bluuebunny/crossref_metadata_2025 huggingface dataset:
To each their own: Disparate abilities of Nrf2 activators to protect against various electrophilic and oxidative insults
Authors: Biesterveld Laura M. , Eggler Aimee L.
Published: May 2022

Electrophilic and oxidative stress contribute to major diseases such as cancer and diabetes. Small‐molecule activators of the Nrf2 cytoprotective pathway show significant promise for prevention and amelioration of chronic diseases by combating these stressors through upregulation of detoxification and antioxidant enzymes. An understudied area is whether distinct small molecular Nrf2 activators can protect cells from all electrophilic and oxidative stressors, or whether certain molecules are better suited to protect against a particular type of stress.

In this study, human keratinocyte HaCaT cells were preincubated for 24 h with one of four Nrf2 activators before different stressors were added for an additional 24 h and cell death was assessed. Three electrophiles were tested: sulforaphane (SFN), the subject of over 70 clinical trials, the multiple sclerosis drug Tecfidera (dimethyl fumarate, DMF), and bardoxolone methyl (BARD), in clinical trials for treatment of chronic kidney diseases and other conditions including COVID‐19. SFN, DMF, and BARD target C151 of the Keap1 protein, a negative regulator of Nrf2. Also tested was KI696, an inhibitor of the Keap1‐Nrf2 interaction. Three chemically distinct stressors—hydrogen peroxide, menadione (a quinone that rapidly generates ROS through one‐electron redox cycling), or chlorambucil (an alkylating chemotherapy agent)—were tested. Stressors were added to the cells at their LC50 concentration after pretreatment with a range of cytoprotective compound concentrations. Unexpectedly, SFN pretreatment showed little to no rescue for all stressors. However, DMF rescued the viability of cells stressed with hydrogen peroxide, menadione, and chlorambucil. BARD showed no rescue for hydrogen peroxide‐treated cells. KI696 improved the cell’s ability to survive chlorambucil. The ability of a given Nrf2 activator (or lack thereof) to protect against a given stressor points to a need to better understand the cytoprotective environment (specific enzymes upregulated, glutathione levels, etc.) that best protect against specific stressors.
Sonicated hyaluronan is a potent inhibitor of Alzheimer's disease progression
Authors: Lee Kuan‐Ting , Chang Nan‐Shan
Published: May 2022

WWOX is a recently defined risk factor for Alzheimer’s disease (AD). Functional deficiency or downregulation of WWOX protein may lead to slow protein aggregation in the brain that contributes to the pathogenesis of AD. Previously, we demonstrated that WWOX deficiency initiates the aggregation of TRAPPC6AΔ, TIAF1, tau and Aβ. TRAPPC6AΔ aggregates or plaques are found in the brain cortex and hippocampus in middle‐aged healthy individuals (aged 40 to 70) and old AD patients (70‐100 years old). Here, we determined that HAson8 (hyaluronan sonicated for 8 hr) significantly retarded AD progression and restored memory loss in triple‐transgenic mouse (3×Tg) model for AD, as determined by novel object recognition test (ORT) and Morris water maze for hippocampus‐dependent, non‐spatial and spatial learning and memory. By immunohistochemistry, HAson8 significantly suppressed the aggregation of Aβ and pT181‐Tau, and reduced the levels of inflammatory microglia and astrocytes in 12‐month‐old 3xTg mouse hippocampi. Also, HAson8 significantly suppressed the expression of AD‐promoting pS14‐WWOX in the hippocampi. HAson8 significantly upregulated neural stem cells, expressing Sox2 and parvalbumin in the subventricular zone and hippocampi. Mechanistically, HAson8 activated Hyal‐2+ Z cells, and that transfer of the activated Z cells to recipient 3xTg enabled these mice to restore memory loss at month 9. Finally, HAson8 altered the ratio of Firmicutes and Bacteroidetes bacteria in the gut, which favors the retardation of AD progression in 3xTg mice. Together, HAson8 is effective in inhibiting AD progression in 3xTg mice via suppression of inflammatory glia cells, upregulation of Z cells and neural stem cells, and reducing AD‐promoting gut bacteria.
IMP1/IGF2BP1 alters extracellular vesicle cargo in colorectal cancer
Authors: Kuhn Madeline , Zhang Yang , Armstrong Randall , Andres Sarah
Published: May 2022

Background

Colorectal cancer (CRC) is the third leading cause of cancer‐related death worldwide. There is an urgent need for new therapeutic targets and methods of early detection to improve patient outcomes. Extracellular Vesicles (EVs) and EV cargo are promising new biomarkers and therapeutic targets. EVs are secreted nanoparticles that carry DNA, RNA, and proteins. EVs can target specific cell types and once taken up by a destination cell, EV cargo can shape cell gene expression and physiology. EVs can shape the tumor microenvironment (TME) (i.e., stromal, immune, and endothelial cells), which plays critical roles in patient survival and disease outcomes. Our current understanding of how EV cargo is determined and how it influences the CRC cell TME is limited. One promising but understudied molecular aspect of EVs is RNA binding proteins (RNAbps). RNAbps are post‐transcriptional regulators of gene expression and could provide a link between host cell gene expression and EV cargo. Insulin‐like growth factor 2 RNA binding protein 1 (IMP1, IGF2BP1) is an RNAbp with a critical role in CRC development and progression. IMP1 is expressed highly in CRC tumors, expression correlates with worse prognosis, and IMP1 binds/potently regulates tumor‐associated transcripts. How EV cargo is influenced by IMP1 and effects on EV targeting to destination cells and on target cell gene expression and function is not known. Understanding how IMP1 shapes EV cargo and which TME cells are targeted, will identify new mechanisms of cancer development/progression through IMP1‐EV‐mediated alteration of the TME and novel EV cargo as candidates for early detection biomarkers.

