bluuebunny/crossref_metadata_2025 huggingface dataset:
Long Non‐coding RNAs Mediate Heart Failure with Preserved Ejection Fraction Associated with Thyroid Dysfunction
Authors: Bounds Curren , Chakraborty Sankalpa , Dickerson Bryce , Shethwala Ruhi , Rajagopalan Viswanathan
Published: May 2022

Introduction

Heart failure with preserved ejection fraction (HEpEF) carries significantly high morbidity and mortality. HFpEF is associated with preserved contractile function and can present with diastolic dysfunction, cardiac fibrosis and hypothyroidism (low levels of thyroid hormones, THs). We previously demonstrated safe, cardioprotective effects with treatment of active form of THs in multiple rodent models of cardiovascular disorders. Long‐noncoding ribonucleic acids (lncRNAs; >200 nucleotides long) account for large majority of the genome with significant transcriptional potential. However, their role in HFpEF associated with TH dysfunction is unclear. We hypothesized that lncRNAs act as mediators in development of hypothyroid HFpEF.

Methods

Hearts were isolated from obese ZSF1 rats that presented with HFpEF phenotype. Wild type and ZSF1 lean rats served as controls. Cardiac ultrasound and morphometric analyses were performed. RNA was isolated from left ventricular (LV) tissues and studied using Agilent LncRNA Microarray platform and analyzed using Feature Extraction and GeneSpring. Fibroblasts were isolated from wild type adult rat hearts and treated with TH receptor inhibitor. Real‐time quantitative polymerase chain reaction was used to study lncRNA expression.

Results

Studies revealed that total thyroxine TH levels significantly decreased in five‐six‐month old (early; p<0.05) ZSF1 obese rats compared to ZSF1 lean controls (45% decrease) and wild type controls (42% decrease), and this remained low at 13 months (late; p<0.05). Total tri‐iodothyronine TH levels also followed similar pattern in the early time‐point (37%‐42% decrease). ZSF1 obese rats showed increased normalized heart weight, LV weight and liver weight indicating cardiac hypertrophy and development of HF. In early obese rats, echocardiography showed preserved LV EF and significantly elevated E/E’ indicating diastolic dysfunction (p<0.05). LncRNA Microarray analysis including unsupervised hierarchical clustering showed that several unique lncRNAs were significantly altered (p<0.05; >2‐fold change) in obese LVs compared to the lean or wild type control counterparts and select lncRNA expression was validated with qPCR. Furthermore, isolated wild type adult rat cardiac fibroblasts subjected to TH receptor inhibition (mimicking hypothyroidism) in vitro showed significant increase in lncRNA expression that was also increased in obese ZSF1 hearts presenting with hypothyroidism (p<0.05).

Conclusions

These results indicate that lncRNAs may play important roles in in HFpEF pathogenesis associated with hypothyroidism. These studies will serve as significant step in identification of key lncRNAs in cardiac‐thyroid axis offering potentially valuable translational opportunities.
Contribution of Adenosine A2A Receptors in the Rostral Ventrolateral Medulla to Acupuncture Modulation of Hypertension
Authors: Guo Zhi‐Ling , Tjenalooi Stephanie C. , Su Hou‐Fen , Fu Liang‐Wu , Malik Shaista
Published: May 2022

Acupuncture is used to manage high blood pressure (BP) in clinical practice. However, the mechanisms underlying its hypotensive effects remain unclear. Our previous studies have shown that electroacupuncture (EA) at the ST36‐37 acupoints, overlying the deep peroneal nerve, attenuates pressor responses through adenosine A2A receptors (A2AR) in the rostral ventrolateral medulla (rVLM). A2AR activation in the rVLM is associated with the upregulation of nitric oxide synthase (NOS) that contributes to sympathoinhibition and BP reduction. However, it is uncertain whether rVLM A2AR contributes to EA’s action in lowering high BP in sustained hypertension (HTN) in the rVLM. We hypothesized that EA lowers BP in HTN through adenosine A2AR mechanisms associated with the upregulation of neuronal NOS (nNOS) in the rVLM. The nNOS derived NO has been shown to exert central effects on BP. To mimic essential HTN in the clinic, we performed EA in conscious Dahl salt‐sensitive hypertensive rat (DSHR). EA (0.1‐0.4 mA, 2 Hz) was applied at ST36‐37 for 30 min twice weekly for four weeks while sham‐EA was conducted with the same procedures as EA except for no electrical stimulation. Elevated BP was reduced by EA (n=10) but not sham‐EA (n=8) and untreated ones (n=6). The A2AR level in the rVLM after EA treatment was increased (n=5; P < 0.05) compared to the sham‐EA (n=5). EA’s inhibitory effect on elevated BP was reversed transiently by unilateral microinjection of SCH 58261 (1 mM in 50 nl; an A2AR antagonist; n=7; P < 0.05) but not the vehicle (n=5) into the rVLM in EA‐treated DSHRs under anesthesia. Activation of rVLM A2AR in DSHRs treated with sham‐EA using an A2AR agonist, CGS‐21680 (0.4 mM in 50 nl; n=6), decreased BP. Furthermore, we found a co‐localization of A2AR with nNOS in the rVLM of DSHRs. The expression of rVLM nNOS was enhanced in DSHRs treated with EA compared to those treated with sham‐EA (n=5 in each group; P < 0.05). These data suggest that adenosine‐ A2AR in the rVLM contributes to EA’s inhibitory effect on elevated BP in sustained HTN, which is likely relevant to the upregulation of nNOS.
SARS‐CoV‐2 and Hippo Signaling Pathway: A Potential Therapeutic Target
Authors: Arumugaswami Vaithi , Garcia Gustavo , Ramaiah Arunachalam
Published: May 2022

