bluuebunny/crossref_metadata_2025 huggingface dataset:
Fusobacterium nucleatum as a Marker for Epithelial to Mesenchymal Transition in Colorectal Cancer
Authors: Huang Angela , Torres Alejandro , Patel Rohin , Saxena Anjana , Patel Mintoo
Published: May 2022

Colorectal cancer (CRC) is both preventable and curable if diagnosed at early stage, and yet it is the second most common cause of cancer deaths in US. Recent emphasis on the role of human microbiome in cancer led us to investigate new microbial diagnostic marker for CRC. CRC originates in the colon or rectum as a polyp that may invade into surrounding tissue and metastasize to secondary sites through epithelial to mesenchymal transition (EMT) of the cancer cells. Fusobacterium nucleatum, an abundant opportunistic bacterium, is often found in the gut and oral cavity, and implicated in promoting colorectal carcinogenesis. F. nucleatum is known to bind E‐Cadherin, an epithelial cell surface adherence protein, through amyloid‐like adhesin fadA, which initiates EMT. FadA mediates the attachment and invasion of F. nucleatum and activates β‐catenin signaling, causing inflammatory and oncogenic responses. This study sought to assess F. nucleatum and EMT biomarkers in CRC. De‐identified formalin fixed paraffin embedded tissue samples of different stages in CRC, classified as normal, precancerous, and cancerous, were immunostained for E‐Cadherin and Vimentin as markers for epithelial and mesenchymal cells respectively. We used amyloid‐like adhesin fadA immunostaining as a marker for F. nucleatum. As expected, normal tissue specimens expressed E‐Cadherin exclusively in the mucosal epithelial cells while mesenchymal cells in the stroma specifically stained for Vimentin expression. However, E‐Cadherin was found to be downregulated in cancer tissue specimens, signifying a partial EMT. Further, some precancerous and cancerous specimens showed Vimentin‐positive foci within epithelial cells that lacked E‐Cadherin expression implicating EMT. We also observed the presence of F. nucleatum secreted amyloid‐like fadA in cancer tissue specimen. Amyloid‐like fadA plays a key role in transformation of F. nucleatum from commensal to pathogen, hence, its detection in the colon may aid in early detection of CRC and provide an opportunity for targeted intervention. Its role in EMT, however, requires detailed molecular analyses. Supplementing the routine histopathological evaluation with these specific biomarkers would provide more predictive and detailed analysis of cancer initiation and progression in tissue samples to aid in personalized treatment plan with the goal of improved disease outcome.
Dynamic Compartmentalisation of Intracellular Sodium in Collecting Duct Cells
Authors: Mullins Linda , Assmus Adrienne , Sherbourne Colin , Early Jason , Claeyssens Frederik , Haycock John , Hunter Robert , Mullins John
Published: May 2022

In Principal cells (PC) of the cortical collecting duct (CCD), the highly regulated and co‐ordinated reabsorption of sodium occurs through the epithelial sodium channel (ENaC) at the apical membrane and Na/K ATPase at the basolateral membrane. However, it is not known how sodium ions (Na+) are transported across the cell and if it is a regulated mechanism. We investigated trans‐cellular transport in the mouse collecting duct cell line, mCCDcl1 cells using a fluorescent sodium dye, Corona Green AM. The dye was found to accumulate in fluid‐filled domes forming between the single cell layer and the plastic dish, indicating that the cells are polarised and that Na+ ions are transported across and concentrated beneath the principal cells. Dye uptake was stimulated by aldosterone, blocked by amiloride (an ENaC inhibiter), and basolateral transport was prevented by ouabain (an Na/K ATPase blocker) thus validating the dye’s apparently faithful replication of sodium transport across the cells. Live cells exhibited a consistent pattern of sodium‐containing vesicles, of various sizes, surrounded by cytoskeleton and lipid membrane. While the smallest vesicles (~0.5µm) co‐stained with lysotracker, larger vesicles (up to 6.4µm) did not co‐stain with either lysosomal‐ or mitochondrial‐specific dyes and appeared to have internal structure, suggesting that they were multivesicular bodies. Time‐lapse confocal imaging of cells cultured on a 3D polyHYPE scaffold and subsequent movies of intracellular dynamics showed a subset of these multivesicular bodies release or take up sodium dye in a controlled manner, over the course of a few minutes. Our novel data reveal intracellular sodium compartmentalisation, which may offer new insights into intracellular sodium dynamics, possibly in response to stress, in the collecting duct.
Multifunctions of α‐Synuclein Explained by Its Dynamic Heterogeneous Conformations with a Hierarchy of Transition Times
Authors: Chen Jiaxing , Zaer Sofia , Drori Paz , Zamel Joanna , Joron Khalil , Kalisman Nir , Lerner Eitan , Dokholyan Nikolay V.
Published: May 2022

α‐Synuclein plays essential roles in synaptic vesicle homeostasis and neurotransmitter release through the interaction with membrane, while α‐synuclein aggregates have been associated with several neurodegenerative diseases, especially Parkinson’s disease. The different roles of α‐synuclein could be attributed to the heterogenous conformations adopted by this intrinsically disordered protein. To demystify the physiological functions of α‐synuclein and understand its pathogenic mechanism of Parkinson’s disease, characterizing the monomeric structural ensemble and identifying the aggregation‐prone and non‐aggregation‐prone structures are of particular importance. Here, we use inter‐residue distance distributions derived from time‐resolved FRET experiments as constraints to guide discrete molecular dynamics simulations of α‐synuclein monomer. We explore the conformational space of α‐synuclein and verify the generated conformational ensemble by additional experiments including far‐UV circular dichroism spectrum and cross‐linking mass spectrometry. We find that some conformational states of α‐synuclein are surprisingly stable displaying dynamic transitions less than milliseconds. A comprehensive analysis of the conformational ensemble uncovers important structural features and potential conformations that are critical to stabilize the monomeric state or induce different oligomerization pathways.
Isoform switching regulates Swi/Snf activity in response to nutrients
Authors: Thurlow Lauren , Johnson Tracy
Published: May 2022