Hypothesis

We hypothesize that IMP1 facilitates the packaging of tumor‐promoting EV cargo that interact and communicate with the TME to enhance tumor progression.

Methods

EVs were isolated from IMP1 null and IMP1‐overexpressing (IMP1OE) CRC cell lines, HT‐29, SW480, as well as wildtype Caco2 cells (express high levels of IMP1) by size‐exclusion chromatography. EVs were assessed by nanoparticle tracking analysis, western blot, electron microscopy, and qPCR.

Results

We found that IMP1 enters EVs from Caco2 and SW480 IMP1OE cell lines, but not HT‐29 IMP1OE or corresponding null controls. IMP1 did not alter EV secretion in CRC cell lines. We found that IMP1 target mRNAs, such as KRAS, cMYC are present within EVs, but not the IMP1 target PTGS2.

Conclusions

Our novel findings suggest that IMP1 enters the EV to directly influence EV cargo in specific CRC cell lines. Our findings have implications for the development of novel early detection biomarkers and therapeutic approaches in CRC.
Cardiomyocyte‐specific deletion of Med13 & Med13L results in dysregulated gene expression and lethal systolic heart failure
Authors: Gardner Richard N. , Oppman Alexis M. , Martins Ines , Grueter Chad E.
Published: May 2022

The Mediator co‐activation complex is an important component in RNA Pol II‐dependent gene expression that functions to integrate intracellular signals, allowing for optimized cellular response to (patho)physiological states. The Mediator kinase submodule functions to fine‐tune these molecular transitions. The kinase submodule consists of four proteins, including Med13, Med12, Cdk8, and Cyclin C. Also involved is the Med13 paralog, Med13‐like (Med13L), which is both mutually exclusive in the submodule and partially redundant. Approximately 22% of reported human mutations in MED13 or MED13L have congenital heart defects often resulting in dysregulated cardiac metabolism and aberrant cardiovascular physiology. Therefore, we hypothesize that the acute loss of Med13 and Med13L in cardiomyocytes results in disrupted Mediator kinase submodule formation leading to altered gene expression and subsequent pathogenesis of heart failure. To investigate this, we created a Tamoxifen‐inducible cardiomyocyte‐specific Med13/Med13L double knockout mouse model and treated mice for two weeks with Tamoxifen beginning at eight‐weeks‐old. Following treatment, mice had significant progressive decreased ejection fraction consistent with severe heart failure. Survival studies demonstrate rapid mortality—50% of Med13/Med13L cardiac knockout mice die within four weeks. Post‐mortem analysis showed increased heart weight and heart‐weight‐to‐body‐weight ratio as well as gross histology consistent with dilated hypertrophy. To begin elucidating the mechanistic processes underlying these findings, mRNA sequencing was performed prior to observed declines in cardiac function and showed that cardiac stress response pathways were differentially expressed. Specifically, the Wnt‐β‐catenin pathway was activated, while basal transcription was unchanged. Taken together, Med13 and Med13L are critical for normal Mediator‐regulated RNA Pol II‐dependent transcription and subsequent cardiac physiology. Therefore, disruptions cause significant cardiac dysfunction and death as sequelae of heart failure.
BET proteins as novel epigenetic targets for the treatment of Opioid Use Disorder
Authors: De Almeida Suzannah , Sartor Gregory
Published: May 2022

Recent research has revealed that targeting epigenetic proteins is an effective strategy to reduce drug‐seeking behaviors in animal models of addiction. We have found that expression of epigenetic reader proteins, called Bromodomains and Extra terminal Domain (BET), are increased in the nucleus accumbens following cocaine self‐administration and that pharmacological inhibition of BET proteins reduced cocaine‐seeking behaviors in rodents. Although BET inhibitors show promise in animal models of cocaine use disorder, the effectiveness of BET inhibitors in animal models of Opioid Use Disorder (OUD) remains unknown. To assess a potential role for BET proteins in OUD, we measured time‐dependent changes in BET mRNA expression in the prefrontal cortex (PFC), nucleus accumbens (NAc), and dorsal striatum (DS), following oral oxycodone self‐administration. In these experiments, female and male mice received home cage water bottles with or without oxycodone (0.5 mg/ml) for 10 consecutive days. The volume consumed and the weight of the mice was recorded at the same time each day. At the end of the 10th day, all mice were given regular drinking water. After 4 h, 24 h, or 7 days of abstinence, expression of BET genes (Brd2, Brd3, and Brd4) were measured via quantitative PCR (qPCR). Our results showed that mice consumed more oxycodone water compared to regular water and that oxycodone consumption (mg/kg and ml/g) in females were higher compared to males. In qPCR studies, expression of Brd2 was significantly increased in the NAc after 4 h of abstinence; Brd3 was significantly increased in the NAc after 24 h and 7 days of abstinence and Brd4 was significantly increased in the NAc after 7 days of abstinence and after 24 h in the PFC. These data indicate that BET genes are altered following abstinence from oxycodone. Ongoing studies are exploring a functional role for BETs in oxycodone consumption and oxycodone‐induced withdrawal‐like behaviors. Together, these experiments may lead to more effective therapies for the treatment of opioid use disorder.
Set1, Set2, and the Clock‐cycle complex are necessary to prevent age‐related retinal degeneration in Drosophila melanogaster
Authors: Bakhle Kimaya M. , Jauregui‐Lozano Juan , Weake Vikki M.
Published: May 2022