SARS‐CoV‐2 is responsible for the ongoing COVID‐19 pandemic, which causes respiratory failure and damage to multiple organ systems. Emergence of new variants of concern (VOCs), including Omicron can potentially render the current vaccines ineffective. However, our understanding of COVID‐19 pathophysiology and molecular basis of SARS‐CoV‐2 infection is very limited. The role of the Hippo signaling pathway, an evolutionarily conserved organogenesis circuitry, in tissue inflammation and innate immune response is beginning to be understood. Given the complexity of COVID‐19 associated cell injury and immunopathogenesis processes, we investigated this Hippo pathway dynamic in SARS‐CoV‐2 infection by utilizing COVID‐19 lung samples, transcriptome and human cell models based on pluripotent stem cell‐derived cardiomyocytes (PSC‐CMs) and human primary lung air‐liquid interface (ALI) culture. The SARS‐CoV‐2 infection resulted in stoppage of cardiomyocyte beating and extensive apoptotic cell death. Especially the infection caused activation of Hippo signaling pathway in cardiomyocytes, as shown by increased level of phosphorylated form of YAP, a downstream transcriptional co‐factor involved in tissue growth, mitochondrial biogenesis and innate immunity. Similar activation was noted in SARS‐CoV‐2 infected lung ALI epithelial cells and COVID‐19 lung autopsy samples. The shRNA‐mediated partial knockdown and pharmacological inhibitor of YAP/TAZ resulted in significantly reduced SARS‐CoV‐2 replication, whereas inhibition of Hippo pathway upstream LATS1 and MST1 kinases led to enhanced virus replication. These results indicate a direct role of Hippo signaling in SARS‐CoV‐2 mediated disease pathogenesis and this pathway can be pharmacologically targeted to treat COVID‐19.
Characterization of Periplasmic Iron Transport in Mycobacterium tuberculosis
Authors: de Miranda Rodger , Chao Alex , Sieminski Paul , Abdul‐Hafiz Sumer , Goulding Celia
Published: May 2022

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), results in 10 million infections and 1.5 million deaths annually. Current TB treatments are typically a cocktail of up to five antibiotics that needs to be administered for a 9‐month duration; unfortunately, several of these drugs illicit severe side effects. These long, harsh treatments lead to patient non‐compliance resulting in a rise in multiple‐drug resistant Mtb strains ‐ MDR‐TB. Due to these factors new treatment strategies are needed. As host iron acquisition is essential for Mtb’s survival, elucidating key players in the Mtb iron uptake pathways may yield good drug targets. Mtb predominately acquires host iron by the siderophore‐mediated uptake pathway, in which small molecules with high affinity for iron are secreted to scavenge for host iron. We seek to shed light on the mechanism by which ferric‐siderophores are transported into the Mtb cytosol. At present there is little known about the proteins required to shuttle ferric‐siderophores across the outer membrane, cell‐wall environment, and periplasmic space to the inner membrane. There are two putative Mtb periplasmic binding proteins (PBPs), FecB and FecB2, and we hypothesize that one or both of these PBPs shuttle ferric‐siderophores across the periplasmic space. To test this, we examined the affinity of FecB and FecB2 for the Mtb secreted ferric‐siderophore, ferric‐carboxymycobactin (Fe‐cMB), by tryptophan fluorescence quenching titration assays. The affinity of FecB for Fe‐cMB was in the high nanomolar range (Kd = 355 ± 146 nM); in contrast, the affinity of FecB2 was in the high micromolar range. This result suggests that FecB is involved in transporting Fe‐cMB across the periplasmic space. To further probe the FecB residues involved in Fe‐cMB binding, we carried out a comprehensive mutational analysis. In an attempt to decipher the protein‐protein interaction network of FecB, we utilized co‐immunopreciptation (co‐IP) of FLAG‐tagged FecB in the non‐pathogenic Mtb model organism Mycobacterium smegmatis, followed by mass spectrometry analysis. The co‐IP FLAG‐FecB experiments resulted in identification of known proteins in the import of Fe‐cMB and surprisingly, also proteins thought to be involved in the export of apo‐siderophores. Together, these data clearly place FecB in the siderophore‐mediated iron‐uptake pathway, and will be discussed in more detail. Finally, this work has broadened our understanding of the Mtb iron acquisition pathway, which may lead to the identification of new therapeutic targets.
Age‐related impairment in dynamic cerebrovascular compliance responses to standing despite preserved cerebrovascular resistance responses
Authors: Moir M. Erin , Sarkisian Savana C. , Shoemaker J. K.
Published: May 2022