Eukaryotic organisms have evolved complex gene regulatory networks to launch coordinated responses to external conditions and stimuli. Under environmental stress, such as nutrient depletion, these responses involve reallocation of cellular resources away from growth and cell cycle stimulating genes and towards stress‐responsive genes. This occurs through several mechanisms including changes at the transcriptional level, repression of ribosome biogenesis factors, altered translation initiation, and RNA features which modulate translation efficiency and transcript stability. Upon nutrient starvation in Saccharomyces cerevisiae, diploid cells undergo meiosis (sporulation in yeast). We have previously shown that the catalytic component of the Swi/Snf chromatin remodeling complex, Snf2, is responsible for shifting gene expression away from intron‐rich ribosomal protein genes (RPGs) to enhance splicing of meiotic intron‐containing genes (ICGs) during sporulation. A similar process occurs during the transition from growth to quiescence known as the diauxic shift in yeast. During both of these complex cell‐state transitions, Snf2 levels change dramatically while SNF2 mRNA levels remain relatively stable. Although critical to its activity, the mechanism by which Snf2 protein levels change remains unknown. Here we describe temporally‐regulated alternate transcription start sites (TSS) under batch growth conditions, which produce SNF2 transcripts with different 5’ leaders affecting downstream translation propensity. Specifically, a long isoform of SNF2 contains three upstream open reading frames (uORFs), which we hypothesize inhibits the translation of the downstream protein‐coding ORF. I used 5’ RACE to identify the previously unannotated TSS of the long isoform, and RNA analysis suggests that the relative levels of each transcript are dynamic and affected by the binding of the highly‐conserved transcriptional regulators Ume6 and Cbf1 at the SNF2 locus. Protein analysis via Western blotting shows that this regulation indeed affects Snf2 expression. In light of the conservation of Snf2, investigating its regulation in response to environmental changes in S. cerevisiae may carry important implications for Snf2 chromatin remodeling activity in higher eukaryotes.
Portability of Small Molecule Binding Sites in Intrinsically Disordered Proteins
Authors: Meyer Ella , DeSilva Sachith R. , Metallo Steven J.
Published: May 2022

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure. Because they are associated with a range of diseases, IDPs are desirable therapeutic targets and have been targeted by small molecules. The transcription factor c‐Myc (Myc) is an IDP that is implicated in over 70% of cancers. Previously, we have shown that several small molecules can bind to short, contiguous sequences on Myc. These sequences remain unstructured when binding to small molecules, potentially indicating only a limited dependence on residues outside of the contiguous binding residues. This mode of binding differs from that common in structured proteins where contact residues are often noncontiguous and scaffolded by extensive protein structure. Here we test the portability of a small molecule‐IDP interaction implied by its dependence on only a short, linear sequence. We compare the binding of a small molecule to its specific recognition sequence in a native Myc context, in a minimalist peptide context, and by porting the binding sequence into the context of a different IDP. An ability of short, disordered sequences to bind small molecules in a context agnostic manner would have implications for the prediction and discovery of IDP binding sites broadly.
Case Study: Superficial Ulnar Artery and its Clinical Significance
Authors: Pattani Sager , Irshad Sumbal , Sewo Lolade , Dacquel Maxwell , Narayan Vinayak , Dhiman Shiv
Published: May 2022

We present a case with an atypical variation of the ulnar artery discovered while examining a cadaver in our routine Gross Anatomy lab dissection. A superficial ulnar artery was found in a 55‐year‐old male cadaver originating directly from the brachial artery. This is a rare variation where the route of the ulnar artery runs superficially to the flexor compartment in the forearm. Based on previous cadaver studies, the incidence of such variation in the adult population appears to be approximately 3.75% 1. Deviations in the ulnar artery can lead to a potential risk of injury to patients during surgery 2. The superficial ulnar artery can mistakenly result in the administration of medicines into the artery as opposed to the median cubital vein during the injection. Hemorrhaging as well as other potential traumas can occur. We will also discuss the potential complications that could arise from this variant and the clinical recommendations for treatment.

1. Fadel RA, Amonoo‐Kuofi HS. The superficial ulnar artery: Development and surgical significance. Clin Anat. 1996;9(2):128‐132. Accessed Jan 27, 2022. doi: 10.1002/(SICI)1098‐2353(1996)9:2<128::AID‐CA5>3.0.CO;2‐D.

