Cycle Phase
Authors: Weggen Jennifer B. , Hogwood Austin C. , Decker Kevin P. , Darling Ashley M. , Chiu Alex , Richardson Jacob , Garten Ryan S.
Published: May 2022

Purpose

Vascular responses to passive movement represent a novel assessment of vascular function and serves as a critical component for adequate blood flow regulation during exercise. Although alterations to estradiol (E2) throughout the menstrual cycle, as well as sex differences, are known to impact vascular function, limited research exists in determining whether vascular responses to passive movement are altered in premenopausal females when compared across sex or menstrual cycle phase. We hypothesized that there would be no differences in leg blood flow responses to passive leg movement (PLM) in premenopausal females when compared to age‐matched males or when evaluated across follicular menstrual cycle phases, when E2 fluctuates the most.

Methods

Vascular responses to one‐minute of PLM were assessed in eleven premenopausal females (n = 21; 23 ± 4 yrs) and age‐matched males (n = 21; 24 ± 4 yrs). All the females completed the PLM assessment during the early (EF) and late follicular (LF) phase of their menstrual cycle, based on self‐reporting of menses. Lower limb microvascular function was assessed during PLM via changes in leg blood flow measured as peak change from baseline (LBFΔPEAK) and area‐under‐the‐curve (LBF AUC). Thigh volume, a known modulator of PLM responses, was also assessed in the passively moved limb in all participants via anthropometric measurements. Blood samples were obtained at rest from the antecubital vein of the arm to determine differences in E2 between the EF and LF phases of the menstrual cycle.

Results

Thigh volume differed significantly between the females and age‐matched males (F: 4.9 ± 1.5 L; M: 8.2 ± 1.6 L; p < 0.01) and, when examined in all participants, thigh volume was significantly correlated to PLM‐induced hyperemia evaluated as both LBFΔPEAK (r = 0.6; p < 0.01) and LBF AUC (r = 0.4; p = 0.014). When the group differences in thigh volume were accounted for in the PLM analysis by employing thigh volume as a co‐variate, no significant differences in LBFΔPEAK (F: 542 ± 444; M: 794 ± 444 ml; p = 0.15) and LBF AUC (F: 164 ± 227; M: 249 ± 227 ml; p = 0.3) were revealed. The females reported no significant differences in PLM‐induced blood flow responses across menstrual cycle phases (EF vs LF) evaluated as LBFΔPEAK (EF: 385 ± 186; LF: 386 ± 154 ml; p = 0.9) or LBF AUC (EF: 123 ± 100; LF: 58 ± 84 ml; p = 0.11). Plasma E2 levels were significantly higher in females in the LF phase (215 ± 149 pg/ml) when compared to the EF phase (149 ± 98 pg/ml) of the menstrual cycle (p = 0.01).

Conclusion

This study revealed no differences in PLM‐induced LBF responses in premenopausal females when compared to age‐matched males or when assessed across follicular menstrual cycle phases.
MKP3 Overexpression and ERK1/2 Phosphorylation in Dopaminergic Neurons Regulate DAT Trafficking and Function
Authors: Besada Christina , Lewandowski Stacia I. , Mortensen Ole V.
Published: May 2022

Dopamine is a neurotransmitter that plays a critical role in movement, motivation, and reward. Dopamine neurotransmission is regulated by the dopamine transporter (DAT), which translocates dopamine from the extracellular space to the presynaptic neuron. DAT is a target for psychostimulants such as cocaine, giving DAT a key role in psychostimulant‐use disorders. DAT cell‐surface expression regulated by trafficking to and from the plasma membrane is an important mechanism for controlling DAT function. Two signaling pathways that contribute to DAT trafficking are the protein kinase C (PKC) and mitogen activated protein kinases (MAPKs), including the extracellular signal‐regulated kinase (ERK1/2) and p38 MAPK. ERK1/2 is inactivated by the MAP kinase phosphatase MKP3, which dephosphorylates and inactivates ERK1/2. Previous data from the lab demonstrated that activation of PKC by administration of phorbol 12‐myristate 13‐acetate (PMA) in an in vitro system results in decreased DAT cell‐surface expression, and overexpression of MKP3 attenuates this effect. Furthermore, other studies have shown that ERK1/2 activation, downstream of PKC activation, may lead to phosphorylation of DAT on residue threonine 53 (Thr53), however the significance of this phosphorylation is not completely understood. In order to characterize the contributions of MKP3 and ERK1/2 to DAT phosphorylation and trafficking in vivo, we have developed two viral constructs based on adeno‐associated viral (AAV) vectors that enable cre recombinase (cre)‐dependent expression. One vector cre‐dependently expresses MKP3. The other vector is based on a genetic tool named OptoSOS. This vector enables cre‐dependent, blue‐light induced activation of ERK1/2, and as a result, can spatiotemporally activate intracellular ERK1/2 signaling. Both viral vectors are employed with transgenic rats that express cre in tyrosine hydroxylase‐positive cells (TH‐Cre rats) to drive expression in dopaminergic neurons. From these studies, we find that MKP3 overexpression and resulting ERK1/2 inactivation increases DAT surface expression by blocking the internalization and degradation of the transporter leading to an accumulation of DAT on the cell‐surface. Interestingly, MKP3 overexpression also decreases phosphorylation at Thr53. While we have shown that ERK inactivation results in increased DAT cell‐surface expression, we have employed the OptoSOS system to activate ERK1/2 and assess resulting changes in DAT trafficking. We have confirmed that ERK1/2 is activated by blue‐light in a cre‐dependent manner, and similar to what we observe with MKP3 overexpression, ERK1/2 activation results in changes in DAT cell‐surface expression and phosphorylation. Taken together, our results indicate that MKP3 and ERK1/2 serve critical physiological roles in DAT regulation and can be utilized to investigate the mechanistic relationship between DAT trafficking, phosphorylation, and transporter activity.
Young Healthy Men and Women Have Similar Arterial Stiffness at Rest and Post‐Acute Inflammation
Authors: Eagan Lauren E. , Mascone Sara E. , Chesney Catalina M. , Ranadive Sushant M.
Published: May 2022