During transient reductions in blood pressure induced by standing upright, a rapid increase in cerebrovascular compliance represents a fundamental response in addition to reductions in cerebrovascular resistance. Age‐related reductions in central arterial compliance are known but how ageing affects the cerebrovascular compliance response to standing remains unknown. Therefore, the present study tested the hypothesis that older adults would demonstrate attenuation of cerebrovascular compliance during standing relative to young adults. Ten young healthy adults (24 ± 3 years, 5 females) and 10 older healthy adults (57 ± 11 years, 4 females) completed a sit‐to‐stand protocol involving at least three minutes of sitting and at least two minutes of standing while brachial artery blood pressure (BP; Finapres) and middle cerebral artery blood velocity (BV; Multigon) were collected. Individual BP waveforms were time aligned to corresponding BV waveforms and were input into a four‐element lumped parameter Windkessel model to calculate an index of cerebrovascular compliance (Ci). Cerebrovascular resistance (Ri) was calculated as the quotient of BP and BV. Beginning 20 beats prior to standing and continuing 40 beats following the stand, every other heartbeat was extracted so that a total of 30 beats were analyzed (i.e., 10 beats during sitting, 20 beats during standing). Seated baseline Ci was calculated as the average of the 10 beats of sitting. Peak Ci (maximum value) minus seated baseline Ci determined the change in (∆) Ci. In the sitting posture, older adults demonstrated similar baseline Ci compared with young adults (7.2e‐4 ± 2.2e‐4 vs. 7.3e‐4 ± 1.5e‐4 cm∙s‐1·mmHg‐1, P= 0.97). Upon standing, Ci increased in all participants, but peak Ci was attenuated in older adults relative to young adults (1.4e‐3 ± 3.2e‐4 vs. 2.2e‐3 ± 4.8e‐4 cm∙s‐1·mmHg‐1, P< 0.001, d= 1.96). Therefore, the change in Ci from sitting to standing was less in older adults compared with young adults (7.1e‐4 ± 2.9e‐4 vs. 1.5e‐3 ± 4.3e‐4 cm∙s‐1·mmHg‐1, P< 0.001, d= 2.15; 109 ± 56 vs. 205 ± 71 %, P= 0.004, d= 1.50) despite both groups showing no difference in the drop in BP (older: ‐21 ± 7 mmHg, young: ‐22 ± 8 mmHg; P= 0.76). Further, no differences were observed between groups in the change in Ri between sitting and standing (older: ‐0.4 ± 03 mmHg∙cm‐1∙s‐1, young: ‐0.3 ± 0.2 mmHg∙cm‐1∙s‐1; P= 0.67). Therefore, older adults demonstrate similar alterations to cerebrovascular resistance upon standing but attenuated cerebrovascular compliance responses. As such, age‐related changes to the cerebral vasculature may be masked by evaluating cerebrovascular resistance alone.
Sterol metabolism regulates clathrin‐mediated endocytosis and intracellular trafficking within isogenic stem cell models
Authors: Francis Kevin , Vuppula Harika , Kerkvliet Jason , Hoppe Adam , Anderson Ruthellen
Published: May 2022

Clathrin‐mediated endocytosis (CME) is a critical cellular pathway for regulation of intracellular signaling, vesicular recycling, and receptor sensitization. While CME is a molecularly complex process which was previously shown to be inhibited by cholesterol depletion, the precise impact of sterol metabolism on CME, the cellular mechanisms involved, and the relevance to disease pathology remain unclear. To determine the impact of sterol homeostasis on CME and possible roles for CME within diseases impacted by cholesterol metabolism, CRISPR/Cas9 gene editing was utilized to fluorescently label clathrin light chain A and dynamin 2 in both immortalized and induced pluripotent stem cell (iPSC) models. To model how cholesterol levels and disease‐relevant sterol changes observed in cholesterol biosynthetic disorders (characterized by the substitution of cellular cholesterol for sterol intermediates) impact endocytosis, CME was analyzed under varying biochemical states. Live cell imaging demonstrated clathrin immobilization at the cell membrane and functional CME impairment occurred following either cholesterol depletion or upon accumulation of sterol species incapable of supporting ordered lipid domains. Polarized total internal reflection fluorescence microscopy demonstrated prolonged clathrin lifetimes associated with endocytic pits with variable curvature in correlation with sterol abundance and support of phase separation. To address the impact of sterol‐mediated CME on neurodevelopment, human iPSCs were analyzed at various stages of differentiation for trafficking deficits following sterol depletion or cholesterol metabolism disruption. CME deficits in iPSCs preceded loss of pluripotency, morphological changes, or aberrant differentiation events observed following cholesterol depletion or sterol intermediate accumulation. CME deficits were also cell‐stage specific, CME activity corresponded to the rate of intracellular sterol metabolism, and organellar deficits with disease relevance resulted from CME deficiencies. Ongoing studies are investigating the consequences of sterol depletion and sterol substitution on developmental signaling pathways and cellular function to provide insight into the mechanistic requirements for sterol homeostasis in CME‐mediated cellular trafficking.
Soluble Protein Oligomers Induce Endoplasmic Reticulum Stress and Exacerbated Vasodilation via Nitric Oxide Release in Resistance Arteries from Male and Female Mice
Authors: Waigi Emily W. , Castaneda Thaddeus R. , Bearss Nicole R. , Edwards Jonnelle M. , Singh Gagandeep , McCarthy Cameron G. , Wenceslau Camilla F.
Published: May 2022