Age‐related eye disease is a prevalent problem that is exacerbated by the rising, aging population. Drosophila melanogaster serves as a powerful model organism for studying age‐related eye disease, as they share 60% of their genetic material with humans. Here, we seek to understand how changes in gene expression regulate photoreceptor neuron survival during aging. A preliminary RNA interference screen was carried out to identify genes necessary for cell survival throughout aging. Microscopy imaging and reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR) were used to further investigate the role of histone methyltransferases Set1 and Set2 and the transcription factor Clock in photoreceptor neuron survival. Set1 and Set2 add methyl groups to lysine residues of histone H3, while Clock forms a heterodimer with the transcription factor cycle to activate transcription of genes involved in the circadian rhythm. Microscopy imaging was used to qualitatively and quantitatively assess loss of rhabdomeres, compartments of photoreceptor cells responsible for phototransduction, in flies lacking expression of either Set1, Set2, or a functional Clock‐cycle complex. Flies expressing RNA interference against Set1 and Set2 show retinal degeneration at or before the age of 30 days. Flies that fail to form a functional Clock‐cycle complex due to expression of a dominant negative form of Clock (ClkDN) show progressive retinal degeneration starting at the age of 5 days. Interestingly, this retinal degeneration is rescued when flies are raised in the dark. These phenotypes were validated by measuring target gene expression using RT‐qPCR. RNA interference against mCherry and overexpression of LacZ were used as controls for Set1 and Set2 RNA interference and overexpression of ClkDN, respectively. These findings suggest that histone methylation and Clock‐dependent gene expression play a role in age‐related eye disease in both Drosophila and humans.
Defining the Critical DNA Features Targeted by RNA Polymerase I Core Factor
Authors: Munoff Nathan , Decatur Wayne , Zeberl Brian , Palmer Mathew , Szemere Zsuzsa , Knutson Bruce
Published: May 2022

RNA Polymerase I(Pol I) is one of three essential DNA dependent RNA polymerases in eukaryotes and is responsible for synthesizing ribosomal RNA. A critical and essential Pol I transcription factor in yeast is Core Factor (CF) which binds to a ~24 bp region in the rDNA promoter called the Core Element (CE). CF plays fundamental roles in the Pol I transcription process helping to recruit Pol I and open Pol I promoter DNA before initiation. Previously, it was not yet known how CF precisely recognized the CE. When interacting with DNA, proteins use two main mechanisms: i) base‐pair readout, and ii) shape/structural readout. Base‐readout is the most common and is dominated by hydrogen bonding between the amino acid residues and base‐pair hydrogen bond donors and acceptors. The second mechanism centers around DNA shape readout where proteins target specific DNA features such as curvature, bendability, and groove width. Previous studies from our lab have shown that CF and its human orthologue, Selectivity Factor 1 (SL1), use an evolutionarily conserved mechanism to target DNA which is governed by interactions with the GC minor groove, a unique surface rarely targeted by DNA‐binding proteins. To further understand the extent of structural recognition as well as specific DNA features CF may be using to interact with CE, we have employed a variety of selection‐based methods to resolve the structural rules governing CF’s interaction with DNA. Our findings are consistent with a model that CF‐CE interaction is governed primarily by DNA shape‐based structural features rather than sequence.
Critical Environmental Limit Protocol: Validity and Reliability (PSU HEAT)
Authors: Cottle Rachel M. , Wolf S. T. , Lichter Zachary S. , Kenney W. L.
Published: May 2022

With increasing average global temperatures and greater frequency, duration, and severity of heat waves, there is an urgent need to better understand the specific environmental temperatures that place humans at greater risk of heat‐related morbidity and mortality. An incremental heat stress protocol can be used to establish critical environmental limits for human heat exchange, i.e., those combinations of ambient temperature and humidity above which heat stress becomes uncompensable and, therefore, unsafe. However, no studies have rigorously investigated the reliability and validity of this experimental paradigm.

PURPOSE

Here, we assessed the (1) between‐visit repeatability and (2) validity of an experimental paradigm that either holds ambient vapor pressure (Pa) constant and incrementally increases dry‐bulb temperature (Tdb) or vice versa.

METHODS

Twelve subjects (5M/7W; 25±4 yr) completed a progressive heat stress protocol during which they walked on a treadmill (2.2 mph, 3% gradient) in a controllable environmental chamber while gastrointestinal temperature (Tgi) was continuously monitored. After an equilibration period, the progressive heat stress protocol involved increasing Tdb every 5 min while Pa was held constant (Tcritexperiments), or increasing Pa every 5 min while Tdb was held constant (Pcritexperiments), until an upward inflection and continuous rise in Tgi was observed. The Tcrit or Pcrit was identified as the average Tdb or Pa, respectively, across the 2 min preceding the Tgi inflection point. For repeatability experiments, 11 subjects returned to the lab to repeat the same protocol as their first visit. For validity experiments, 10 subjects returned to the lab for a progressive heat stress trial in which Tdb or Pa was held constant at the Tcrit or Pcrit value from their first visit.