2. Rodríguez‐Niedenführ M, Vazquez T, Parkin IG, Sanudo J. Arterial patterns of the human upper limb: Update of anatomical variations and embryological development. European Journal of anatomy, ISSN 1136‐4890, Vol 7, Nº 1, 2003 (Ejemplar dedicado a: Supplement 1), pags 21‐28. 2003;7. Accessed Jan 26, 2022.
Mapping Out Histone Post‐Translational Modifications in a Yeast Prion Model
Authors: Cobos Samantha N. , Son Elizaveta , Paredes Jailene , Rana Navin , Bennett Seth A. , Torrente Mariana P.
Published: May 2022

Prions are proteins with the ability to self‐template into alternative folds, allowing them to operate outside the canonical steps of the central dogma of molecular biology. Interestingly, while these infectious protein species are responsible for causing several diseases in humans and animals alike, they have also been postulated to provide positive cellular outcomes in yeast. Because of proteins propensity to misfold and aggregate, prions could allow for environmental adaptiveness without directly affecting an organism’s genome. However, the exact mechanisms by which these proteins could potentially lead to gene utilization changes are not known, and the extent of their roles in normal cellular biology has not been adequately investigated. Rnq1 and Swi1 are two such yeast prion proteins, each responsible for the propagation of the [PIN+] and [SWI+] prion states, respectively. Due to the ease with which prion states exchange, we hypothesize that they could be connected to particular changes in histone post‐translational modifications (PTMs) to alter the way that genes are expressed, and thus could be the key to understanding how prions impact cellular phenotypes. Through previously published methods, we have begun to map out the changes in histone PTM levels between yeast prion states. Overall, [PIN+] yeast showed increased levels of H3K9ac, H3K18ac, and H3K56ac compared to [pin‐]. Interestingly, all these modifications are correlated with increased gene transcription. In agreement, we find higher levels of total RNA in [PIN] yeast. Furthermore, [SWI+] yeast have shown a decrease in H3K56ac and H3K36me2 levels compared to [swi‐] cells. Decreases in these marks generally represent decreased gene expression, which agree with the decrease in global RNA levels in [SWI+] compared to [swi‐] samples. Furthermore, treatment with 1mM guanidinium hydrochloride (GuHCl) ‐ to shut off or “cure” the prion state in yeast‐ reversed the observed histone PTM level changes. Here, we have effectively shown that the yeast epigenome is directly linked to prion states, further confirming the role of prions as another form of epigenetic modulation.
Creatine Nitrate and Caffeine Alone and Co‐ingested on Cognition, Readiness to Perform, and Sleep Quality
Authors: Newton Andrew , Koozehchian Majid S. , Mabrey Gina , Forbes Scott , Haddad Monoem , Naderi Alireza
Published: May 2022

BACKGROUND

Caffeine and creatine nitrate are two purported supplements that may alter cognitive function. The primary purpose was to examine the impacts of each supplement ingested individually and co‐ingested on cognitive performance. A secondary purpose was to explore the effects of caffeine and creatine nitrate alone and co‐ingested on subjective feelings of readiness and sleep quality. METHODS: In a double‐blind, randomized, placebo‐controlled cross over study, 12 resistance‐trained participants (22±1 y; 180.0±9.5 cm; 85.9±15.1 kg) completed 4 conditions in random order: creatine nitrate (CN: 5 g/day + 0.675 g/day of maltodextrin), caffeine (CAF: 400 mg/day + 5 g/day of maltodextrin), CN+CAF (5 g/day of creatine nitrate + 400 mg/day of caffeine), and placebo (PL: 5.4 g/day of maltodextrin). Supplements were ingested for 7 days with a 7‐day washout period between conditions. Before and after supplementation, participants completed a Stroop color‐word test, in addition to a readiness to perform a visual analog scale and a sleep quality questionnaire (sleep latency and duration). The readiness to perform the questionnaire assessed sleep, vigor, optimism, energy, appetite, and soreness. Data were analyzed using a two‐way (within‐within) analysis of variance (p≤0.05) with a Tukey’s post hoc when appropriate. RESULTS: There was a significant interaction for the Stroop color‐word interference (p=0.049), with the post hoc revealing that CN+CAF was superior to CN alone (CN+CAF: 65.9 ± 17.3; CN: 56.8 ± 10.8, p=0.007); however, no other conditions were significantly different. There was no interaction or main effects for the readiness to perform questionnaire or sleep quality (p>0.05). CONCLUSION: CAF+CN appears to be superior for cognitive performance compared to CN alone. Overall, neither supplement ingested individually or together altered subjective readiness to perform or sleep quality.
Using Caffeine to Enhance the Therapeutic Effects of Respiratory Training After Cervical Spinal Cord Injury
Authors: Randelman Margo , Johnson Max , Fortino Tara , Zholudeva Lana , Lane Michael
Published: May 2022

Cervical spinal cord injury (SCI) often leads to damage of the phrenic motor circuit that innervates the diaphragm, resulting in life‐threatening respiratory deficits. While there is some limited spontaneous plasticity after a cervical SCI, severe respiratory deficits persist. Previous research has shown that non‐invasive respiratory training with intermittent hypoxia (IH) can enhance respiratory plasticity after SCI. There are two prominent plasticity promoting pathways associated with IH training, dependent on serotonin or adenosine. Prior work suggests there is greater activation of the serotonergic plasticity‐promoting pathway after inhibiting the adenosine pathway. Therefore, this work aims to use a clinically relevant adenosine antagonist – caffeine – in combination with IH respiratory training to further enhance the respiratory plasticity and diaphragm recovery of adult female rats after a mid‐cervical (C3/4) contusion injury. We hypothesized that 4 weeks of daily IH training primed with 8mg of caffeine, delivered 30 minutes before the training, will enhance the serotonergic plasticity‐promoting pathway to stimulate both anatomical and functional phrenic plasticity and improve respiration post‐SCI. To label phrenic circuitry, 72 hours before terminal experiments, animals were traced with a retrograde, transsynaptic tracer, pseudorabies virus (PRV), applied to the ipsilateral hemidiaphragm to label phrenic moto‐ and interneurons. Anatomical plasticity was assessed using immunohistochemistry to quantify interneuronal connectivity and density of descending serotonergic input to the phrenic motor network. Specifically, this study also investigated the connectivity of a cholinergic interneuron subpopulation within phrenic circuitry after IH training and caffeine delivery. Functional plasticity and respiratory recovery after IH training primed with caffeine were assessed with weekly whole‐body plethysmography and terminal diaphragm electromyography. The results of this study using caffeine as a primer for respiratory training can be easily translated to improve training outcomes and promote greater plasticity and recovery after SCI.
Hydrogen Peroxide Produced in Subcellular Compartments of Lung Adenocarcinoma Cell Line Induces Epithelial to Mesenchymal Transition
Authors: Rahman Md. A. , Livesey John C.
Published: May 2022

