bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex Differences and Cardiovascular Diseases in Down Syndrome
Authors: Flores Laura D. , Pryakhina Yana , Tomasson Michael H. , Bates Melissa L. , DeRuisseau Lara R.
Published: May 2022

Congenital heart disease and cardiovascular disease are the leading causes of death for individuals with Down Syndrome (Ds) (Zhu, J.L., et al, 2013). A systematic review was conducted in June 2021 to address the research question “Are there reported sex differences for cardiovascular disease and cardiovascular physiological function in Ds”. Since there are significant sex differences in the risk of congenital heart and cerebrovascular disease in the general population, we hypothesized that these differences are present in the Ds population. Four domains were developed: congenital heart disease, baseline physiology and risk factors, heart disease and hypertension, and cerebrovascular disease. Exclusion criteria were applied to identify studies that addressed sex differences. The systematic review shows that women with Ds have a higher rate of congenital heart disease, coronary and cerebrovascular events and Moyamoya. Following the systematic review, a retrospective medical chart review of the University of Iowa Hospitals and Clinics was conducted using the platform TriNetX to identify the incidence of congenital and cardiovascular diseases in males and females. Relative risk was computed comparing Ds to general patient counts as well as men and women within each population. Ds patients did not share most of the sex differences in risk factors observed in the general population, however there are important sex differences in each domain that may contribute to earlier mortality in women with Ds. Several studies report women have an increased probability of developing risk factors for cardiovascular disease, such as obesity. Therefore, future research to understand the mechanism underlying these sex differences is necessary.

References

Zhu, J.L., et al., Survival among people with Down syndrome: a nationwide population‐based

study in Denmark. Genetics in Medicine, 2013. 15(1): p. 64‐69.
Hypoxic Vasodilation is Augmented During the High versus Low Estrogen Phase of the Menstrual and Oral Hormonal Contraceptive Pill Cycle
Authors: Jacob Dain , Harper Jennifer , Ivie Clayton , Limberg Jacqueline
Published: May 2022

Background

Hypoxia elicits peripheral vasodilation to preserve oxygen delivery to metabolically active tissue. Hypoxic vasodilation, predominantly mediated by β‐adrenergic and nitric oxide mechanisms, is greater in adult female participants compared to adult male participants. Greater circulating estrogen in adult females has been purported to contribute to observed sex differences in hypoxic vasodilation. Nearly 80% of adult females in the United States will take oral hormonal contraceptive pills (OCPs) in their lifetime. Adult females taking OCPs exhibit greater β‐adrenergic receptor mediated vasodilation and nitric oxide production relative adult females with natural menstrual cycles (NC). Strategically utilizing the fluctuations of circulating estrogen that occur within a NC and OCP cycle, we aimed to examine the contribution of endogenous and exogenous estrogen on hypoxic vasodilation in female participants. We hypothesized female participants taking OCPs would exhibit greater hypoxic vasodilation relative to female participants with a NC. We further hypothesized hypoxic vasodilation would be greatest during the high estrogen (HE) phase of the menstrual or pill cycle compared to the low estrogen (LE) phase.

Methods

Ten female participants with a NC (25±1 yrs, 23±1 kg/m2) and ten female participants taking monophasic OCPs (24±1 yrs, 21±1 kg/m2) completed two study visits (IRB #2011312, NCT03606434). Participants were studied once during the LE phase of the menstrual or pill cycle (Cycle day: NC 3±1; OCP 5±1) and once during the HE phase of the menstrual or pill cycle (Cycle day: NC 15±1; OCP: 17±1). Forearm blood flow (venous occlusion plethysmography) and blood pressure (BP, finger plethysmography) were measured during normoxia and hypoxia (80% SpO2). Blood flow was normalized for BP and expressed as forearm vascular conductance (FVC).