RESULTS

The between‐visit repeatability for critical environmental limits was excellent (ICC = 0.98). Similarly, there was excellent agreement between original and validity trials for Tdb (ICC = 0.95) and Pa (ICC = 0.96) at the Tgi inflection point. Furthermore, the wet‐bulb temperature at the Tgiinflection point was not different during reliability (p = 0.78) or validity (p = 0.32) trials compared to original trials.

CONCLUSION

Together, these data provide strong evidence that this experimental paradigm is rigorous, reliable, and valid, supporting its use to investigate critical environmental limits to the maintenance of heat balance.
Model for Calcium‐Dependent Activation of a Type I Metacaspase From the Fungus Schizophyllum commune
Authors: Coyle Timothy F. , Fox Kristin M.
Published: May 2022

Apoptosis or programmed cell death (PCD) is one of the most important cellular processes which functions to avoid the accumulation of mutations or damage within a cell. PCD has been extensively studied in eukaryotic cells, demonstrating that caspase proteins have a significant role in this process. However, unlike mammalian cells, caspase genes are absent in plants, fungi, and protozoa. Instead, these organisms contain homologous proteins known as metacaspases which have demonstrated similar proteolytic functions in aiding PCD. Determining an accurate model for how these metacaspases are activated could provide insight into the methods behind this protease‐dependent cell death which could lead to further strategies for the regulation of fungal infections. This research focuses on the model of activation for the Type I metacaspases from the fungus Schizophyllum commune. The S. commune Type I metacaspases are dependent on calcium ions for activation; however, the molecular mechanism behind how calcium activates this protease remains unknown.

It has been proposed that when calcium binds to the metacapsape, these ions induce a structural rearrangement of the active‐site region leading to activation. To further understand how calcium and other divalent metallic cations influence the activation of this protease, we used kinetic activity assays to quantify metacaspase activity and differential scanning fluorimetry to measure divalent metallic cation‐metacaspase binding.

In addition to strong activation by calcium, manganese ions have demonstrated a moderate effect on metaspace activity, while magnesium, copper, samarium, and cobalt don’t affect activity. We will present evidence that calcium binds to two sites on the protein, while manganese binds to only a single site. Furthermore, calcium binding to the Type I metacapses occurs at two sites with affinities that differ by several orders of magnitude. Initial results suggest that it is necessary for calcium to saturate the high affinity site prior to binding to the low affinity site. Thus, it has been predicted that calcium’s initial saturation of the high affinity site induces slight conformation changes to the active‐site; however, once the low affinity site has been saturated, an active‐site loop undergoes further rearrangements resulting in metacaspase activation. Further analysis of binding interactions between the Type I metacapse and calcium (along with other divalent cations) will provide insight into how these structural rearrangements lead to increased metacaspase activity.
Intestinal Zip14 ablation dysregulates tight junction protein expression, inflammatory genes and lncRNAs in intestinal epithelial cells and enteroids
Authors: Jimenez Felix R. , Ruggiero Courtney H. , Cousins Robert J.
Published: May 2022

The mechanisms by which Zip14 ablation mediates mild intestinal inflammation and decreased barrier function are not completely understood. We used RNA sequencing to profile the transcriptome of intestinal epithelial cells (IEC) isolated from Zip14 Cre/flox and control flox/flox mice. RNA‐seq analysis identified 1,345 differentially expressed genes in Zip14 Cre/flox mice compared with control mice. In‐silico analyses revealed that Zip14ablation promoted dysregulation of specific proinflammatory genes (e.g. IL‐6 and Ifn‐γ) and genes influencing intestinal barrier homeostasis (specifically, Claudins 1‐and‐2). Highly dysregulated genes were selected for confirmation by qPCR, and proteins by western analysis, using extracts from isolated IECs from mice of the two genotypes. Highly upregulated genes in the Zip14 Cre/flox mice included Fabp6, Cldn2and Cldn8, while downregulated genes included Cldn1. To explore the possibility that Zip14ablation may alter transcriptional activity, ChIP assays of selected promoters were conducted. Increased binding of Stat3, FXR and NF‐kB was observed in Cldn2, FABP6, and Cldn8promoters, respectively, with chromatin from the Zip14 Cre/floxmice compared to control mice. By contrast, Cdx2 binding was decreased for the Cldn1promoter site of the knockout mice. Zip14 ablation also altered expression levels of long noncoding RNAs (lncRNAs). Specifically, the lncRNAs H19, U90926, and Meg3 were highly upregulated, and Gata4 was downregulated as validated by qPCR. Using organoids derived from intestines of mice of both genotypes, it was demonstrated that growth was not impaired with Zip14 ablation. Other biochemical markers of Zip14ablation were recapitulated in organoid cultures. This included indices of enhanced Zn2+ trafficking and differential expression of specific tight junction genes. Decreased HDAC3 activity in IECs from the Zip14 knockout mice suggest that epigenetic modifications account in part for the genomic changes observed. We conclude zinc transported by Zip14 controls intestinal permeability through epigenetic modifications, thus representing a novel health‐promoting role for this micronutrient.
Theoretical Model of Extracellular Potassium Diffusion as a Signaling Mechanism in Neurovascular Coupling
Authors: Djurich Sara , Secomb Timothy W.
Published: May 2022