Epithelial to mesenchymal transition (EMT) is a process by which cancer cells increase invasiveness and motility by changing their phenotype from epithelial to mesenchymal. The mechanism of hydrogen peroxide (H2O2)‐induced EMT remains elusive. We hypothesize that H2O2 production in different subcellular compartments will trigger EMT at different intensities. We used a chemo‐genetic approach to produce hydrogen peroxide in the cytoplasm, nucleus and mitochondria of A549 lung adenocarcinoma cells to see how EMT progresses in each case. Cells were transfected with one of three different plasmids containing specific localization sequences for cytoplasm, nucleus and mitochondria. These plasmids have the peroxide generating enzyme D‐Amino‐Acid Oxidase (DAAO) which generates H2O2 by metabolizing D‐Alanine. We also used the inhibitors of peroxide metabolism, 3‐aminotriazole (3‐AT) and auranofin (Au) to increase H2O2 concentrations in these compartments. Preliminary studies show that cytoplasmic peroxide generation induces EMT significantly after 7 days. The epithelial marker E‐Cadherin was reduced and the mesenchymal marker N‐Cadherin was increased significantly in our immunoassays. The extent of change in EMT markers was greater upon the addition of 3‐AT and Au. However, there was no significant change in EMT markers when H2O2 was generated in the nucleus or mitochondria even in the presence of peroxide metabolism inhibitors. We are investigating the molecular mechanisms responsible for cytoplasmic peroxide‐induced EMT.
Reduced perception of pain via baroreceptor unloading in patients with chronic back pain
Authors: Liu Jennifer , Iwakuma Yuto , Clonch Davina , Gangwish Megan E. , Holwerda Seth W.
Published: May 2022

Nociceptive transmission at the spinal cord is modulated by descending pain inhibitory systems originating in the brainstem that interact with neural cardiovascular pathways, such as baroreceptor input. However, data regarding antinociceptive properties of cardiopulmonary baroreceptors are largely mixed, and no studies have examined cardiopulmonary baroreceptor modulation of pain perception in patients with chronic pain. Therefore, we tested the hypothesis that cardiopulmonary baroreceptor unloading would reduce pain perception in chronic back pain (CBP) patients and healthy participants. Mechanical pressure pain threshold (algometer on the upper trapezius) and pain perception of a repetitive heat stimulus (thermode on the anterior forearm) was tested in 12 CBP patients and 8 healthy controls during supine lower body negative pressure (LBNP) of ‐10 mmHg and 0 mmHg (control condition). Pressure pain threshold during LBNP was significantly increased compared with the control condition in CBP patients (270 ± 31 vs. 240 ± 27 kPa, P=0.04) and healthy controls (293 ± 59 vs. 259 ± 50 kPa, P=0.02), indicating reduced pain perception. Similarly, the average pain rating (scale 0‐100) of the repetitive heat stimulus was significantly reduced during LBNP compared with the control condition in CBP patients (42 ± 6 vs. 48 ± 6, P<0.01). However, no significant change was observed in average pain rating of the repetitive heat stimulus during LBNP in healthy controls (38 ± 5 vs. 37 ± 7, P=0.89). Together, these preliminary findings suggest reduced pain perception during cardiopulmonary baroreceptor unloading with a potentially greater effect in patients with CBP compared with healthy individuals.
Investigation of the Adhesion‐Modulating Properties of Bovine Serum Albumin (BSA) in Dictyostelium discoideum
Authors: Saljanin Palina , Artemenko Yulia
Published: May 2022

Dictyostelium discoideum is a social amoeba commonly used as a model organism in cellular migration studies. Dictyostelium adhesion is mediated by nonspecific interactions, such as Van der Waals forces, as well as specific protein‐mediated interactions between the cell and the substrate. Since adhesive properties of this organism affect its motility, finding ways to manipulate its nonspecific or specific interactions can be useful for future migration experiments. We tested the effects of bovine serum albumin (BSA), which is known to affect nonspecific binding via its interference with electrostatic interactions, on Dictyostelium adhesion. Bacterially‐grown wild‐type cells had increased adhesion when plated on 3% BSA, while axenically‐grown cells exhibited a decrease in adhesion on BSA. To explore why BSA has differential effects on adhesion of Dictyostelium grown under different conditions we tested adhesion of cells lacking one of the specific mediators of attachment, Talin A (TalA). Unlike wild‐type cells, both axenically and bacterially‐grown TalA‐null cells had decreased adhesion on BSA. These data suggest that BSA effects on adhesion may be dependent on TalA in bacterially‐grown cells. Alternatively, since TalA‐null cells have an impaired ability to phagocytose, they may behave more like axenically‐grown cells even when grown on bacteria. In this case, differential effects of BSA may be due to the inherent differences in the adhesion mechanism of cells grown under axenic vs. bacterially‐fed conditions. We are currently examining these possibilities.
A Pilot Anatomy‐based Teacher Training Curriculum for Medical Students
Authors: Proffitt James V.
Published: May 2022