Amyloid b (Ab) protein which gives rise to small, toxic, and soluble Ab oligomers (SPOs) is capable of interfering with synaptic function and integrity, thus memory loss in Alzhemier’s disease (AD). Further their accumulation induces endoplasmic reticulum (ER) stress, leading to cell death in several cardiovascular diseases. Our previous data showed for the first time that in arteries from both male and female mice, SPOs exert their vasoactive effects by inducing ER stress in acute conditions, resulting in pathologically exacerbated endothelium‐dependent vasodilation due to loss of vascular autoregulation, edema formation and chronic, irreversible vascular collapse. ER stress was also confirmed with anti‐KDEL antibody staining, specific for ER resident chaperones Grp78/94 and visualized with multiphoton fluorescent confocal microscopy. Additionally, we showed that ER stress induced further release of SPOs in arteries from female mice, showing a positive feedforward mechanism. Based on these prior research, we hypothetized that SPOs lead to exacerbated and deleterious vasodilation via nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activation. Mesenteric resistance arteries (MRA) were isolated from male and female C57BL/6J mice (~14 weeks old) (n=4‐7). The arteries were pre‐incubated for 30 minutes in the presence or absence of specific hydrogen peroxide (H2O2) inhibitor, catalase (500u/ml); NOS inhibitor, L‐NAME (100µM); cyclooxygenase (COX) inhibitor, Indomethacin (10µM); NADPH oxidase‐1 (Nox1) inhibitor (10 µM) and Nox 1/4 inhibitor (GKT136901,10μM), followed by incubation with SPOs (0.1 μM) for 45 minutes. Subsequently, concentration response curves to acetylcholine (1 nM‐30 µM) were performed after contraction with phenylephrine (10 µM). No differences were observed in the presence of catalase, indomethacin or GKT136901. However, L‐NAME was able to eliminate the SPOs ability to increase vasodilation in arteries form both male and female mice (Fig 1. A and B, Two‐way ANOVA). We therefore concluded that the mechanism that SPOs use to pathologically exacerbate endothelium‐dependent vasodilation in the resistance arteries is NO dependent, and was observed in arteries from both male and female mice. This could be one of the mechanisms that AD leads to loss of myogenic tone and edema in the microvasculature.
α1B‐Adrenoceptor is Required for Chemokine (C‐X‐C Motif) Receptor 4 Mediated Chemotaxis in THP1 Cells
Authors: Enten Garrett , Gao Xianlong , Majetschak Matthias
Published: May 2022

Introduction

Chemokine (C‐X‐C motif) receptor 4 (CXCR4) is a key regulator of leukocyte trafficking under physiological conditions and in numerous inflammatory disease processes. Previously, we demonstrated that CXCR4 hetero‐oligomerizes with α1B‐adrenoceptors (ARs), through which the receptors cross‐talk. The existence and function of these hetero‐oligomers in leukocytes, however, is unknown. Thus, we tested whether such heteromeric complexes are detectable in the human monocytic cell line THP1 and in freshly isolated human monocytes, and evaluated how α1B‐AR knockdown by CRISPR/Cas9 gene editing in THP1 cells affects CXCR4‐mediated chemotaxis.

Methods

Peripheral blood mononuclear cells were isolated by density gradient centrifugation from human whole blood; monocytes were then isolated via negative selection using MACS LS, an indirect magnetic labeling system. Proximity ligation assays (PLA) were used to visualize individual receptors and receptor‐receptor interactions in THP1 cells and freshly isolated monocytes. CRISPR/Cas9 gene editing was utilized to create a α1B‐AR knockout strain (ADRA1BKO); wild type THP1 cells were used as a negative control (ADRA1Bwt). Cell migration toward the cognate CXCR4 agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) was tested employing a 96 well Boyden chamber. Transmigrated cells were counted automatically utilizing high contrast bright field and post‐imaging particle analyses. The chemotactic index (ChI) was calculated as the ratio of cells that transmigrated in the presence versus the absence of CXCL12. Data were analyzed with ANOVA/Tukey’s post‐hoc. Data are mean ± SE from n=4 experiments performed on different days. A 2‐tailed p<0.05 was considered significant.

Results

PLA confirmed the presence of endogenously and constitutively expressed CXCR4:α1B‐AR heteromers in freshly isolated monocytes and THP1 cells. PLA signals for α1B‐AR and CXCR4:α1B‐AR heteromers were not detectable in ADRA1BKO cells. CXCR4 signals were not affected by CRISPR/Cas9 gene knockout. ADRA1Bwtcells showed a bell‐shaped chemotactic dose response to CXCL12 with a maximal ChI of 9.1±1.1 at 100 nM. CRISPR/Cas9 knockout of α1B‐AR reduced the chemotactic response to CXCL12 by 89±52% (p<0.05). ADRA1BKO results were replicated in an additional α1B‐AR knockout strain to rule out clonal effects.

Conclusion

Our observations suggest that CXCR4 heteromerizes with α1B‐AR in human monocytes and that CXCL12‐mediated chemotaxis depends on the formation of CXCR4:α1B‐AR heteromers. These data provide evidence for a novel molecular mechanism by which CXCR4‐mediated chemotaxis in monocytes/leukocytes is controlled.
Direct targeting of epigenetic regulator Menin inhibits neuroblastoma growth
Authors: Chilamakuri Rameswari , Agarwal Saurabh
Published: May 2022

Histone modifiers such as methyltransferases and demethylases have recently emerged as an attractive target to treat different cancers. Menin is a tumor suppressor protein and an essential oncogenic co‐factor of MLL1 COMPASS complex. Menin is a highly specific binding partner of the N‐terminus of MLL1 complex that have H3K4 methyltransferase activity. Menin‐MLL1 as an epigenetic regulator regulates the expression of target oncogenes, including HOXA, MEIS1, and AF9. Blocking menin‐MLL1 interaction destabilizes the COMPASS complex and hinders the oncogenic potential of cancer cells and cancer stem cells (CSCs). Neuroblastoma (NB) is an aggressive pediatric solid tumor develops during the early embryonic stage from extracranial sympathetic nervous system, and accounts for 10‐15% of all pediatric cancer‐related deaths. In the present study, we hypothesized that disrupting menin‐MLL1 interaction by using a specific small molecule inhibitor could disrupt COMPASS complex and overall inhibit NB growth, metastasis, and relapse. In the present study, we analyzed 1235 NB patients from different NB patient datasets and found that the upregulation of menin inversely correlate with overall survival of NB patients. We used six NB cell lines, including MYCN‐ amplified and ‐non‐amplified cell lines and performed cytotoxic assays. Results showed that inhibition of menin‐MLL1 interaction by MI‐503 significantly inhibits NB proliferation and colony formation capacity in contract to control treatments. Furthermore, MI‐503 significantly and in a dose‐dependent manner blocked NB cell cycle progression at S phase and enhanced apoptosis in treatment groups compared to controls. Additionally, we developed a 3D spheroidal tumor model for NB to mimic in vivo tumor conditions and tested the ability of MI‐503 in inhibiting tumor growth. Results showed significant inhibition of spheroidal tumor growth by enhancing tumor cell death in response to MI‐503 treatment. To further determine the effect of MI‐503 on inhibition of H3K4me3 levels, a key function of menin‐MLL1 COMPASS complex, we performed Western blot assays and observed a significant and dose dependent inhibition of H3K4me3 levels in response to MI‐503 treatment. Overall, our study highlights the role of menin‐MLL1 interaction in NB. Inhibition of menin‐MLL1 interaction by a specific small‐molecule inhibitor MI‐503 leads to the inhibition of epigenetic regulator functions and inhibits NB growth. Our future efforts will focus on further elucidating the role of menin in NB and to develop effective therapeutic strategies incorporating epigenetic inhibitors for NB patients.
Mitochondrial Telomerase Prevents Chemotherapy‐Induced Cardiovascular Toxicity
Authors: Brandt Lukas , Hader Shelby N. , Norwood Toro Laura E. , Gutterman David D. , Beyer Andreas M.
Published: May 2022