Results

Hypoxia elicited an increase in FVC (% change from baseline) that was greater during the HE phase (NC: 21±10%, OCP: 41±6%) of the menstrual/pill cycle compared to the LE phase (NC: 5±8%, OCP: 10±8%) in both NC and OCP participants (Main effect of phase, p<0.01; Main effect of group, p=0.16; Interaction of phase and group, p=0.30).

Conclusion

Hypoxic vasodilation is augmented in the HE phase of the menstrual and pill cycle in healthy young female participants, independent of whether the hormone is endogenous (NC) or exogenous (OCP). These data further our understanding of the role of estrogen in hypoxic vasodilation. These findings may have clinical implications for the vascular response to hypoxia in conditions of low estrogen (i.e., postmenopause) and/or estrogen excess, and warrant further investigation.
The Effect of a Family History of Hypertension on Left Ventricular Mass When Controlling for Physical Activity Frequency
Authors: Matthews Evan L. , Callaghan Kerry N. , Hosick Peter A. , Brian Michael S.
Published: May 2022

A positive family history of hypertension (+FHH) is associated with non‐exercise induced increases in left ventricular mass (LVM) and high‐normal blood pressure (BP). Regular physical activity (PA) can lead to non‐pathological increases in LVM, but evidence shows adults with elevated BP can decrease LVM with PA.

PURPOSE

To determine if a +FHH is associated with a greater LVM indexed to body surface area (BSA) compared to a negative family history of hypertension (‐FHH) group within a sample of young, mostly active healthy adults.

METHODS

Healthy young (18‐32yrs) participants self‐reported family history of hypertension (FHH) status and habitual moderate and vigorous PA frequency as days per week with at least 10 minutes of recreational activity. Participants then underwent an echocardiogram. Participants were excluded from this retrospective analysis if there was a prior diagnosis of cardiovascular‐related disease or hypertension. Within‐group normality of each variable was tested using the Shapiro‐Wilk test. Simple bivariate comparisons between groups were made using unpaired t‐tests for normally distributed variables and Mann‐Whitney tests when normality was violated. LVM/BSA was logarithmically transformed prior to ANCOVA testing due to non‐normality.

RESULTS

Of the 61 participants, 32 (M=11, W=21; non‐active=8) reported ‐FHH and the remaining 29 (M=13, W=16; non‐active=2) reported a +FHH. While still in the normal range, the +FHH group displayed greater systolic BP (‐FHH 112±12, +FHH 117±12 mmHg, p=0.043), but similar values for all other demographic information when compared to the ‐FHH group (age ‐FHH 23±3, +FHH 23±2 yrs, p=0.116; BSA ‐FHH 1.74±0.20, +FHH 1.74±0.21 m2, p=0.903; diastolic BP ‐FHH 68±6, +FHH 69±8 mmHg, p=0.287; resting heart rate ‐FHH 66±13, +FHH 64±9 bpm, p=0.578; moderate exercise frequency ‐FHH 1.8±1.8, +FHH 2.6±2.1 days/week, p=0.117; vigorous exercise frequency ‐FHH 2.5±2.2, +FHH 3.4±2.0 days/week, p=0.105). The +FHH group had greater LVM (‐FHH 129.5±41.8, +FHH 155.2±42.6 g, p=0.015) and LVM/BSA (‐FHH 73.5±17.4, +FHH 88.4±17.3 g/m2, p=0.004). Separate ANCOVA models accounting for moderate and vigorous PA found that FHH status independently predicted LVM/BSA and PA frequencies were a significant modifier (ANCOVA controlling moderate PA: FHH status p=0.004, partial η2=0.133; moderate PA p=0.020, partial η2=0.089), (ANCOVA controlling vigorous PA: FHH status p=0.004, partial η2=0.132; vigorous PA p=0.007, partial η2=0.117).