Background

Numerous medical education training programs are currently implemented internationally to train residents and future academic physicians for teaching duties, many of which use peer‐teaching in anatomy as a major component. The length of these programs and the degree to which they provide both teaching practice and generalized training in medical educational competencies varies widely. Conversely, teacher training programs aimed at anatomy graduate students have a robust balance of in‐class practice and out‐of‐class professional development but take place over a longer timescale. With unscheduled curricular time at a premium in most medical programs, developing comprehensive training courses that can be administered over an accelerated timescale and provide generalizable professional skills in medical education is essential.

Intervention

In summer of 2021 I implemented a 6‐week pilot program for developing practical teaching and medical education skills in undergraduate medical students. The program consisted of an in‐class component where students performed daily peer teaching in a summer graduate‐level anatomy course and out‐of‐class professional development including training on educational methods, peer evaluations of student‐made materials, and reflective exercises.

Assessment

5 students were enrolled in the pilot program, 3 of which completed a 1‐5 Likert Scale, IRB‐approved survey after completion. The 22‐question survey measured pre‐and‐post program perceived confidence in 4 skill categories: anatomy knowledge (4 questions), teaching and assessment (5 questions), communication (9 questions), and educational research and innovation (4 questions). Although current survey sample lacks enough statistical power for formal nonparametric tests, initial student responses indicate improved self‐reported confidence in all 4 areas. Educational research and innovation showed the highest average increase (+2.4), followed by teaching and assessment (+2) anatomy knowledge (+1.58) and communication (+1.47).

Significance & Conclusion

With the caveat that limited data are available, students’ perceived confidence increased in all skill areas following participation in the training program. Additional longitudinal follow up will be needed to further determine the utility derived from the course pre‐and‐post residency. If successful, this course may serve as a model for both medical student and anatomical educator training courses that can be deployed on shorter timescales.
Structural basis for the activation of Arf1 at the Golgi complex by its GEF Gea2
Authors: Muccini Arnold , Fromme J. C.
Published: May 2022

In eukaryotes, vesicle formation at the Golgi complex is initiated by the conserved small GTPase Arf1. Arf1 is responsible for various trafficking pathways at the Golgi including retrograde recycling to the ER, intra‐Golgi tracking, and exit from the Golgi at the TGN. These various pathways occurring at different stages of the Golgi require precious regulation of Arf1. In budding yeast, Arf1 localization and activity is controlled by three separate guanine nucleotide exchange factors (GEFs): Gea1, Gea2, and Sec7. The paralogues Gea1 and Gea2 are responsible for activating Arf1 at the early Golgi to commence COPI vesicle formation which is essential for retention of resident proteins at the endoplasmic reticulum and early Golgi. Yet, the mechanism by which Gea1 and Gea2 are recruited to the Golgi at the right place and time to activate COPI vesicle formation is unclear. Having a structure of the full‐length protein would facilitate these efforts, however past attempts at solving the crystal structure have failed. In this poster I will present my work in determining the full‐length structure of Gea2 and Gea2 bound to Arf1 using cryo‐EM to 3.5Å resolution. This data has enabled me to build atomic models of these proteins of which I have been able use to test various hypotheses of how these proteins are regulated and controlled.
Early life stress drives sustained reductions in circulating butyrate and propionate short‐chain fatty acids in pre‐pubertal and adult mice
Authors: Kemp Keri M. , Colson Jackson , Maynard Craig L. , Pollock Jennifer S.
Published: May 2022