The cardioprotective effects of estrogen on arterial function and inflammation are well‐documented; however, it is unclear whether arterial stiffness and compliance vary between healthy premenopausal women and age‐matched men following an acute inflammation. The objective of the current study was to evaluate central and local arterial stiffness and compliance at rest and in response to an acute inflammatory stimulus, specifically, when estrogen levels are thought to be similar between young healthy women and men. It was hypothesized that sustained effects of estrogen throughout the lower‐hormone phase of a natural menstrual or oral contraceptive pill cycle would induce lower central and local stiffness and higher compliance in women as compared to men at rest and in response to an acute inflammatory stimulus. Concentrations of interleukin‐6 (IL‐6) and assessments of central and local arterial stiffness and compliance were obtained from 29 young healthy adults (21±3 y; female, n=15) at baseline (BL), and 24‐ (24H) and 48‐hours (48H) following administration of a seasonal influenza vaccine. To capture the lower‐hormone phases of female participants, women were assessed within the early follicular phase if naturally menstruating or the placebo pill phase if using oral contraceptives. Central stiffness was assessed via carotid‐to‐femoral pulse wave velocity (cfPWV) and local stiffness was measured using ultrasonography at the carotid artery to obtain β‐stiffness, elastic modulus (Ep) and arterial compliance (AC). IL‐6 concentrations were expressed as change‐values from BL and were compared between sexes and timepoints (24H‐BL vs. 48H‐BL). Indices of central and local stiffness and AC were compared between sexes and individual timepoints (BL vs. 24H vs. 48H). Men and women exhibited similar changes in concentrations of IL‐6 (effect of sex: P=0.60), with significant elevations at 24H‐BL as compared to 48H‐BL (effect of time: P=0.0002), indicating that the influenza vaccine successfully induced an acute inflammatory response in both groups. However, there were no effects of sex or time on cfPWV, β‐stiffness or Ep (P>0.05 for all). While men displayed greater AC as compared to women at BL (1.09±0.25 vs. 0.88±0.17 mm2/kPa, P=0.01), there were no sex‐differences at 24H and 48H (P=0.10 and 0.70, respectfully), suggesting that the women were able to compensate for their lower resting AC values when exposed to an acute inflammatory stimulus. In conclusion, when estrogen levels are similar between young healthy women and men, there are no sex‐differences in central or local arterial stiffness at rest nor in response to an acute inflammatory stimulus.
Exercise Training Alleviates Alzheimer’s Disease‐Associated Aortic Endothelial Dysfunction
Authors: Lee Junghoon , Zarezadehmehrizi Aliasghar , Hong Junyoung , Lee Jonghae , Park Yoonjung
Published: May 2022

Backgrounds

A strong association between cardiovascular disease and Alzheimer’s disease (AD) has been reported, but its possible mechanism has been unknown. We have tested whether aortic endothelial function in AD is impaired and investigated the protective effect of exercise training (EX) on endothelial function in aorta of AD mice.

METHODS

Wild type (C57BL/6, WT) and AD (APP/PS1dE9, AD) mice underwent treadmill training (WT‐EX and AD‐EX) or remained sedentary for 12 weeks. The expression of total‐and phosphorylated‐endothelial nitric oxide synthase (t‐eNOS and p‐eNOS), beta‐amyloid, and superoxide dismutase 2 (SOD2) were quantified by Western blot analysis.

RESULTS

t‐eNOS and p‐eNOS expressions in AD were decreased compared to WT but EX increased both t‐eNOS and p‐eNOS in AD (P < 0.01 and P = 0.09). Beta‐amyloid levels in AD were higher compared to WT but EX decreased it (P < 0.05). SOD2 levels in AD were increased by EX (P < 0.05) whereas there was no change in WT.

CONCLUSION

Regular aerobic exercise alleviates e‐NOS‐mediated endothelial dysfunction in aorta of AD mice possibly via reduced beta‐amyloid and increased SOD2 levels.
Characterizing a Canine Urothelial Carcinoma Model of Acquired MEK1/2 Inhibitor Resistance
Authors: Duval Dawn L. , Idate Rupa , Das Sunetra , Ackart David , Gustafson Daniel L. , Cronise Kathryn
Published: May 2022