Neurovascular coupling (NVC) is the process by which neuronal activity causes changes in cerebral blood flow within seconds of the onset of activity. The goal of this study is to explore the potential role of extracellular K+ diffusion as a mechanism for NVC. A mathematical model is used to simulate K+ kinetics in the extracellular space (ECS), including effects of K+release during neuronal activity, K+ uptake by astrocytic and neuronal Na+/K+ ATPase, and K+ diffusion in the ECS. The model consists of a partial differential equation in time and one spatial dimension. Parameter values are derived from experimental literature or from previous models. The effective K+ diffusivity is 937.5 μm2/s. Astrocytic and neuronal K+ uptake is assumed to have Michaelis‐Menten kinetics with maximum rate 84.7 mM/s and Michaelis constant 1.5 mM. The average distance from a neuron to the closest microvessel is 15 μm. A domain of length 15 μm is therefore assumed, with one end representing the neuron and the other the blood vessel. At rest, ECS K+ concentration is 3 mM. One spike is estimated to change the extracellular K+ concentration by 0.11 mM from rest. The neuron is assumed to fire continuously for 10 s at 100 Hz. Equilibrium values of K+ in the ECS are reached within 10 s, with values of 11 mM at the neuron and 9.6 mM at the vessel. The increase in extracellular K+ concentration at the vessel lags approximately 0.3 s behind the increase at the neuron. These results suggest that K+ diffuses fast enough to the vasculature to be a signal for NVC. Although uptake of K+ by Na+/K+ ATPase limits the concentration that reaches the vasculature, the increase in K+ concentration around the vasculature is sufficient to elicit a vascular response. Limitations of the model are that a simplified, one‐dimensional geometry is assumed and possible effects of K+ release at astrocytic endfeet are not included.
Identification of NUAK1/2 Regulators in the Hippo Signaling Pathway
Authors: Song Youchen , Gill Mandeep K. , Song Siyuan , Attisano Liliana
Published: May 2022

The Hippo signaling pathway plays a central role in regulating cell proliferation, differentiation, and tissue size. In response to various upstream regulators, the mammalian STE20‐like protein kinase 1/2 (MST1/2) and large tumor suppressor kinase 1/2 (LATS1/2) phosphorylate Yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ) to promote their cytoplasmic retention and degradation. Unphosphorylated YAP/TAZ translocate into the nucleus and interact with several transcription factors to regulate gene expression that drives cell growth and migration. Dysregulation of the Hippo pathway causes tissue overgrowth and tumorigenesis and elevated YAP/TAZ levels or activities are positively correlated with progression of almost all solid cancers. The NUAK family kinase 1/2 (NUAK1/2) are members of the AMP‐activated protein kinase‐related kinase family. Our lab showed that NUAK2 negatively regulates the Hippo pathway by preventing LATS‐mediated YAP/TAZ phosphorylation and cytoplasmic sequestration. Moreover, this work showed that loss of NUAK2 attenuates in vivo mammary tumor growth. However, how NUAK1/2 are regulated is poorly understood.

Here, we will undertake screens in cancer cell lines to identify upstream and downstream NUAK1/2 regulators and partners. Since, abrogation of NUAK expression blocks cell growth in a context‐dependent manner, my current efforts are being directed towards generating cell lines expressing inducible NUAK1/2 shRNAs. Independent clones have been selected and validated for their knockdown efficiencies and effects on YAP/TAZ downstream target gene expression. Once validated, the selected lines will be subjected to genome‐wide CRISPR screens. We hypothesize that the screens will identify components that enhance or attenuate the effect of NUAKs and will reveal any differential regulators and partners for NUAK1/2. Discovering novel cancer‐relevant NUAK regulators will enhance our understanding of the tumor promoting roles of NUAKs and provide insights into how to target these kinases for better therapeutic outcomes.
MED12 is a Transcriptional Mediator of Cardiac Contractile Function
Authors: Montgomery Kathlyene R. , Mark Christopher , Baskin Kedryn
Published: May 2022

The Mediator complex plays an essential role in transcription for nearly all genes, and functions by transmitting physiological and developmental signals to RNA polymerase II. The Mediator complex is regulated by a kinase submodule made up of four proteins: MED12, MED13, CDK8, and Cyclin C. MED12 is a pivotal member of this submodule as it is required for CDK8 activity, which regulates Polymerase II‐driven transcription. The objective of this study is to identify the role of MED12 in the heart and determine how it regulates transcription through interactions with transcription factors. MED12 protein levels are increased in human heart failure biopsies, and in the mouse heart after transverse aortic constriction (TAC)‐induced heart failure. Therefore, we hypothesize that increased levels of MED12 contributes to development of heart failure. To test this hypothesis we generated cardiac‐specific MED12 transgenic (cTg) mice by driving increased MED12 expression in cardiomyocytes using the aMHC promoter. We performed serial echocardiography and observed that increased MED12 expression leads to decreased cardiac function, measured as fractional shortening. Decreased cardiac function was accompanied by increased cardiac chamber size and dilation, without evidence of cardiac fibrosis. We performed RNA sequencing on cTg and control cardiomyocytes, to investigate mechanisms of MED12‐induced cardiac dysfunction, and found that increased MED12 levels lead to dysregulated calcium handing gene expression. MED12 is a transcriptional regulator, but does not directly bind DNA. To determine how MED12 regulates expression of calcium handling genes we used cardiomyocytes from our mouse models to screen for transcription factors that interact with MED12. We immunoprecipitated MED12 and identified interacting proteins by mass spectrometry, many of which are regulators of transcription. In follow‐up experiments we demonstrated that MED12 interacts with the transcription factor (TF) MEF2 to regulate calcium handling genes. Preliminary experiments suggest that MED12 also interacts with TFs CREB and SRF. Additional mechanistic studies are ongoing to determine how MED12 coordinates gene expression through multiple transcription factors in cardiomyocytes. Collectively, our data demonstrate that tight regulation of MED12 levels is necessary to maintain normal cardiac function and regulate gene expression in the heart, and increased MED12 expression leads to the development of heart failure.
Analysis of Crassostrea Virginica Protein Metal Complexes after Exposure to Toxic Environmental Pollutant Cadmium
Authors: Wellek Jacob B. , Sherrer Shanen M.
Published: May 2022