Exposure to early life stress (ELS) is associated with a greater risk of developing cardiovascular disease (CVD) later in life. Using a mouse model of ELS, we have recently showed that 4‐week‐old pre‐pubertal mice exposed to ELS have lower microbial diversity and reduced abundances of Lachnospiraceae and Ruminococcaceae taxa, which are important producers of short‐chain fatty acids (SCFAs). An essential mechanism by which gut microbes influence host physiology is through the production of SCFAs that act as vasoactive mediators, histone deacetylase inhibitors, and immunomodulators. Therefore, we hypothesized that ELS‐induced changes in the gut microbiota would result in reduced circulating SCFAs. To test this hypothesis, we subjected mice to maternal separation (MaSep) in which pups underwent daily separation for 4 h on postnatal days (PDs) 2–5 and 8 h on PDs 6–16 until weaning on PD17. Normally reared (NR) mice remained undisturbed with dams until weaning on PD21. All mice were maintained on standard chow (NIH‐31) following weaning. Plasma was collected at PD28 (n=17 MaSep mice from 7 litters and 13 NR mice from 7 litters) and at PD84 (n=8 MaSep from 3 litters and 11 NR mice from 3 litters) for analysis of lactate and the SCFAs acetate, butyrate, isovalerate, propionate, and succinate by gas chromatography/mass spectrometry. Acetate, succinate, and lactate were not different between MaSep and NR mice at either timepoint. However, ELS exposure resulted in significantly reduced butyrate plasma concentrations at PD28 (14.38 ± 0.50 µM in MaSep mice compared to 18.58 ± 1.87 µM in NR mice; P = 0.02) and PD84 (11.94 ± 0.18 µM in MaSep mice compared to 15.03 ± 0.21 µM in NR mice; P < 0.001). ELS also reduced propionate plasma concentrations at PD28 (35.96 ± 1.47 µM in MaSep mice compared to 43.70 ± 1.44 µM in NR mice; P = 0.001) and PD84 (32.16 ± 0.92 µM in MaSep mice compared to 42.53 ± 1.05 µM in NR mice; P < 0.001). Isovalerate plasma concentration was greater in MaSep mice at PD84 (1.11 ± 0.13 µM) compared to NR mice (0.45 ± 0.11 µM; P = 0.002), while there was no difference in isovalerate between MaSep and NR mice at PD28. These data indicate that exposure to stress in early life can result in sustained reductions in butyrate (~22%) and propionate (~21%) concentrations in circulation. Recent advances have determined that butyrate and propionate are involved in blood pressure regulation and vascular physiology. Much less is known about the branched SCFA isovalerate. Future studies will determine if decreases in circulating butyrate and propionate, or increases in circulating isovalerate, play a role in ELS‐induced increased risk of CVD.
Triiodothyronine Improves Contractile Function of Hypoxic/Reoxygenated Human Right Atrial Tissue
Authors: Kuthan Philipp , Eickelmann Chantal , Thielmann Matthias , Ruhparwar Arjang , Heusch Gerd , Kleinbongard Petra
Published: May 2022

Background

In patients with acute myocardial infarction, the one‐year mortality remains at 7‐15% despite successful interventional reperfusion. Thus, innovative treatment strategies beyond reperfusion are needed. In isolated perfused rat hearts, triiodothyronine (T3), when given during early reperfusion in a supraphysiological concentration, improves left ventricular function and reduces infarct size (Basic Res Cardiol 2021;116:27).

Aim

To investigate whether or not such acute, non‐genomic effects of T3 also protect human myocardium.

Methods

We isolated right atrial trabeculae from patients undergoing routine coronary artery bypass grafting and investigated the effect of T3 on contractile function during hypoxia/reoxygenation. Trabeculae (≥ 3 mm length, ≤ 1 mm diameter) were isolated in cardioplegic buffer and transferred to Tyrode buffer. Electrically initiated (1 Hz) developed force of contraction (mN/mm2) was recorded for 10 min at baseline (95% O2 – 5% CO2, glucose). Hypoxia/reoxygenation (60 min/30 min) was induced by changing the buffer gas supply and composition and by increasing the stimulation rate (95% N2 – 5% CO2, choline chloride, 3 Hz) and returning back to baseline. T3 (500 µg/l) or NaOH (as solvent control), respectively, was administered A) during hypoxia and reoxygenation (n=11 vs. n=10) or B) 15 min before and during reoxygenation (n=12 vs. n=14).

Results

At baseline, before T3 or NaOH treatment, the contractile function of trabeculae was comparable (mN/mm2: 15.4±3.5, 13.8±4.4 in protocol A, and 14.5±4.1, 13.7±4.0 in protocol B, respectively). T3 improved the contractile function during reoxygenation, irrespectively of whether given continuously (A) or just before and during reoxygenation (B; see Figure).

Conclusion

T3 protects patient myocardium: contractile function is improved during reoxygenation in right atrial trabeculae. This bioassay will now permit an identification of causally involved cardioprotective signaling molecules of such non‐genomic protection in human tissue by T3.
Calcium Signaling in Trauma: Increased Intracellular Calcium Flux in the Severely Injured
Authors: Schaid Terry , Mitra Sanchayita , DeBot Margot , Cralley Alexis , Erickson Christopher , Dabertrand Fabrice , Fontaine Jackson , D'Alessandro Angelo , Hansen Kirk , Jones Kenneth , Sauaia Angela , Banerjee Anirban , Silliman Christopher , Moore Ernest , Cohen Mitchell
Published: May 2022

Introduction

The pathogenesis of organ dysfunction following traumatic injury involves endothelial barrier disruption, and plasma from severely injured patients increases endothelial permeability in vitro. Many endothelial cell (EC) functions entail changes in intracellular calcium (Ca2+) concentrations, and multiple inflammatory mediators have been shown to induce EC barrier disruption in a Ca2+ dependent manner. However, study of Ca2+ signaling in trauma remains limited. Our objective was to investigate the role of intracellular Ca2+ signaling in trauma pathology. We hypothesized that ex vivo plasma from injured patients would induce intracellular EC Ca2+ flux and do so to a greater magnitude in more severely injured patients.

Methods

Blood was collected from injured patients at a single, urban Level I Trauma Center. Patients were categorized by minimal injury without shock (Group 1: ISS<15 + base excess >‐6mEq/L; n=3) and severe injury with shock (Group 2: ISS>15 + base excess <‐6mEq/L; n=4). Human cerebral microvascular endothelial cells were grown on culture inserts and pre‐incubated with Fluo‐4 Ca2+ indicator. Plasma was diluted to concentrations previously used for in vitro permeability assays and flowed over EC’s under physiologic conditions for five minutes. Confocal microscopy was used to visualize and quantify intracellular Ca2+ flux. Thrombin was used as a positive control for inducing Ca2+ flux. We used Prism software to calculate areas under curves (AUC) and unpaired t‐tests to compare group means.