Activating BRAF mutations are drivers of oncogenesis in several human cancers and in over 70% of canine urothelial carcinomas (cUC). Selective BRAF inhibitors are effective alone and in combination with MEK inhibitors in approximately half of BRAF mutant melanoma cases, but the majority of these patients ultimately develop resistance. Thus, intrinsic and acquired resistance to MAP kinase pathway targeted agents remains a challenge requiring the development of novel therapeutic strategies. Using canine urothelial carcinoma as a spontaneous cancer model to explore treatment of BRAF mutant cancers and acquired drug resistance, we sought to identify determinants of MEK1/2 inhibitor sensitivity and resistance. We applied the human‐derived MAPK Pathway Activity Score (MPAS), a predictor of sensitivity to MEK inhibition in human cancers based on expression levels of 10 downstream targets, to a panel of 32 canine cancer cell lines and found that MPAS correlates with trametinib sensitivity in this canine dataset. MPAS genes with significant (p<0.05) Pearson correlations to sensitivity were CCND1, DUSP6, ETV4, ETV5, and SPRY2. We also identified 30 non‐MPAS genes whose expression levels are significantly correlated with trametinib sensitivity, suggesting their role in de novo sensitivity to trametinib. Each of the 5 urothelial carcinoma lines had high MPAS scores and trametinib IC50 values <10nM. CRISPR‐Cas9 knockouts in two canine urothelial carcinoma lines also identified MEK1 as a critical dependency. To investigate mechanisms of acquired resistance, we generated trametinib‐resistant (TramR) clonal derivatives of the BRAF mutant Tyler1 canine urothelial carcinoma cell line (Tyler1‐TramR), with trametinib IC50 values greater than 500 nM versus 1 nM in parental Tyler1. Tyler1‐TramR cell lines exhibited sustained inhibition of ERK1/2 phosphorylation when treated with trametinib and were also resistant to pharmacologic inhibition of ERK1/2. Collectively, these data suggest an ERK‐independent mechanism of resistance. Gene expression analysis of two Tyler1‐TramR clones identified dramatic downregulation of epithelial markers accompanied by an increase in expression of mesenchymal genes and transcription factors that regulate the epithelial‐to‐mesenchymal transition (EMT). One Tyler1‐TramR clone exhibited metabolic alterations including decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and reduced glycolytic capacity. Overall, the findings in this study suggest that the mechanisms of acquired and intrinsic MEK inhibitor resistance are conserved between man and dog, further elucidating the translational value of cUC as a model to optimize therapies for Ras/Raf/MAP kinase driven cancers.
Genesis of Antibiotic Resistance (AR) LXXXVI: Turbulence Modeling(TM) of Hemodynamics in Simplified Severe Sepsis Protocol‐2 (SSSP‐2)‐NCT01663701: Mechanism(s) of ISP Afloat Transient APG Thrust VKVS free shear flow augmented cerebral vessel collapse, GCS3, & IHM
Authors: Montoya Hector , Flores Jorge , Kannan Subburaj
Published: May 2022

Based on the theoretical inferences from the CFD pertaining to VKVS/vortices/shedding frequency/vortex‐induced vibration/ hysteresis effect, Shed Vortex, and Karman/symmetric vortex shedding, Courtesy: Flow‐3D HydroÒ, COMSOL Multiphysics®, © 2021, NASA‐U.S: https://earthobservatory.nasa.gov/images/85989/canary‐islands‐kick‐up‐von‐karman‐vortices, Harvard Natural Sciences Lecture Demonstrations http://sciencedemonstrations.fas.harv… https://www.youtube.com/watch?v=_Hbbkd2d3H8, Vortex shedding; https://www.youtube.com/watch?v=ahohd8ceGR4, and hemodynamic data from NCT01663701, here we present a plausible physiological basis for the effect of fluid bolus augmented VKVS, eliciting ISP, thrust skewed viscosity/velocity profile rupturing and cause the cerebral hemorrhage, that leads to precipitous ihm in the usual care cohort of SSSP‐2 (PMID: 28973227). Hypothesis:Fluid bolus augmented VKVS augment incompressible free shear flows in cerebral arteries ensue decomposition of glycocalyx/endothelium as a function of time, viscosity and velocity profiles. Geometry:RBC, WBC, platelets, p47; p53; Table 2.1 p63, Table 2.2, p64; Fig 2.15 p 78; ISBN:9780128024089. Governing Equation, Derivation and Prospects (Physiological Connotation): Subsequent to fluid bolus induced fluid volume expansion is a function altered viscosity (hematocrit) dependent velocity profile as a function of time augment five different free shear flow: a. far wake, downstream of ZoO, b. mixing layer, downstream of ZoO occurring between two parallel streams with different velocity profile, c. plane jet, d. round jet, occur downstream of ZoO, and e. constrained radial jet, occur when two jets of similar velocity profile, (possibly different viscosity) in a turbulent flow; Assumptions based on the hemodynamic data from NCT016637001: I. Aforesaid free shear flow has self‐similarity, self‐preserving; Velocity component: Equation, 3.22; II. two velocity profiles located downstream at locations x1 and x2 are not same: equation, U (x, y)/uo(x) [or (U‐U (x, y)/U0(x) for the wake); III. Boundary layer approximations are not valid for aforesaid free shear flows, IV. molecular transport momentum differs at different location; V: pressure not the same thought vessels, VI: no continuity (equation of motion refer equation 3.23; 3.24; VII: closure coefficient differs for different points in the cerebral/ coronary circulation, applicable governing equations for the eddy‐viscosity model, Eq:3.25, p60‐61; ISBN‐13: 978‐1928729082. Taken together, fluid bolus induced fluid volume expansion augment cyclical anaerobic/aerobic milieu conferring altered generation time(G): G=t/n, where time (t) per generation (n = number of generations) for ClPr exacerbating toxic shock expedite irreversible loss of vasculature penultimate ihm in usual care group of NCT01663701.
Functional Inferences Derived from Defining the Interactome of H3K9me2 Writers and Readers
Authors: Pollin Gareth , de Assuncao Thiago Milech , Lomberk Gwen , Urrutia Raul
Published: May 2022