Cadmium is a metal found in the parts per million of the earth’s crust, and it has been shown to have toxic effects on various plants, animals and humans. Exposure to cadmium occurs mainly through use of tobacco‐based products or industrial pollution. Once ingested, it is observed to take 20 to 30 years for cadmium concentration to reduce in half, which means it is difficult to clear from the body. A protein that is involved in the detoxification of harmful metals in cells is metallothionein. Having a strong binding affinity to divalent metals, metallothioneins allow cells to shuttle cadmium and other toxic metals away from important biological processes. Our current research aims to determine the effects of cadmium on the proteins within Crassostrea Virginica (oysters) living in the Chesapeake Bay area and how fast these changes occur. After homogenizing oyster samples and testing the amount of total protein, the protein structural differences will be analyzed using circular dichroism spectroscopy and Western blot techniques with metallothionein and other proteins as well. The quantification of cadmium uptake in oysters and final destinations of cadmium within protein complexes of samples will also be examined. Knowing the impacts of this heavy metal on oysters is directly useful for increasing insights into the toxic effects from industrialization on our ecosystem, as well as elucidating the functional aspects of macromolecular complexes containing cadmium.
Acidity and nucleophilic reactivity of persulfides
Authors: Benchoam Dayana , Cuevasanta Ernesto , Möller Matías N. , Alvarez Beatriz
Published: May 2022

Persulfides(RSSH/RSS‐) are chemical compounds related to thiols (RSH) and hydrogen sulfide (H2S). They participate in sulfur trafficking and processing, and are proposed to be transducers of the signaling effects of H2S in mammals. Despite the increasingly recognized relevance of persulfides, their biochemical properties are poorly understood. In this work, we studied the acidity of six low molecular weight persulfides, as well as their nucleophilic reactivity towards the synthetic probe monobromobimane. Kinetic determinations were performed at different pHs exploiting that monobromobimane does not accept or release protons within the pH range studied and produces fluorescent products. The persulfides of glutathione, cysteine, homocysteine, cysteamine, cysteine methyl ester and β‐mercaptoethanol, presented pKa values of 4.7‐6.5 and pH‐independent rate constants of 3.2‐9.0 × 103 M‐1s‐1. The persulfides were more acidic than the corresponding thiols, which have pKa values of 6.5‐9.6. The ranges of pKa values suggest that the substituents in persulfides affect the acidity to a lesser extent than in thiols, probably due to the longer distances to the acidic sulfur atom because of the additional sulfur. We constructed a Br⊘nsted plot (log k vspKa) which showed a correlation between the nucleophilicity and basicity of persulfides, which resulted weaker than the observed for thiolates. In addition, the persulfides presented higher nucleophilicity than the expected for thiolates with the same pKa values. This can be rationalized in terms of the alpha effect: the increased reactivity of a nucleophile when the adjacent atom has high electron density. Overall, our work suggests that, at physiological pH, persulfides have higher reactivity compared to thiols due to two factors: the increased availability of the ionized species and the enhanced intrinsic reactivity. We are currently investigating protein persulfides to rationalize how the protein environment could affect the biochemical properties of the persulfides. Our study contributes to the understanding of the biochemistry and the biological functions of persulfides.
The Role of Social Group and Matriline on Fluctuating Asymmetry in Female Rhesus Macaques
Authors: Romero Ashly N. , Terhune Claire E.
Published: May 2022

Fluctuating asymmetry (FA), or random deviations from symmetry, is typically thought to reflect stress during development. Therefore, FA has been used as a proxy for developmental instability in a wide variety of bilaterally symmetric organisms. The literature on FA explores a variety of environmental stressors that invoke mechanisms allowing developmental instability (temperature, food, etc.), but the role of heredity on developmental instability should not be discounted. We aimed to clarify the role of social group and matriline on FA, and thereby developmental stability, in female rhesus macaques. The Cayo Santiago macaques provide a unique opportunity to investigate the nuances of FA because of the wealth of information about the lives and deaths of each individual from the island. Further, in most cases, female rhesus macaques remain in the social group they were born into for their entire lives. We hypothesized that individuals in the same social group would exhibit similar levels of FA because they occupy similar geographical territory and are more closely related than females between social groups. Additionally, we hypothesized that individuals from higher ranking social groups will exhibit less FA than other social groups due to greater access to resources. Lastly, we hypothesized that individuals in the same matriline would exhibit more similar levels of FA to one another than other matrilines. We calculated craniofacial FA using 3D geometric morphometric techniques for 112 female macaques with known matrilines, 77 of which had known social groups. We used ANOVAs to compare mean FA between social groups and matrilines. No significantly different FA levels were observed between either (p=0.398 and p=0.752, respectively). These results indicate that genetic influence via relatedness may not have an impact on FA levels in a population and/or that FA is not distributed by social group or social group rank (though individual rank cannot be ruled out here). This work contributes to the FA literature by further parsing out the environmental and genetic influences affecting developmental instability.
The Influence of Activin on the Tumor Microenvironment in Colon Cancer Metastasis
Authors: Wiley Mark B. , Bauer Jessica , Mehrotra Kunaal , Zessner‐Spitzenberg Jasmin , Kone Lyonell , Quiao Guilin , Castellanos Karla , Church David N. , Kerr Rachel S. , Kerr David J. , Grippo Paul , Maker Ajay , Jung Barbara
Published: May 2022