Results

Compared to Group 1 plasma, Group 2 induced greater calcium flux, which was sustained for a longer period of time, corresponding to greater AUC for total intracellular calcium flux (P<0.05, Figure).

Conclusions

Ex vivo plasma from more severely injured trauma patients increases intracellular Ca2+ flux in EC’s. These data suggest Ca2+signaling may play a role in trauma‐induced endotheliopathy and present as a therapeutic target. Our study lays the groundwork for additional investigation to elucidate sources of Ca2+ in this setting as well as identify plasma mediators of both Ca2+ flux and endothelial permeability.
The Plastoglobule‐localized AtABC1K6 is a Mn2+‐dependent Protein Kinase Necessary for Timely Transition to Reproductive Growth
Authors: Lundquist Peter K. , Espinoza‐Corral Roberto
Published: May 2022

The Absence of bc1 Complex (ABC1) is an ancient, atypical protein kinase family that emerged prior to the archaeal‐eubacterial divergence. Loss‐of‐function mutants in ABC1 genes are linked to respiratory defects in microbes and humans, and to compromised photosynthetic performance and stress tolerance in plants. However, demonstration of protein kinase activity remains elusive, hampering their study. Here, we investigate a homolog from Arabidopsis thaliana, AtABC1K6, and demonstrate in vitro protein kinase activity as autophosphorylation, which we replicate with a human ABC1 ortholog. We show that AtABC1K6 protein kinase activity requires an atypical buffer composition, including Mn2+ as divalent cation co‐factor and a low salt concentration. AtABC1K6 associates with plastoglobule lipid droplets of A. thaliana chloroplasts, along with five paralogs. Protein kinase activity associated with isolated A. thaliana plastoglobules was inhibited at higher salt concentrations, but could accommodate Mg2+ as well as Mn2+,indicatingsalt sensitivity, but not the requirement for Mn2+, may be a general characteristic of ABC1s. Loss of functional AtABC1K6 impairs the developmental transition from vegetative to reproductive growth. This phenotype is complemented by the wild‐type sequence of AtABC1K6 but not by a kinase‐dead point mutant in the unique Ala‐triad of the ATP‐binding pocket, demonstrating the physiological relevance of the protein’s kinase activity. We suggest that ABC1s are bona fide protein kinases with a unique regulatory mechanism. Our results open the door to detailed functional and mechanistic studies of ABC1s and plastoglobules.
ADGRF1 promotes tumorigenesis via the activation of STAT3 pathway in HER2+ and triple‐negative breast cancers
Authors: Abdulkareem Noor M. , Bhat Raksha , Trivedi Meghana V.
Published: May 2022

Adhesion G protein‐coupled receptors (aGPCR) regulate many cellular processes involved in cancer. ADGRF1, a class VI aGPCR, is overexpressed and/or predicts poor survival in many cancer types. We previously reported that in breast cancer (BC), ADGRF1 is highly expressed in human epidermal growth factor receptor‐2 positive (HER2+) and basal‐like subtype, 85% of which are triple‐negative. Also, higher ADGRF1 expression predicts worse BC‐specific survival in both HER2+ and triple‐negative BCs patients. We previously showed that inducible ADGRF1 overexpression promoted and knockdown inhibited tumorigenesis in HER2+ BC cells. Consistently, ADGRF1 overexpression promoted tumor growth in vivo. We also reported that ADGRF1 coupling to Gαs was responsible for its pro‐tumorigenic effects. However, the downstream mediators of tumorigenesis promoted by ADGRF1 signaling remain largely unknown in cancer. Since STAT3 is a well‐known mediator of tumorigenesis in many cancers, and previous studies have shown that ADGRF1 modulates STAT3 activation in some cancers, we aimed to evaluate the effects of genetic and pharmacologic modulation of ADGRF1 on STAT3 phosphorylation (Y705) in HER2+ and triple‐negative breast cancers models. In a HER2+ BC cell line model (BT474), we found that ADGRF1 overexpression increased STAT3 phosphorylation (Y705) in vitro and in vivo. ADGRF1 knockdown also decreased STAT3 phosphorylation in BT474 cells and MDA‐MB‐231 cells, a triple‐negative BC cell line with high endogenous ADGRF1 gene expression and amplification. ADGRF1 knockdown inhibited cell growth as well as mammosphere and colony formation (a measure of tumorigenesis) in triple‐negative BC cell lines. Synaptamide increased secondary mammosphere formation, STAT3 phosphorylation (Y705), and cAMP but not IP1 in MDA‐MB‐231 cells while it had no effect on BT474 cells with inducible ADGRF1 overexpression. Synaptamide‐induced STAT3 phosphorylation was reversed by SQ22536, an adenylyl cyclase inhibitor. Our data suggest that ADGRF1 promotes tumorigenesis by coupling to Gas, which in turn activates STAT3 in HER2+ and triple‐negative BCs.
Potential Signaling Regulations of Stereisomeric DNA Interstrand Crosslinks Produced by Mitomycins in K562 Cells
Authors: Harun Kameza , Zacarias Owen , Clement Cristina C. , Cheng Shu Yuan , Champeil Elise
Published: May 2022