The H3K9me2‐driven histone code pathway has emerged as key for cancer initiation, progression, and a potential target for therapeutic strategies. The H3K9me2 mark is written by the lysine methyltransferases, G9a and GLP, and read by HP1 proteins. The goal of the current work was to develop a comprehensive interactome for these proteins to reveal linked protein partners and complexes, in which these writers and readers function during cell proliferation in human cervical cancer epithelial cells. We immunopurified each of these proteins individually and performed mass spectrometry of the associated proteins at two different phases of the cell cycle, namely G1/S and G2/M. We validated and modeled the H3K9me2‐interacting protein networks using several data science‐based algorithms. Our analyses reveal known and novel interacting proteins for both writers and readers of the H3K9me2 mark. In addition, we show that each of these proteins form distinct protein networks in a cell cycle‐specific manner. The protein networks formed by the writers and readers, which are all known regulators of the same histone mark, overlap in their protein complexes less than previously anticipated. Moreover, we show that these proteins not only formed networks within chromatin but also with other non‐chromatin proteins, such as nuclear receptor coactivator proteins, as well as mediator and polymerase subunits. Furthermore, we discovered that many of the non‐chromatin proteins have the presence of a H3K9me‐like, histone mimicry‐based recognition domain, suggesting another level of interaction and regulation with the H3K9me2‐driven histone code pathway. Mining of large‐scale mass spectrometry data show this H3K9me‐like domain in several G9a‐interacting proteins, including CDC73, ZNF644, and GTSE1. Ontological analyses and network reconstruction suggest that these H3K9me2‐associated interacting protein networks regulate various functions, including gene silencing, gene activation, elongation, DNA replication, DNA repair, and chromosome structure. Thus, these findings extend the current paradigm on pathways that link, directly or indirectly, to the H3K9me2 mark through computational modeling of proteomic‐derived networks. These results extend our understanding of the biochemistry underlying cell cycle‐associated epigenomic processes that, like the H3K9me pathway, are relevant to human cancer.
MicroRNA Profiling in West Nile Virus Carrying Mosquito, Culex tarsalis
Authors: Becker Olivia , Mitchell Jon C.
Published: May 2022

West Nile virus (WNV), is a serious mosquito‐borne zoonotic disease. There is currently no vaccine or treatment. The prevalence of WNV is particularly high in South Dakota with the primary species of carrier being Culex tarsalis. MiRNAs are a class of non‐coding RNAs that modulate a wide range of cellular processes (Chugh PE, Damania 2014). In particular, miRNAs have been shown to actively participate in an antiviral defense mechanism in mosquitoes, however, they appear to differ per virus‐vector combination (Göertz GP et al., 2019). Research surrounding the production of miRNAs in response to viral infections has discovered >70 different miRNAs depending upon genus/species of mosquito vectors are produced (Göertz GP et al., 2019). A critical question then becomes “why do some mosquito species produce certain miRNAs in response to WNV infection while other species do not?”. In order to begin understanding this phenomenon, it is important to examine what specific miRNAs are produced in vivo following WNV infection. The following research describes the utilization of total RNA isolated from Cx. tarsalis (+/‐ WNV determined via envelope/probe‐specific qRT‐PCR). Viral load/mosquito pool was determined following TaqMan™ miRNA profiling. Amplified miRNA products were arrayed into libraries, sequenced, and analyzed through NCBI analysis tools (BLAST™, BowTie2™). Results presented detected different viral infections (viral load within pools of Cx. tarsalis) corresponding to specific miRNAs. This knowledge could be key to understanding why certain species of mosquitoes transmit WNV compared to others and hopefully aid health care procedures surrounding WNV infections in both animals and humans.
Chronic AICAR treatment enhances anabolic signaling in sarcopenic skeletal muscle
Authors: Calixto Jouber , Johnson Connor , Smith Andrew , Brodowski Kole , Wilcox Shalene H. , Hill Jonathon T. , Thomson David M.
Published: May 2022

Many fields of study have grappled with the challenge of decelerating the effects of aging. Physical exercise plays a vital role in attenuating the impact of the aging process. While many exercise‐induced chemical signaling pathways are not fully understood, AMP‐activated protein kinase mediates many beneficial adaptations to exercise training. AMPK is activated by the accumulation of AMP (and ADP) during muscle contraction‐induced ATP breakdown. The prodrug AICAR mimics AMP accumulation and leads to AMPK activation by increasing the concentration of the AMP analog, ZMP. The primary purpose of the present study is to determine the effect of chronic (31 days) AICAR treatment on muscle anabolic signaling in old (24 mo.) mice. AICAR improved treadmill running performance and attenuated muscle atrophy in the aged mice. Surprisingly, given AICAR's known acute effects on anabolic signaling, AICAR treatment increased the skeletal muscle concentrations of insulin‐like growth factor‐1 and apelin, both of which are known to activate anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Accordingly, ribosomal protein S6 phosphorylation was also elevated in AICAR‐treated muscles. Our results suggest that in addition to its other well‐established metabolic effects, chronic AICAR treatment may enhance anabolism in sarcopenic muscle and potentially in other muscle‐wasting conditions.
Integrative Modeling, Molecular Mechanics, and Molecular Dynamics Evaluation of Genomics Variants in KMT2C (MLL3), a Gene Involved in Kleefstra Syndrome Type 2
Authors: Jorge Salomao D. , Chi Young‐In , de Assuncao Thiago Milech , Mathison Angela J. , Volkman Brian , Smith Brian , Lomberk Gwen , Zimmermann Michael T. , Urrutia Raul
Published: May 2022