CONCLUSION

This preliminary analysis suggests that young adults with a family history of hypertension have elevated left ventricular mass compared to their counterparts without a family history of hypertension. This finding is independent of their habitual moderate and vigorous physical activity patterns.
Rescuing Behavioral Impairment after Doxorubicin Chemotherapy in Rats
Authors: Poudel Bristi
Published: May 2022

Chemotherapy regimens have been extensively used for the treatment of various types of cancers. Despite the effectiveness of this treatment, clinical use of chemotherapeutic agents often produces negative side effects like deficits in cognition and memory, delayed processing speed, and executive function. This condition is widely recognized as chemotherapy‐induced cognitive impairment or “Chemo brain.” Our study aimed to identify the neurobiological mechanisms possibly responsible for this cognitive impairment. We examined the effects of chronic treatment with the chemotherapeutic drug Doxorubicin on cognitive impairment. Doxorubicin is a reactive oxygen species‐producing antineoplastic Anthracycline agent that adversely affects hippocampal neurogenesis, causes neuro‐inflammation, and induces apoptosis. Baseline activity data were collected and then twelve young female Long Evans hooded rats were randomly assigned to receive either Doxorubicin (6mg/kg) or 0.9% saline given intraperitoneally, once weekly for four weeks. After treatment, rats were assessed on the string‐pulling task, the bar walk test, and open field test. Two individuals blind to the treatment calculated the average time duration, number of attempts to pull the string, number of misses, total number of mouth contacts before and after treatment for the string‐pulling task, number of deep slips, slight slips, and the total number of slips for the bar walk test. Similarly, the number of entries to the center, number of entries to the corners, time spent at the center vs corner, and latency to first enter and exit the center was calculated for the open field test and compared between two groups. No significant differences were detected between Dox‐treated and Control rats on any measure, which suggests that the Dox‐treatment produced little or no significant peripheral neuropathy that disrupted performance on the motor tasks we used and did not induce significant anxiety in our rats. Immuno‐histochemical analyses of brain tissues is ongoing to examine markers of neurogenesis and neuro‐inflammation.
Inhibition of Na+/H+ exchanger‐3 (NHE3) results in hyperphosphatemia without major alterations in Ca2+ homeostasis
Authors: Poulsen Søren B. , Murali Sathish K. , Thomas Linto , Rieg Timo , Fenton Robert A.
Published: May 2022

Pharmacological and genetic inhibition of renal Na+/H+ exchanger‐3 (NHE3) may lower blood pressure in hypertensive mouse models, but potential side effects are not well‐characterized. NHE3 is proposed to play a major role in renal Ca2+ reabsorption, which is regulated by the parathyroid hormone (PTH). Furthermore, PTH also regulates phosphate (Pi) balance. Therefore, the aims of this study were to examine the effects of NHE3 inhibition on Pi and Ca2+ homeostasis. Mice with NHE3 deletion in the kidney tubule (NHE3loxloxPax8) were used to assess chronic effects, whereas mice administered the NHE3 inhibitor S3226 were used to examine acute effects. NHE3loxloxPax8 mice had increased plasma Pi and PTH levels that coincided with reduced abundances of the Na+‐Pi cotransporters (NaPi) ‐2a and ‐2c, but urinary Pi excretion was normal. Feeding mice a low Pi diet reduced PTH and increased both NaPi‐2a and ‐2c protein abundances in NHE3loxloxPax8 and control mice, but NaPi‐2c levels remained lower in NHE3loxloxPax8 mice. NHE3loxloxPax8mice were normocalcemic with normal urinary Ca2+ excretion, but had greater furosemide‐induced calciuresis, suggesting increased Na+‐K+‐Cl‐ cotransporter (NKCC2) activity. NHE3loxloxPax8 mice had reduced calciuresis subsequent to a thiazide diuretic. Treatment of wild‐type mice with the NHE3 inhibitor S3226 reduced creatinine clearance, increased plasma Pi and fibroblast growth factor 23 (FGF‐23) and reduced the abundances of NaPi‐2a and ‐2c. Plasma Ca2+ was not significantly altered by S3226 whereas urine Ca2+ excretion was transiently reduced. Collectively, inhibition of NHE3 results in hyperphosphatemia without major alterations in Ca2+ homeostasis. Data suggest that NHE3 inhibitors may constitute significant health risks due to alterations in renal function and Pi homeostasis.
IL‐10 Attenuates ER‐Stress Induced Apoptosis in Doxorubicin Treated Cardiomyocytes
Authors: Malik Akshi , Bagchi Ashim K. , Jassal Davinder S. , Singal Pawan K.
Published: May 2022