Background and Aims

TGFβ and activin A are key molecules regulating tumor growth, inflammatory responses in the TME, and have differential effects which are receptor and cell specific. Identification of the source of activin A/TGFβ production and site of signaling will be critical for directing therapeutic development. Here, we employ both mouse models and human tissue samples to 1) identify pathways in which SMAD4 mutations exacerbate tumor growth and metastasis, 2) recognize how TGFβ and activin A signaling are influencing immune responses in the TME, and 3) to use the cellular source and signaling of activin A, rather than the amount, to predict patient outcomes in CRC. We have previously shown elevated levels of circulating activin A in stage IV CRC patients and that activin signaling promotes epithelial to mesenchymal transition and migration. We now hypothesize that activin A signaling pathways have distinct effects on immune cells and tumor cells which regulate tumor development and metastasis of CRC.

Methods

Knockdown of activin A was assessed in a surgical colon cancer liver metastases mouse model. The influence of the SMAD4‐dependent, canonical pathway was elucidated in a Ts4‐Cre; cApcflox;Smad4flox (TcAS) mouse model for colon cancer. Western blotting and migration assays were performed on human colon cancer cells with mutations in receptors for TGFꞵ and/or activin to determine functional interactions of the downstream pathways. We used a TMA of 1054 CRC patients with stage II and III colon cancer from the QUASAR2 cohort to correlate activin and TGFb expression with outcome.

Results

The non‐canonical pathway pAKT is activated in ACVR2 restored HCT116+chr2 colon cancer cells, but not in ACVR2 mutated cells. Ablation of the the activin A canonical pathway target SMAD4 in mice leads to decreased survival in colon cancer mouse model and pERK is increased in SMAD4‐/‐ knock out mice and correlates with dysplasia grade. Mice harboring colonic lesions with high stroma content were positive for activin A and CAF marker α‐SMA and have shorter median survival. Activin knockdown in mouse metastasis model leads to decreased tumor growth. Patient data revealed that a high ratio of activin A relative to TGFꞵ in the tumor epithelium, but not in the stroma, is associated with an increased survival rate. Ongoing studies are being conducted to determine if activin A is being localized to immune cells interacting with the tumor epithelium which may be promoting anti‐tumor activity in these patients.

Conclusion

Recent clinical trials explored the feasibility of activin A and TGFβ blockade in solid tumors. Data presented here supports complex and intertwined roles for activin and TGFβ signaling in colorectal cancer, both in immune activation as well as metastatic spread of advanced cancers.
A Novel Peroxin is Required for Mitochondrial Morphology: Implications for Resolving a New Peroxisome‐Mitochondrial Contact Site
Authors: Knight Emily W. , Gianos Andrew , Crowe Logan , Morris Meredith T.
Published: May 2022

Peroxisomes are dynamic and ubiquitous organelles that house many metabolic pathways and interact with other organelles such as the endoplasmic reticulum, lipid droplets, and mitochondria. One mechanism for organelle interaction is through membrane contact sites. While contact sites between multiple organelles have been identified, little is known about the proteins that serve as molecular tethers in such sites. We study organelle dynamics using peroxisome‐like organelles called glycosomes in the early diverging organism Trypanosoma brucei and have identified a novel peroxin (protein involved in peroxisome biogenesis) that is essential for mitochondrial morphology. Silencing this protein leads to a significant growth defect and swollen mitochondria. Multiple mitochondrial membrane transport channels have been identified in immunoprecipitation studies. Based on these findings, we hypothesize that this protein that we have named a putative peroxisome‐mitochondrial contact protein (PPMCP), localizes to glycosomes and mitochondria at contact points, which facilitate the transfer of metabolites between the two organelles. Disruption of this connection results in “leaky” mitochondria and cell death. Current work is focused on resolving the metabolic defects in PPMCP‐deficient cells and identifying additional molecular components of these contact sites. This work forwards our understanding of how contact sites are established and the role they play in interorganelle communication.
Endothelial PP2AC prevents endotoxemia‐induced acute lung injury by suppressing STAT3 activity
Authors: Siddiqui M. R. , Machireddy Narsa , Faridi Hafeez M. , Shahid Mohd , Akhtar Suhail , Shanley Tom
Published: May 2022