Anti‐cancer therapy (including doxorubicin (Dox)) is frequently used to combat growth of tumors. Chemotherapy (CTx) induced cardiotoxicity manifests as cardiomyopathy and can ultimately progress to heart failure and significantly contributes to morbidity and mortality among cancer survivors. Limited evidence demonstrates the impact of Dox on vascular endothelial function and possible contribution to progression of heart failure. TERT, the catalytic subunit of telomerase, has been increasingly recognized for its benificial effects on vascular quiescence via suppression of mitochondrial ROS to maintain mitochondrial integrity. Anthracycline treatment (e.g., Dox) correlates with both, suppressed TERT expression and activity as well as increased mitochondrial DNA damage. These combined effects of inadequate TERT levels and loss of mitochondrial integrity may contribute to CTx induced cardiotoxicity.

The goal of this study was to define the contribution of TERT to CTx‐induced endothelial dysfunction and to cardiac dysfunction. We hypothesize that TERT gain of function prevents, while TERT loss of function augments CTx‐induced microvascular and cardiac dysfunction. We modulated TERT activation with a known telomerase activator cycloastragenol (CAG) via dietary supplement.

In TERT WT animals, treatment with Dox significantly reduced endothelial dependent dilator function as early as two weeks after first dose. Dietary supplementation with CAG prevented Dox induced decrease in endothelial function (A). In contrast, in TERT‐specific KO animals Dox induced vascular damage was increased and CAG treatment failed to prevent endothelial dysfunction (B). Cardiac function followed similar patterns. In WT rats Dox caused significant reductions in left ventricular ejection fraction (LVEF) six weeks after the first dose of Dox, which was prevented by dietary supplementation with CAG. The reduction in LVEF was augmented in TERT KO animals and not rescued by CAG (C). Collectively, our data showed that Dox induced endothelial dysfunction was prevented by the pharmacological activation of TERT via CAG and furthermore suggests, that Dox induced endothelial dysfunction manifests before or independent of a cardiac damage. Likewise, CAG administration was sufficient to prevent reductions in LVEF relative to baseline in Dox treated WT animals. Specificity of a TERT mediated phenotype via CAG was established by failure of rescue in TERT KO rats. Ultimately, our results indicate that CAG has the potential as therapeutic to counteract the detrimental effects of CTx in the cardiovascular system via activation of TERT.
Molecular Mechanism by which Coumestrol Exerts its Anticancer Activity in Triple‐Negative Inflammatory Breast Cancer
Authors: Rodríguez‐Mártir Keishla M. , Colón‐Ortiz Ayeisha N. , Fuxa‐Rey Verónica , Negrón‐Figueroa Dalissa , Morales‐Rivera Carlos S. , Carrasquillo‐Dones Emmanuel A. , Peterson‐Peguero Esther A.
Published: May 2022

Coumestrol (Cou) is a phytoestrogen present in soy and clover sprouts which is structurally similar to estrogen (E2) and has a cytotoxic effect in breast cancer cell lines. Nevertheless, the molecular mechanism by which Cou can exert its effect in the most aggressive breast cancer subtype, triple‐negative inflammatory breast cancer (TN‐IBC), is still unknown. Several studies show that TN‐IBC cell lines are unresponsive to hormonal therapies, but E2 can activate signaling pathways involved in pro‐oncogenic phenotypes such as motility and invasion. Therefore, the objective of this study was to determine if Cou has an anti‐cancer effect in TN‐IBC cell lines and the molecular mechanism that Cou exerts for its anti‐cancer activity using 2D cell culture models. For this, a dose‐response curve of Cou in 2D models of TN‐IBC cell lines was generated to determine the half‐maximal inhibitory concentration (IC50). Also, relative cell viability was performed in ER‐positive, triple‐negative, TN‐IBC, and HER2‐amplified IBC cell lines. Functional assays were performed in TN‐IBC cell lines to determine the effect of E2 alone, Cou alone, and in a combination of treatments on cell viability, migration, and invasion. Also, the effect on phosphorylation of downstream kinases was analyzed by a proteome profile human phospho‐kinase array. An RNA‐seq was performed in TN‐IBC cell lines to identify transcriptome changes after Cou treatment. Preliminary observations demonstrated that Cou treatment using the IC50 (13µM) caused a decrease in cell viability in TN‐IBC cell lines. Similarly, Cou treatment reduces triple‐negative non‐IBC viability. Also, in comparison with E2, Cou decreases migration and invasion in TN‐IBC cell lines. Finally, Cou treatment reduces the phosphorylation of kinases that promote pro‐oncogenic phenotypes and upregulate genes involved in breast cancer phenotype suppression. In summary, the data gathered in this study will guide us in elucidating a signaling pathway affected by Cou’s anti‐cancer activity. More importantly, it will open the opportunities to design more effective targeted therapeutic strategies to improve the prognosis and survival rates of IBC patients.
Aspects of research and teaching that impact anxiety and depression among life sciences Ph.D. students
Authors: Cooper Katelyn , Wiesenthal Nicholas , Gin Logan , Busch Carly
Published: May 2022