Kleefstra Syndrome (KS) is a genetic, neurodevelopmental disorder characterized by intellectual disability, infantile hypotonia, severe expressive language delay, and characteristic facial appearance, with a spectrum of other distinct clinical manifestations. Pathogenic mutations in the epigenetic modifier, type 2 lysine methyltransferase (KMT2C), are attributed to cause KS core features in individuals who are Euchromatic Histone Lysine Methyltransferase 1, EHMT1, mutation negative. These individuals are designated as having KS type 2. In this study, we made use of multidimensional approaches, including conventional genomic bioinformatics, molecular modeling, molecular mechanics, and molecular dynamics simulations, to understand and enhance the annotation and potential mechanisms by which disease‐associated missense variants affect KMT2C function. In addition, we report a scoring system based on statistical integration and modeling of data derived from the structure and dynamics of KMT2C, which allows us to classify germline variants into SV (Structural Variants), DV (Dynamic Variants), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Moreover, these scores of disease‐associated variants reflect alterations in molecular fitness when compared with tolerated, benign variants, used as controls. Thus, these studies provide data that is more refined, both structurally and functionally for each variant, compared to current annotation tools used in human genomics databases and reveal mechanisms for their dysfunction not predictable from clinical genomic methods alone. This new knowledge extends our understanding of molecular mechanisms underlying the dysfunction of KS type 2‐associated genomic mutations. Lastly, by identifying KMT2C mutation‐specific druggable conformations, this study builds the trajectory toward the future development of small molecules to ameliorate the symptoms of this disease.
Formulation and Testing of Alcohol‐based Hand Sanitizers
Authors: Bowers Stefan , Galloway Monea , Van Stry Melanie , Jones Carter Candace , Gaber Farag , Ude Mba
Published: May 2022

The pandemic caused as a result of Covid‐19 brought awareness to the use of hand sanitizers around the world. Maintaining hand hygiene has been confirmed to be critical for diminishing the colonization and incidence of infectious diseases. The objective of this study was to create a formulation of alcohol‐based hand sanitizer that is effective at reducing the presence of microorganisms. A comparative assessment of antimicrobial efficacy of different hand sanitizers was used to compare two leading brand hand sanitizers to the 2 formulations made in the experiment. A modified Kirby‐Bauer zone of inhibition method was used to monitor the reduction of living E. coli cells in the presence of each hand sanitizer. Formulation 1 was proven to be the most efficient having exhibited the highest zone of inhibition within the study. Formulation 1 consisted of: Isopropyl Alcohol 75%, Glycerol 1.45%, Hydrogen Peroxide 0.125%, and water. Hydrogen peroxide in the formulation was found to be the missing ingredient in comparison to most of the leading brand hand sanitizers. The formulation will be tested further to discover what microorganisms may not be affected by this solution.
Sex‐specific cholinergic regulation of dopamine release mechanisms through nicotinic receptors in the nucleus accumbens
Authors: Brady Lillian J. , Thibeault Kimberly C. , Lopez Alberto J. , Tat Jennifer J. , Nolan Suzanne O. , Siciliano Cody A. , Calipari Erin S.
Published: May 2022

Biological sex has been pinpointed as significant biological factor impacting the prevalence and prognosis of Substance Use Disorder (SUD). Despite the overwhelming utilization of male subjects in SUD research, epidemiological evidence shows that women are the most susceptible population. At the hub of female vulnerability to SUD is significant dysfunction in the mesolimbic dopamine system connecting the ventral tegmental area (VTA) to the nucleus accumbens (NAc). Importantly, an important characteristic of dopamine release from axon terminals in the NAc is that it is rapidly modulated by local regulatory microcircuit mechanisms independent of somatic activity in the VTA. In the NAc, dopamine is released in tonic (slow and regular) and phasic (short, burst/spikes) patterns that are subject to heavy modulation by cholinergic (ChAT) interneurons signaling through α4β2*‐containing nicotinic acetylcholine receptors (nAChRs) located directly on dopamine terminals. Previous work suggests that ChAT regulation of dopamine release through nAChRs is fundamentally different between males and females, yet the processes and mechanisms that underlie these sex differences are largely unknown. In this project, we took a multifaceted approach to determine sex‐dependent neurochemical mechanisms that underlie ChAT regulation of dopamine release through nAChRs in male, naturally cycling and ovariectomized (OVX) female mice. Using ex vivo fast scan cyclic voltammetry (FSCV) paired with pharmacological applications, we found that ChAT regulation of dopamine release through α4β2*‐nAChRs is not present in female mice under most conditions. Deficits in nAChR modulation of dopamine release in intact females were not affected by the estrous cycle; however, they were rescued by ovariectomy – indicating that ovarian hormones play significant a role in this process. Critically, we find that 17β−Estradiol (E2) increases dopamine release acutely, an effect that is blocked by antagonism of α4β2*‐nAChRs. Additionally, we observed reduced nAChR agonist effects on dopamine release in females; which is what would be expected for desensitized receptors. Finally, Gq‐DREADD behavior studies revealed that male mice learned at a faster rate than intact females when ChAT interneurons were activated. Overall, we show that circulating ovarian hormones, regardless of the hormone cycle, alter the ability of α4β2*‐nAChRs on dopamine terminals to modulate dopamine release in the NAc. This suggests that sex differences in ChAT regulation of dopamine neurotransmission underlies sex‐dependent differentiation in reward learning. Moving forward, it will be critical to directly link these sex differences to reward learning for the development of sex‐specific pharmacotherapies to treat SUD.
Viperin Catalyzes the Conversion of Cytidine Triphosphate (CTP) to 3′‐deoxy‐3′,4′‐didehydro‐CTP by Radical S‐adenosylmethionine
Authors: Calderon Irene , McDowell Danny , Suder Karen C. , Replogle Jessica B.
Published: May 2022