Despite its antitumor efficacy, use of Doxorubicin (Dox) is limited by its cardiotoxic side effects. In Dox‐induced cardiomyopathy (DIC), a causal role of oxidative stress (OS) has gained a lot of traction. Among other subcellular changes, Dox causes vacuolization of the cytoplasm and dilation of endoplasmic reticulum (ER). Dox also causes an excessive production of reactive oxygen species as well as imbalance in redox state both of which can affect ER homeostasis and protein folding. An accumulation of unfolded/misfolded proteins triggers the unfolded protein response (UPR) in the ER, which activates transcriptional and translation pathways involving: a) Protein‐kinase like endoplasmic reticulum kinase (PERK); b) activating transcription factor 6α (ATF6α); and c) inositol requiring kinase1α (IRE1α) (Fig.1). Thus leading to the activation of ER chaperons, protein foldases and induce the expression of various genes, including X‐box binding protein 1 (XBP1). UPR plays an important role in the attenuation of protein translation, upregulation of chaperones and antioxidant expression, and thus maintains ER homeostasis. However, if UPR fails to restore ER homeostasis, ER stress‐initiated apoptotic signaling pathways are activated via CCAAT/enhancer homologous protein (CHOP), caspase‐12 activation, phosphorylation of c‐JUN NH2‐terminal kinase (JNK) and pro‐apoptotic gene expression. Understanding the role of these pathways of the ER under Dox treatment will highlight potential targets for new interventions as an adjunct therapy to restore ER functioning.

In this study on isolated rat cardiomyocytes, we analyzed ER‐stress markers, spliced XBP1 mRNA, and ER‐apoptotic proteins. We also assessed protective effects of Interleukin‐10 (IL‐10) against Dox initiated ER‐induced apoptosis and overall ER‐stress. Dox significantly decreased the viability of cardiomyocytes and upregulated ER stress markers (GRP78, GRP94, and PDI). Interestingly, the exogenous administration of IL‐10, one hour prior to the exposure of cardiomyocytes to Dox for 24hrs, was found to be effective in suppressing the expressions of ER‐stress markers as well as ER‐related apoptotic proteins and preserved cell viability. Additionally, significant changes in downstream targets of IRE1α, XBP1 in IL‐10 treatment were recorded. IL‐10 significantly increased the expression of unspliced XBP1 (U), an inhibitor of spliced XBP1(S) particularly in Dox‐treated cells. This was followed by a reduction in the UPR mediated cell death in cardiomyocytes confirmed by a decrease in expression of GRP78, GRP94, and spliced XBP1 (S). IRE1 α arm of UPR initiates phosphorylation of JNK, which was reduced by IL‐10 in Dox treated cells. Dox also initiated activation of procaspase‐12, which is known to activate caspase‐3 and apoptosis. IL‐10 significantly reduced the activation of procaspase‐12 and cell death. These data suggest that IL‐10 provides protection against Dox‐induced cardiotoxicity by restoring ER homeostasis.
Integrin subunit beta 8 contributes to lenvatinib resistance in liver cancer
Authors: Qiu Wei , Hou Wei
Published: May 2022