In 2018, researchers declared a graduate student mental health crisis after finding that graduate students are significantly more likely to report depression and anxiety compared to their counterparts in the general public. Calls to improve the mental health of science graduate students followed, but no studies examined how science Ph.D. programs specifically affect student mental health. In response, we conducted a set of interview studies; we interviewed 83 life sciences Ph.D. students from over 30 institutions to identify what specific aspects of research and teaching affect Ph.D. student mental health. In turn, we examined how students perceive their depression or anxiety disorders affect their graduate research and teaching. Using inductive coding, we identified factors that either alleviate or exacerbate student anxiety, as well as student depression. With regard to depression, graduate students more commonly mentioned factors related to research that exacerbated their depression and factors related to teaching that helped their depression. This was also found to be true for student anxiety. We identified four overarching aspects of graduate school that commonly influenced student mental health: the amount of structure in teaching and research, positive and negative reinforcement, success and failure, and social support and isolation. Graduate students reported that depression had an exclusively negative effect on their research, primarily hindering their motivation and self‐confidence, but that it helped them to be more compassionate teachers. Students described anxiety as something that caused them to be meticulous in their research and teaching, but sometimes caused them to spend excessive time on research and teaching tasks. We built upon these interview studies by conducting a national survey study of Ph.D. students with depression and anxiety disorders, the results of which will be highlighted. This work pinpoints specific aspects of graduate school that PhD programs can target to improve mental health among life sciences graduate students.
Variation in ORF3a Protein of SARS‐CoV‐2 Decreases The Severity of Host Cell Damage
Authors: Landherr Maria , Cypress Michael W. , Chaput Isabel , Jhun Bong Sook , O‐Uchi Jin , Polina Iuliia
Published: May 2022

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) primarily targets the respiratory system. However, direct SARS‐CoV‐2 infection and viral protein expression have also been reported in other organs, which potentially contributes to multi‐organ dysfunction and increased mortality in COVID‐19. Since the original Wuhan‐type genome was sequenced, several genetic variants of SARS‐CoV‐2 have emerged with differing pathophysiological properties such as the levels of transmissibility, disease severity, and mortality. We previously reported that the protein encoded by open reading frame 3a (ORF3a), a critical protein for SARS‐CoV‐2 replication and release, is found in the mitochondria of host cells and could increase oxidative stress and apoptotic signaling. ORF3a‐Q57H is a highly recurrent variation and the most commonly found variant of ORF3a. Interestingly, the Q57H variant is associated with increased transmissibility, but lower mortality.

Aim

To investigate the impact of the ORF3a‐Q57H variant on host cell damage.

Methods

Plasmids carrying ORF3a‐Q57H were generated by PCR‐based site mutagenesis using Wuhan‐type ORF3a (ORF3a‐WT) as a template. Whole cell lysates were prepared from HEK293T cells and H9c2 cardiac myoblasts expressing ORF3a‐WT or the mutant ORF3a‐Q57H and used for biochemical assays. Live cell imaging for assessing subcellular localization of ORF3a/ORF3a‐Q57H, mitochondrial reactive oxygen species (mROS), and caspase 3 activity in H9c2 cells was performed by confocal microscopy.

Results

In whole cell lysates, we found that ORF3a‐Q57H exhibits significantly higher protein expression compared to ORF3a‐WT. However, there is no significant difference in the ability of mitochondrial trafficking between ORF3a‐WT and ORF3a‐Q57H assessed by live cell imaging using GFP‐tagged ORF3a/ORF3a‐Q57H with mitochondria‐targeted DsRed. Next, we investigated the effects of ORF3a expression on apoptotic and mitophagy signaling by quantifying the caspase 3 activity and LC3A/B ratio. We found that ORF3a‐Q57H has significantly lower apoptosis and mitophagy signaling compared to WT despite its higher protein expression levels. ER‐stress signaling was not activated in either ORF3a‐WT or ORF3a‐Q57H, as assessed by markers including Glucose‐regulated protein 78/94 and C/EBP‐homologous protein. Lastly, live cell imaging using the mitochondrial superoxide‐sensitive dye Mitosox Red revealed that ORF3a‐WT significantly increases mROS levels, but ORF3a‐Q57H expression does not.