Virus inhibitory protein, endoplasmic reticulum‐associated, interferon‐inducible (viperin) is a member of the radical S‐adenosylmethionine (SAM) superfamily of enzymes. Its expression is induced by interferon, and it inhibits a variety of DNA and RNA viruses such as influenza, HIV, dengue virus, West Nile virus, hepatitis C virus, and rabies virus. For flaviviridae especially, it catalyzes the conversion of cytidine triphosphate (CTP) to 3′‐deoxy‐3′,4′‐didehydro‐CTP (ddhCTP). When ddhCTP is incorporated by the viral RNA‐dependent RNA polymerase, RNA polymerization stops because the growing RNA strand lacks a 3’‐hydroxyl group. For other virus families, viperin has been found to interfere with mitochondrial metabolism and interact with a variety of cellular proteins. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of Mus musculus viperin bound with CTP. Viperin is a globular protein that has been shown to be highly conserved and is made up of three regions: N‐terminal extension, central domain, and the C‐terminal extension. The N‐terminal extension localizes viperin to the endoplasmic reticulum and lipid droplets where flaviviruses assemble. The central binding domain contains four sequence motifs associated with the radical SAM superfamily, including the tricysteine motif (CX3CX2C), responsible for binding the catalytic [4Fe‐4S] cluster. The C‐terminal extension binds to CTP and is involved in the installation of the [4Fe‐4S] cluster by the iron‐sulfur cluster–installing protein CIAO1. Binding of CTP to viperin results in conformational changes such as ordering of the C‐terminal extension and formation of an eight residue β‐hairpin loop that binds the γ‐phosphate of CTP. When bound, the 4’‐hydrogen of CTP is ideally positioned for abstraction by the 5′‐deoxyadenosyl radical generated by the reductive cleavage of SAM in the central domain. This is followed by the loss of the 3’‐hydroxyl group leading to the formation of the chain terminator, ddhCTP. The structural insights of CTP‐bound viperin provides an understanding of the role of the C‐terminal extension and its importance as an anti‐viral protein.
Effects of different types of resistance exercise failure training on the methylation status of genes that drive skeletal muscle hypertrophy
Authors: McIntosh Mason , Sexton Casey , Godwin Josh , Ruple Bradley , Osburn Shelby , Hollingsworth Blake , Agostinelli Philip , Kavazis Andreas , Ziegenfuss Timothy , Lopez Hector , Smith Ryan , Young Kaelin , Dwaraka Varun , Mobley Christopher , Sharples Adam , Roberts Michael
Published: May 2022

Objective

We sought to determine how one bout of resistance training to failure with either higher repetitions (30FAIL) or lower repetitions (80FAIL) affected the promoter methylation statuses of genes that drive skeletal muscle hypertrophy.

Hypotheses

We hypothesized that a bout of 80FAIL training would lead to a more robust hypomethylation of genes that regulate skeletal muscle hypertrophy compared to 30FAIL training.

Methods

Eleven previously‐trained college‐aged men (age: 23 ± 4 years, 11.42 ± 6.38 percent fat, 4 ± 3 years training experience) volunteered for this study. Each participant underwent two training bouts (spaced one week apart) involving either: i) 30FAIL training; 4 sets of back squats and 4 sets of leg extensors to failure at 30% of one‐repetition maximum (1RM), or: ii) 80FAIL training; 4 sets of both exercises at 80% of 1RM. Muscle biopsies from the vastus lateralis were collected prior to each bout (PRE), 3 hours following each bout (3hPOST), and 6 hours following each bout (6hPOST). Following the conclusion of the study, tissue was batch‐processed for DNA isolation, and DNA was subjected to the Illumina MethylationEPIC array. In an a priori fashion, genes that have been shown to induce skeletal muscle hypertrophy in genetic mouse models were the target of this investigation (Verbrugge et al. Frontiers Physiol, 2019).

Results

Total training volume (sets x reps x load) between the 30FAIL and 80FAIL bouts were not significantly different (p= 0.571). Differentially methylated region changes for the following genes from PRE to 3hPOST and PRE to 6hPOST are presented herein: SKI, FST, AKT1, ACVR2B, MSTN, KLF10, RHEB, IGF1, PAPPA, PPARD, IKBKB, FSTL3, ATGR1, UCN3, MCU, JUNB, NCOR1, GPRASP1, GRB10, MMP9, DGKZ, PPARGC1A, SMAD4, LTBP4, BMPR1A, CRTC2, XIAP, DGAT1, THRA, ADRB2, ASB15, CAST, EIF2B5, BDKRB2, TPT1, NR3C1, NR4A1, GNAS, PLD1, CRYM, CAMKK1, YAP1, INHBA, TP53INP2, INHBB, NOL3, and ESR1. Additionally, significant differences between conditions at each time point are highlighted.

Conclusions

This study continues to display how different modalities of resistance training affect the skeletal muscle molecular milieu and furthers our scientific understanding of factors that contribute to training adaptations.
RBM20 regulates muscle hypertrophy through titin and calcium signaling
Authors: Gao‐Hu Jeffrey S. , Guo Wei
Published: May 2022

Study objective

RNA binding motif 20 (RBM20), an RNA binding protein, regulates alternative splicing of titin and calcium handling genes that mediate hypertrophy signaling. The objective of this study is to investigate whether RBM20 regulates muscle hypertrophy through titin and/or calcium signaling.

Methods

Soleus (SOL) and extensor digitorum longus (EDL) were collected from wild‐type (WT) and Rbm20 knockout (KO) rats. Transcriptome profiling was analyzed by RNA sequencing (RNA‐seq). Hypertrophic signaling was examined using HeLa cells as well as differentiated and undifferentiated C2C12 myoblasts. RBM20 was knocked down in C2C12 myoblasts by siRNA transfection. Both immunostaining and western blotting were performed to test expression of hypertrophy associated proteins in both cell lines.