Acute lung injury (ALI) is a critical hypoxemic respiratory failure characterized by alveolar‐capillary damage, unchecked inflammation, vascular leak, and pulmonary edema secondary to endothelial barrier dysfunction. Identifying the candidate molecules targeting mechanisms for excessive production of proinflammatory cytokines and endothelial dysfunction are warranted for therapeutic intervention. Recent studies show that the signal transducers and activators of transcription 3 (STAT3) phosphorylation in critical for inflammatory cytokines production and systemic inflammation. Protein phosphatase 2A (PP2A) is a hetero‐trimeric, ubiquitously expressed Ser/Thr phosphatase. The phosphatase activity of PP2A is regulated by its catalytic C subunit (PP2AC). PP2A plays an essential role in modulating tissue inflammation by maintaining the phosphorylation/de‐phosphorylation balance of multiple proteins. So far, however, no studies have deciphered PP2AC isoform‐a function in regulating pulmonary vascular endothelial permeability and inflammatory signaling. We hypothesize that PP2AC expression in vascular endothelial cell (EC) protects the lung against sepsis‐induced lung injury. Wild‐type (WT) and endothelial specific PP2AC‐deficient mice (PP2ACCKO) were challenged with lipopolysaccharide. We found that lipopolysaccharide‐induced inflammatory response, characterized by up‐regulated level of IL‐1b, IL‐6, MPO activity and pulmonary vascular hyper‐permeability in lung tissue of PP2ACCKO mice was significantly elevated as compared to WT littermate control. Mechanistically, we showed that PP2AC deletion in mouse lung‐EC and in human lung‐EC markedly augmented Ser727‐phosphorylation‐mediated STAT3 activation and MMP9 level in response to lipopolysaccharide. Intriguingly, administration of a STAT3 inhibitor suppressed lipopolysaccharide‐induced production of MMP9 and proinflammatory cytokines IL‐1b, IL‐6, MPO activity and pulmonary vascular hyper‐permeability and thus rescued the augmented inflammatory phenotype in PP2ACCKO mice lung. In conclusion, these data showed the protective role of endothelial‐PP2AC in limiting endothelial barrier dysfunction and inflammation, and thereby it attenuates sepsis‐induced lung injury by preventing hyper‐activated STAT3 signaling.
Skeletal Muscle Oxygen Delivery and Utilization during Exercise in Heart Failure with Preserved Ejection Fraction: Role of Sympathetic (α‐adrenergic) Vasoconstriction
Authors: Alpenglow Jeremy , Bunsawat Kanokwan , Fancisco Michael , Weavil Josh , Broxterman Ryan , Iacovelli Jarred , Harrison Jon , Morgan David E. , Ryan John J. , Ma Chisty L. , Wray D. W.
Published: May 2022

BACKGROUND

Heart Failure with preserved ejection fraction (HFpEF) is characterized by severe exercise intolerance underpinned by low aerobic capacity (VO2peak). It is likely low VO2peak in HFpEF is not only related to deficits in cardiac function, but also peripheral vascular and skeletal muscle dysfunction that may disrupt the balance between O2 delivery and utilization. There is emerging evidence for sympathetic nervous system overactivity in patients with HFpEF that results in excessive vasoconstriction in the peripheral circulation, and may therefore contribute to dysregulation of exercising muscle blood flow. We tested the hypothesis that pharmacological blockade of sympathetic (α‐adrenergic) vasoconstriction would increase blood flow, O2 delivery and utilization of exercising skeletal muscle to a greater extent in HFpEF compared to healthy control subjects.

METHODS

Four healthy control (CON) (2 M, 69±3 yrs) and three HFpEF (1 M, 71±3) volunteers participated in this study. Femoral blood flow (FBF) was assessed by ultrasound Doppler. Catheters were placed in the femoral artery and vein for drug infusions and determination of arterial and venous O2 content (CaO2 and CvO2, respectively). O2 utilization of the exercising leg was calculated via the Fick equation: VO2 = FBF∙(CaO2‐CvO2), and O2 delivery was calculated as FBF∙CaO2. Measurements were made at rest and during dynamic single‐leg knee‐extension (KE) exercise (5 W) before and after intra‐arterial infusion of the α‐adrenergic antagonist phentolamine (PHEN).

RESULTS

Baseline FBF, O2 delivery, and VO2 were similar between groups. At rest, PHEN increased FBF and O2 delivery similarly in both groups, while the increase in VO2 tended to be smaller in patients with HFpEF compared to CON (+3±2 vs. +14±5 ml/min; respectively, P=0.09). During KE exercise, a ≍20‐30% decrement in FBF (2052±261 vs. 2529±230 ml/min; HFpEF vs. CON, P=0.23), O2 delivery (282±27 vs. 392±35 ml/min, HFpEF vs. CON, P=0.07), and VO2 (193±25 vs. 284±31 ml/min; HFpEF vs. CON, P=0.09) were observed in patients with HFpEF. PHEN increased exercising muscle FBF (+243±45 vs. +247±58 ml/min; HFpEF vs. CON) and O2 delivery (+51±9 vs. +34±19 ml/min; HFpEF vs. CON) similarly between groups, while the PHEN‐induced increase in VO2 tended to be greater in patients with HFpEF (+20±12 vs. +6±7 ml/min; HFpEF vs. CON, P=0.32).

CONCLUSION

Taken together, these preliminary data suggest a reduction in FBF, O2 delivery and utilization during small muscle mass exercise in patients with HFpEF that may be, in part but not fully, explained by excessive sympathetic (α‐adrenergic) vasoconstriction in this patient group.