Conclusion

SARS‐CoV‐2‐ORF3a‐Q57H causes lower oxidative stress and cell damage compared to the Wuhan‐type variant, which could help explain the decreased mortality associated with the ORF3a‐Q57H variant. These results provide novel insights on how genetic variations of SARS‐CoV‐2 influence the pathophysiology and clinical severity of COVID‐19.
Ablation of PDE4A Exacerbates Pseudomonas aeruginosa‐Induced Acute Lung Injury in Mice
Authors: Boyd Abigail , Abou Saleh Lina , Aragon Ileana , Fiedler Edward , Irelan Daniel , Spadafora Domenico , Barrington Robert , King Judy , Richter Wito
Published: May 2022

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a family of four isoenzymes, PDE4A to D, that hydrolyze the second messenger cAMP. Three of these enzymes, PDE4A, PDE4B and PDE4D, are widely expressed throughout pulmonary and immune cell types, and their concurrent inhibition with non/PAN‐selective PDE4 inhibitors is well‐known to exert potent anti‐inflammatory benefits. To determine if PDE4 inhibition may represent a therapeutic approach for acute lung injury resulting from severe pneumonia, we explored the effect of genetic ablation of individual PDE4 subtypes, or their concurrent inhibition with PAN‐PDE4 inhibitors, in a model of acute intra‐tracheal Pseudomonas aeruginosa (PA) infection in mice. We have shown recently that selective ablation of PDE4B is sufficient to protect mice from lung injury in this model by reducing pulmonary and systemic levels of pro‐inflammatory cytokines, as well as pulmonary vascular leakage and mortality. With the present study we explored whether selective inactivation of PDE4A or treatment with PAN‐PDE4 inhibitors may provide additional therapeutic benefits.

Methods

Mice deficient in PDE4A (PDE4A‐KO) and their wildtype (WT) littermate controls were infected intratracheally with the PA lab strain PA01 and subjected 16 h later to bronchoalveolar lavage (BAL), cardiac blood draws, and tissue extractions. Alternatively, vascular/alveolar leakage was assessed by measuring Evans Blue Dye extravasation. To test the effect of PAN‐PDE4 inhibition, mice were treated with the PDE4 inhibitor Piclamilast (5 mg/kg, i.p.) at 2 h prior to PA01 infection.

Results

Compared to WT controls, PDE4A‐KO mice displayed a hyper‐inflammatory phenotype characterized by increased levels of pro‐inflammatory cytokines, immune cells (neutrophils) in BAL fluid, and vascular leakage. Despite these hyperinflammatory responses, the bacterial load in lungs was also increased in PDE4A‐KO mice compared to WT controls. Treatment with a PAN‐PDE4 inhibitor produced incongruent effects, with an increased bacterial load mirroring the effect of PDE4A ablation, whereas reduced levels of inflammatory cytokines such as TNFα mimicked the effect of PDE4B ablation.

Conclusions

Ablation of PDE4A produces detrimental effects in this model of acute PA‐lung infection. Current studies aim to determine whether an impaired bacterial killing, and the resulting increase in bacterial load, are responsible for the hyperinflammation, or whether these are independent effects of PDE4A ablation. PAN‐PDE4 inhibition mimics some of the beneficial effects of selective PDE4B ablation, but also some of the detrimental effects of PDE4A ablation. These findings suggest that selective inactivation of PDE4B may be a more effective therapeutic approach, compared to PAN‐PDE4 inhibition, in settings of acute PA‐lung infection.
ARHGAP29 Regulates Keratinocyte Migration through the RhoA/ROCK Pathway
Authors: Rhea Lindsey , Garnica Bailey , Reeb Tanner , Dunnwald Elliot , Dunnwald Martine
Published: May 2022

Genetic variants in Rho GTPase Activating Protein 29 (ARHGAP29) have been associated with non‐syndromic cleft lip and/or palate, one of the most common human birth defects. Orofacial clefting results from the failure of facial processes to fuse during development which can be caused by defects in proliferation, adhesion, and/or migration. ARHGAP29 is widely expressed and preferentially interacts with the small GTPase RhoA, a critical modulator of the actin cytoskeleton required for cell morphology and migration. Therefore, we hypothesized that ARHGAP29 would play a critical role in keratinocyte migration. To test this hypothesis, we knocked down ARHGAP29 in a human keratinocyte cell line using both shRNA and CRISPR technologies. To assess migration, we used an in vitro scratch assay on a confluent keratinocyte monolayer and followed scratch closure over 24 hours. ARHGAP29 knockdown keratinocytes displayed significantly increased cell area and significant delays in scratch closure. By tracing single cells throughout the duration of scratch closure, we found that ARHGAP29 knockdown cells were significantly slower and showed defects in directionality that were rescued with a ROCK inhibitor, Y‐27632, suggesting these migration defects are downstream of the RhoA pathway. Collectively, our results demonstrate that ARHGAP29 regulates keratinocyte morphology and promotes migration speed and directionality through the RhoA/ROCK pathway, providing potential insight into its importance in craniofacial development.
Immersive XR Anatomy Teaching Tools: Review of four custom‐built virtual spaces, their deployments and educational value
Authors: Ramirez Kristen , Dorsainville Greg , Lieber Jillian , Melamud Kira , Clayburn Andrew , Harnik Victoria , Poles Michael , Oh So‐Young
Published: May 2022