Results

RBM20 KO promoted SOL hypertrophy and EDL atrophy in rats. Hypertrophy‐associated hallmark proteins CaN, Erk and p38 were elevated in KO SOL, but not in KO EDL, while atrophy‐associated hallmark protein p62 was increased in KO EDL, but not in KO SOL. RNA‐seq data identified differentially expressed and spliced genes associated with hypertrophy in both EDL and SOL. Titin was switched to largest isoform as a result of alternative splicing in both KO SOL and EDL muscles. To test whether titin is the major mediator that regulatew hypertrophic signaling in RBM20 KO rats, we transfected Rbm20 gene into HeLa cells that do not express titin. The results revealed that the levels of hallmark proteins did not changed with overexpression of RBM20. To further test the role of titin in RBM20‐mediated hypertrophy, Rbm20 was knocked down in undifferentiated C2C12 cells which do not express titin as well, and no change was observed for hallmark proteins. C2C12 myoblasts were then differentiated to myotubes and reduced RBM20 in differentiated C2C12 cells promoted reduced expression of hypertrophic hallmark proteins.

Conclusion

Our results suggest that RBM20 regulates muscle hypertrophy in a muscle type‐dependent manner, and RBM20‐mediated hypertrophy is regulated via titin, a hypertrophic mediator.
The Role of SDF‐1 in Signaling Differences Between CXCR4 and Atypical ACKR3 Chemokine Receptors
Authors: Gorgy Justina , Abrol Ravinder , Caguioa Ronelle
Published: May 2022

SDF‐1 is a ligand that plays a critical role in cancer metastases and is secreted in specialized somatic cells in the body. Its chemoattractant properties attract and induce migration of tumor cells located in other tissues in the body to the site in which SDF‐1 is secreted. This attraction and migration of tumor cells allows a new tumor to form at secondary locations within the body. Although the understanding of the function of SDF‐1 being secreted in certain regions of the body is not clear, we seek to understand the binding patterns of this ligand with its chemokine receptors CXCR4 and ACKR3 (also called CXCR7). Chemokine receptors are a subclass of G‐protein coupled receptors, which are multi‐pass transmembrane proteins with 7 TM domains. GPCR activation initiates signaling cascades within the targeted cell. Understanding which signaling pathway is favored in CXCR4 and ACKR3 can help us understand the function of SDF‐1 and the mechanism of secondary tumor formation and development. ACKR3 is considered an atypical receptor as it has been experimentally shown to not couple to Gi/o protein in many cells whereas CXCR4 can couple to either Gi/oprotein or β‐arrestin. ACKR3 strongly binds to SDF‐1 and can control the local levels of SDF‐1 available for binding to CXCR4. Both receptors can couple to β‐arrestins, which are proteins responsible for receptor internalization and potential G‐protein‐independent signaling. To understand the interplay between these two receptors in the context of their interactions with SDF‐1, Gi protein, and β‐arrestin, we have successfully generated structural models of these two receptors in apo form, as heterodimers (bound to only SDF‐1, only Gi protein, or only β‐arrestin), and as heterotrimers (bound to both SDF‐1 and Gi protein, or both SDF‐1 and β‐arrestin). These structural models have been relaxed in their physiological environment using a series of molecular dynamics (MD) simulations. Binding free energy analysis of the heterodimers is beginning to shed light on the signaling differences between the two receptors, what identifies ACKR3 as an atypical receptor, and how it sequesters excess SDF‐1 to modulate CXCR4 signaling.
Absorption, Distribution, Metabolism, and Excretion of FDA‐approved Antisense Oligonucleotide Drugs
Authors: Migliorati Julia M. , Liu Sunna , Liu Anna , Gogate Anagha , Nair Sreenidhi , Bahal Raman , Rasmussen Theodore P. , Manautou Jose E. , Zhong Xiao‐bo
Published: May 2022

Absorption, distribution, metabolism, and excretion (ADME) are key biological processes for determination of a drug’s pharmacokinetic parameters, which have direct impacts on therapeutic efficacy and adverse drug reactions (ADRs). Drug structures, formulations, and administration sites and routes cause different classes of drugs to have various ADME characteristics and consequent impacts upon efficacy and ADRs. For example, orally administered small molecule drugs are usually absorbed and distributed by uptake transporters, biotransformed by phase I/II drug‐metabolizing enzymes, and excreted by efflux transporters. However, newly developed large molecule biological drugs, including antisense oligonucleotide (ASO) drugs, have substantially different ADME parameters. ASO drugs are single‐stranded antisense nucleic acids with diverse modes of drug action that induce mRNA degradation, exon skipping, and ASO‐protein interactions. ASOs have a great potential to treat certain human diseases that have not been treatable with small molecule drugs. The ADME behaviors of ASO drugs contribute to their unique set of ADRs and toxicity. To better understand their ADME features, ten FDA‐approved ASO drugs were selected, including Fomivirsen, Pegaptanib, Nusinersen, Mipomersen, Inotersen, Defibrotide, Eteplirsen, Golodirsen, Viltolarsen, and Casimersen. A comprehensive meta‐analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization and nucleotide degradation by endonucleases and exonucleases are major contributors to ADME features of the ASO drugs and these differ from the roles of transporters and metabolizing enzymes of small molecule drugs. The summarized information provides the most updated knowledge of ADME characteristics of these ASO drugs, leading to a better understanding of their therapeutic efficacy and potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distributions are identified and future perspective and directions are discussed.
Using Model‐Based Analysis and Physiology‐Informed Machine Learning to Refine the Stratification of Phenotypes in Heart Failure
Authors: Jones Edith , Randall E. B. , Hassavoort Kiley S. , Cameron David M. , Hummel Scott L. , Beard Daniel A. , Carlson Brian E.
Published: May 2022