Mitomycin C (MC) is a DNA alkylating agent broadly used in chemotherapy. MC and Decarbamoyl mitomycin C (DMC), a synthetic MC derivative which lacks the carbamoyl at the C10 position, can form interstrand crosslinks (ICLs) between the exocyclic amines of opposing deoxyguanosine moieties. These ICLs are responsible for the cytotoxicity of mitomycins towards cancer cells since they prevent replication of highly proliferating cells. MC and DMC can crosslink DNA in both 5’‐CpG and 5’‐GpC sequence contexts, and the crosslinking is diastereospecific: At 5’‐CpG, mitomycins produce trans‐ICLs whereas cis‐ICLs are found at 5’‐GpC steps. The major ICL formed by Mitomycin C in culture cells is the trans‐ICL whereas decarbamoylmitomycin C yields the stereoisomeric cis‐ICL preferentially. Our overarching goal is to synthesize oligonucleotides bearing the cisand trans isomeric crosslinks to investigate the relationship between the ICL structure and the cellular response. Our hypothesis is that the trans and cis‐ICLs trigger different cellular responses. There is evidence that the ICL produced by DMC triggers a p53 independent cell death and research uncovering mechanisms through which p53 independent cell death occurs is crucial to identify molecular targets to treat tumors with a mutant p53. These tumors represent at least 50% of all cancers. We present here the different synthetic routes to access cis and trans isomeric interstrand crosslinks formed by mitomycins in their reaction with DNA and preliminary label free proteomic studies to identify the stereoisomeric ICLs molecular targets. Proteomic studies were accomplished from K562 leukemia cells transfected with the purified ICLs for 24 hours and showed several interesting proteins differently regulated by the trans‐ and cis‐ICLs: (1) Histone‐lysine N‐methyltransferase EHMT2 which down‐regulates a cell cycle inhibitory gene expression network (decreased 46% upon treatment with the trans‐ICL, but no change was observed with the cis‐ICL); (2) Target of EGR1 protein 1 which inhibits cell growth rate and cell cycle (increased 69% upon treatment with the trans‐ICL, but decreased 30% with the cis‐ICL); (3) Poly(ADP‐ribose) glycohydrolase DNA repair, involved in DNA repair (decreased 19% upon treatment with the trans‐ICL, but increased 63% with the cis‐ICL); (4) Telomerases Cajal body protein 1, involved in cell cycle regulation (no change observed upon tratment with the trans‐ICL but decreased 75% with the cis‐ICL. In addition, Ingenuity Pathway Analysis was conducted to identify the major molecular pathways impacted by each ICL treatment. In conclusion, we have identified both ICL molecular targets and cellular responses involved in DNA repair and cell cycle regulation in K‐562cell lines. Further validations are needed to confirm the results.
Control of Fibrotic Gene Expression in Cardiac Myocytes and Fibroblasts via the Rap1 GTPase
Authors: McLeod Michael R. , Heywood Jonathan G. , Thomas Sarah M. , Richardson William J. , Dubash Adi D.
Published: May 2022

Fibrosis describes the excessive deposition of extracellular matrix components by cardiac myofibroblasts (CFs), a process which frequently promotes the adverse effects of pathological conditions damaging cardiac muscle, such as myocardial infarction and arrhythmogenic cardiomyopathy. Roughly 1 million Americans suffer a myocardial infarction each year, after which necrotic cardiac myocytes (CMs) are replaced by CF‐derived extracellular matrix (ECM), in response to (a) biochemical signaling cues from necrotic CMs and (b) mechanical cues from the contracting myocardium and ventricle cavity pressure. Accumulation of ECM proteins (such as collagen) within the infarct scar is initially critical to prevent further tissue damage, but chronic and excessive collagen deposition is detrimental to regeneration of CMs and healing of the myocardium. While the physiological outcomes of cardiac fibrosis are well studied, the biochemical and mechano‐responsive signaling mechanisms which regulate fibrotic gene expression remain poorly investigated. The RhoA GTPase signaling pathway has been shown to play a central role in regulating fibrotic gene expression and is activated via many different pro‐fibrotic signaling factors and biomechanical stimuli (i.e., changes in ECM composition, organization and rigidity). The GTPase Rap1 is also very well‐known to mediate both inside‐out and outside‐in signaling via attachment of integrins to ECM proteins. In certain contexts, Rap1 has also been shown to antagonize RhoA signaling, but very little is known about its role in fibrotic gene expression. We therefore hypothesized that expression of Rap1 would inhibit fibrotic gene expression in cardiac myocytes and fibroblasts. Expression of either wildtype (WT) or constitutively active (63E) GFP‐tagged Rap1A constructs in cardiac fibroblasts resulted in a dramatic decrease in mRNA levels of ACTA2 (smooth muscle alpha‐actin), a commonly used marker of myofibroblast activation. Further, expression of WT or 63E Rap1A in either cardiac myocytes or fibroblasts resulted in a significant decrease in mRNA levels of ECM proteins (such as COL1A1, COL3A1, COL4A1 and FN1), as well as other common markers of fibrotic gene expression (CTGF and EGR1). Taken together, these data indicate that Rap1 signaling inhibits fibrotic gene expression and myofibroblast activation. Interestingly, expression of WT or 63E Rap1A in cardiac fibroblasts caused a dramatic increase in inflammatory markers (such as TNF, IL6, NFKB1A) and matrix remodeling enzymes (MMP3 and MMP9), genes which have been shown to play a role in increasing collagen turnover, reducing ECM rigidity/stiffness and repair of damaged myocardial tissue. Altogether, these data point to a significant role for Rap1 GTPase in reducing fibrotic gene expression and myofibroblast activation and suggest possible new therapeutic strategies to combat the damaging effects of cardiac fibrosis.