Use of digital anatomical teaching resources mitigate trends of decreased anatomy contact hours, elimination of cadaveric specimens or student‐led dissections of them, remote or hybrid learning in response to COVID‐19, and to integrate anatomy content with radiology, surgery and other clinical applications. While there are a plethora of atlases built with cadaveric images, radiology, and artist‐rendered 3D models, we often find a disconnect between the complex geometry of many anatomical structures and the 2D screen interfaces used by students to access these digital resources. The open‐source Mozilla Hubs platform allows for the creation of customizable 3D virtual spaces, accessible via URL on a mobile, desktop, or virtual reality (VR) device. This platform was used to create four educational experiences deployed to first and second year medical students during pre‐clerkship courses: a desktop‐based tour of the surface and chambers of the heart; a VR tour of the chambers of the heart; a desktop‐based foregut escape room integrated with questions and multimedia clinical supplements; and a VR tour of the structure and neurovasculature of the pterygopalatine fossa. In each experience, the students are immersed within these anatomical spaces, encouraged to explore and appreciate the spatial relationships and orientations of key internal structures. When applicable, radiological imaging, videos, and animations display physiologic processes, the flow of blood and other fluids, and the functionality of a given structure in vivo. Audio and text instructions, labels, and additional information guide student learning, encouraging directed exploration while immersed in the experience. This presentation will discuss the four experiences, display real‐time video capture of the interactions and spaces, their deployment, educational value, and student reception. Students’ feedback suggests that the context and timing of the deployment of these experiences is crucial for student learning and appreciation, including the role of faculty assistance and training on how to navigate the interface prior to the educational intervention. Qualitative feedback and performance on pre‐ and post‐ experience quizzes show that these experiences are both statistically and perceptually valuable educational resources, and that the novel nature of this experience is engaging and exciting to students when deployed appropriately. This presentation will conclude with suggestions for best practices in choosing, developing, and deploying custom immersive educational interventions to anatomy learners.
Exploring Sex Differences in Renal Sodium Transporters with Four Core Genotype (FCG) Model
Authors: McDonough Alicia A. , Ralph Donna L. , Soong Joanne , Carrisoza‐Gaytan Rolando , Kleyman Thomas R. , Satlin Lisa M.
Published: May 2022

In the kidney, sex‐specific differences in Na+ transporter profiles along the nephron are now evident (“transporters” = co‐transporters, channels, claudins and pumps, their phosphorylation (p)): females (F) exhibit variable differences vs males (M) along the proximal tubule (PT) and higher distal (DT) and collecting duct (CD) Na+ transporter abundance and activity vs. M. Circulating levels of gonadal hormones determine sex differences in many physiologic traits, however, recent studies indicate that sex differences may also be due to the sex chromosome complement (SCC; XX vs.XY). The novel FCG C57BL/6 mouse model dissociates gonadal sex (ovaries (F) or testes (M)) from sex chromosomal complement (SCC; XX, XY). That is, traits influenced by gonadal hormones are similar between MXX and MXY (testes) vs FXX and FXY (ovaries); whereas traits influenced by SCC are similar between MXX and FXX vs. MXY and FXY (independent of gonads).

Method

We implemented the 4CG model with transporter profiling (semi‐quantitative immunoblotting; n = 4‐5 mice/genotype) to reveal the contributions of gonadal hormones vs. SCC on the sexual dimorphisms in transporter protein abundance along the nephron.

Results

(all P<0.05): Along the PT: OAT1 was lower and AQP1 and SGLT2 higher in abundance in gonadal F (FXX) vs M (MXY); SCC also contributed to differences: NHE3 was lower by 15‐30% in MXX vs. MXY and SGLT2 was 15% lower in FXY vs FXX. Inthe medullary thick ascending limb (TAL): alpha and beta NKATPase are 60% higher in FXX vs MXY and 25‐30% lower in FXY vs FXX; alpha was 15% lower in MXX vs. MXY. Along the cortical TAL and DT: NKCC2, NKCC2p, NCC, NCCp, alpha and beta NKATPase, claudin 7, SPAK and SPAKp kinase abundance were 1.4 ‐ 2.6 ‐ fold higher in FXX vs MXY; SCC differences were limited to 15%, 30%, and 20% lower NCC, and NCCp, and claudin 7 in MXX vs MXY, respectively. Along the CD: ENaC subunits were 30‐50% higher, claudin‐8 2‐fold higher, pendrin and UMOD 20% higher in FXX vs. MXY; SCC differences included 15‐20% lower ROMK and Kir 4.1 and ENaC beta and gamma subunits in MXX vs MXY. Results provide evidence for impact of both gonadal hormones and SCC on kidney transporter abundance, in some cases, offsetting the effects of the other. Further progress will come from defining whether X vs Y genes contribute to the differences. These findings have potential impact on electrolyte and blood pressure (patho)physiology in F vs. M.
Modulating Intramuscular Fat Infiltration by Small Molecules
Authors: Zhou Lylybell Y. , Norris Alessandra , Kopinke Daniel
Published: May 2022

Skeletal muscle has a remarkable ability to regenerate upon injury. However, during aging and in diseases such as muscular dystrophies and diabetes, muscle tissue is replaced by fibrotic scar and fat tissue in a process called fatty fibrosis. Currently, there are no known treatments to prevent fatty fibrosis or the decline in muscle mass and function. Our lab and others have shown that ectopic activation of the Hedgehog (Hh) pathway accelerated muscle regeneration after an acute injury and preserved muscle function with age and disease. In addition, our lab demonstrated that Hh is a potent anti‐adipogenic signal and that activating Hh prevents intramuscular fat formation. However, the cellular and molecular mechanisms by which Hh balances fatty fibrosis and muscle regeneration are unclear. In this project, we use small molecules as a novel approach for modulating intramuscular fat infiltration via Hh signaling: Smoothened agonist (SAG) to activate and Vismodegib to inactivate Hh. We found that SAG (HHON) successfully activates the Hh pathway while Vismodegib (HHOFF) inactivates the pathway, as detected by RT‐qPCR analysis for the expression of key Hh target gene Gli1. Excitingly, by modulating the Hh pathway, SAG and Vismodegib decreased and increased fat infiltration, respectively. We are currently evaluating the effects of SAG and Vismodegib administration on muscle regeneration and fibrosis. Future experiments will focus on using small molecules to modulate the Hh pathway at different time points after injury, as well as looking to therapeutic applications in treating fatty fibrosis in conditions such as Duchenne muscular dystrophy. Together, the results of this project will help uncover the regulatory role and dynamics of Hh signaling in intramuscular fat infiltration, ultimately providing a starting point for developing powerful new therapeutics to treat fatty fibrosis.