A model‐based approach was developed to elucidate etiological differences between and within patient groups representing the two dominant heart failure diagnoses: heart failure with reduced (HFrEF) and preserved (HFpEF) ejection fraction. A closed‐loop model of the cardiovascular system informed by patient‐specific transthoracic echocardiography (TTE) and right heart catheterization (RHC) data was used to identify key parameters representing cardiovascular mechanics and hemodynamics. Thirty‐one patient records (10 HFrEF, 21 HFpEF) were obtained from the Cardiovascular Health Improvement Project database at the University of Michigan. Model simulations were tuned to match RHC and TTE pressure, volume, and cardiac output measurements in each patient. The underlying physiological model parameters were compared to model‐based norms and between HFrEF and HFpEF diagnoses. Our results confirm the main mechanistic parameter driving HFrEF is reduced left ventricular (LV) contractility, whereas HFpEF exhibits a more heterogeneous phenotype. To determine subgroups within the HFpEF diagnosis, we conducted a principal component analysis on the optimized parameters, which combined with machine learning techniques including k‐means and hierarchical clustering methods reveal (i) a group of HFrEF‐like HFpEF patients (HFpEF1) that share characteristics with HFrEF, (ii) a HFpEF group that exhibit classical characteristics of patients with diastolic dysfunction (HFpEF2), and (iii) a group of HFpEF patients that do not consistently cluster (NCC) among the machine learning techniques. These subgroups cannot be distinguished from the clinical data alone. However, reanalyzing the clinical data of each of these newly determined subgroups reveals that elevated systolic and diastolic LV volumes seen in both HFrEF and HFpEF1 may be used to identify HFrEF‐like HFpEF patients. Similar HFpEF groupings have been identified by other studies using specialized clinical measures (e.g., from non‐routine echocardiography and heart biopsies) whereas our study analyzes data from standard clinical procedures, such as TTE and RHC. Hence, our methodology has great translational potential to be broadly accepted in the clinic. These results suggest that physiology‐informed model‐based analysis of standard clinical data in conjunction with unsupervised machine learning can distinguish subgroups of HFpEF as separate phenotypes. Moreover, this methodology has the potential to retrospectively assess patient selection in past HFpEF clinical studies, aid in the optimization of prospective HFpEF subgroup selection for future clinical trials, and elucidate patient‐specific treatment strategies.
Structural Elements of ‘Anti‐prion J protein 1’ (Apj1) Required for Efficient Curing of the [PSI+] Prion by Hsp104 Overexpression
Authors: Ganser Samantha J. , McNish Bridget A. , Corpus Bridget A. , Hines Justin K.
Published: May 2022

Prions are self‐propagating aggregates of misfolded protein that often form ordered amyloids. Amyloids are associated with a variety of human neurodegenerative diseases, including but not limited to Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. Amyloid‐forming prions are also present in Saccharomyces cerevisiae, where they are propagated through populations by a fragmentation mechanism that requires three chaperone proteins: the disaggregase Hsp104, the Hsp70 Ssa, and the J‐protein Sis1. When Hsp104 is ectopically overexpressed, however, the prion [PSI+] is specifically eliminated from cell populations in a Sis1‐dependent manner. We previously determined that the J‐protein Apj1 is also required for efficient elimination, or curing, of the prion variants [PSI+]STR and [PSI+]Sc4 by Hsp104 overexpression. Additionally, we showed that Apj1 and Sis1 have overlapping functions in the Hsp104‐mediated curing process, as overexpressing one in the absence of a functional version of the other restores curing. A construct bearing the first 161 residues of Apj1 is sufficient to restore efficient curing in Δapj1cells. Residues 1‐161 can be subdivided based on sequence features and homology into: a J‐domain, a polyglutamine track with an imperfect QA repeat region, a region bearing homology to the G/F region of Sis1, and finally a region rich in glycine, serine, phenylalanine, and asparagine which resembles the amino acid content of many prion‐forming domains. Various constructs produced by mutating and truncating regions of Apj1‐161 were ectopically expressed in Δapj1 cells to determine which regions, if any, were sufficient to replace Apj1 in Hsp104‐mediated curing. We found that constructs containing the polyQ region restored efficient curing and those lacking the polyQ region did not support efficient curing, indicating the importance of the polyQ region in the curing process. The Sis1 homologous region and the SFN rich region proved nonessential for Hsp104‐mediated curing. Ongoing experiments are being conducted to determine which regions of Apj1‐161 are required to replace the function of Sis1 in Hsp104‐mediated curing.
Investigating the role of the ERF104 gene in stress tolerance in model plant Arabidopsis suecica using CRISPR
Authors: Gabriel Kristina E.
Published: May 2022

ETHYLENE RESPOSNE FACTORS are highly conserved genes that have been found to be involved in stress‐related hormone signaling in plants. When these genes are over‐expressed, they can have negative impacts on plant health, leading to inhibited growth and leaf senescence under stressful conditions. Could knocking down the function of an ERF improve plant response in the face of climate change related stressors such as soil salinity? A CRISPR gene knockdown will be performed on ETHYLENE RESPONSE FACTOR 104 in Arabidopsis suecica. A. suecica is an allopolyploid plant which has shown differential expression of ERF104 in genetically different accessions. A CRISPR gene knockout of ERF104 will be performed on two different accessions of A. suecica, which will then be genotyped to look for signs of genetic mutation. Phenotypic observations of mutation will also be performed as the plants grow and are exposed to stressful conditions. Upon successful completion of the experiment, I expect to see that the ERF104gene is no longer being overexpressed in A. suecica, which should result in the plant showing fewer signs of stress when grown on salinity plates.