Lenvatinib is a multi‐kinase inhibitor approved as a first‐line therapy for advanced hepatocellular carcinoma (HCC), a major type of liver cancer. However, the development of drug resistance is common, and the underlying mechanisms are largely unknown. In this study, we established two lenvatinib‐resistant (LR) HCC cell lines and identified integrin subunit beta 8 (ITGB8) as a critical contributor to lenvatinib resistance in HCC. The elevated expression of ITGB8 was observed in LR HCC cells. Furthermore, silencing of ITGB8 reversed lenvatinib resistance in vitro and in vivo, whereas ectopic expression of ITGB8 in lenvatinib‐sensitive parental HCC cells exhibited increased resistance to lenvatinib. Mechanistically, ITGB8 regulated lenvatinib resistance through an AKT‐dependent signaling pathway, in which HSP90 was involved. In supporting this model, either an AKT inhibitor MK‐2206 or an HSP90 inhibitor 17‐AAG re‐sensitized LR HCC cells to lenvatinib treatment. Collectively, our results establish a crucial role and novel mechanism of ITGB8 in lenvatinib resistance, and suggest that targeting the ITGB8/HSP90/AKT axis is a promising therapeutic strategy in HCC patients showing lenvatinib resistance.
Alterations in hepatic albumin phosphorylation in patients with alcohol‐associated hepatitis and cirrhosis
Authors: Hardesty Josiah , Day Le , Warner Jeffrey , Zirnheld Kara , Warner Dennis , Jacobs Jon , McClain Craig , Kirpich Irina
Published: May 2022

Background

Alcohol‐associated liver disease (ALD) encompasses a range of disease states including alcohol‐associated hepatitis (AH) and cirrhosis (AC), two severe clinical manifestations with high mortality due to compromised liver function. One key function of the liver is to synthesize and secrete albumin (ALBU), which plays a critical role in maintaining overall homeostasis. Hypoalbuminemia is a common feature of AH and AC; however, the contributing mechanisms are not established. Recently, S82‐ALBU phosphorylation via FA20A/C was shown to be required for hepatocellular release of ALBU. In this study, we hypothesized that hypoalbuminemia in AH and AC may occur in part due to hepatocellular retention secondary to compromised ALBU phosphorylation.

Methods

Proteomic and phosphoproteomic analyses were conducted using LC/MS/MS on liver biopsy samples from AH patients (n=34), AC patients (n=10), and non‐ALD controls (n=10). Further, AH patients were stratified by MELD score (Model for End‐stage Liver Disease) as follows: AH1 (MELD 17‐20), AH2 (21‐25), AH3 (26‐29), and AH4 (30‐37). Significantly changed proteins were correlated with MELD and compared between non‐surviving and surviving AH patients. One‐way ANOVA was used for comparisons between three groups (parametric), unpaired Student’s t‐test was used for two groups (parametric), and Pearson’s correlation was used for regression analysis. A p<0.05 was considered statistically significant.

Results

Expression of hepatic ALBU was elevated in AH and AC patients relative to non‐ALD controls and was positively associated with MELD in AH patients. Among AH severity groups, liver ALBU was further elevated from AH3 to AH4. We further observed that pS82‐ALBU, and its allosteric regulator FA20A (molecules involved in ALBU release), were reduced in AH and AC relative to non‐ALD controls. This would suggest that one mechanism of increased expression of hepatic ALBU may be due to inhibition of its release. These results for AH were confirmed in an independent cohort.

Conclusion

This study demonstrated that hepatic ALBU levels are elevated in AH and AC, likely due to the loss of pS82 phosphorylation by FA20A/C, compromising hepatocellular ALBU release. Altered ALBU phosphorylation and subsequent release could be a key pathogenic mechanism of hypoalbuminemia in AH and AC. Further studies are warranted to test this hypothesis.
Experiential factors affecting the empathy of students in their pre‐clinical year(s) of 21 universities
Authors: Vigoda Joseph , Adeniyi Adedeji , Tudor Lisl , Brassett Cecilia , McWatt Sean , Sagoo Mandeep G. , Wingate Richard , Chien Chung‐Liang , Traxler Hannes , Waschke Jens , Vielmuth Franziska , Sigmund Anna , Sakurai Takeshi , Yamada Yukari , Zeroual Mina , Olsen Jorgen , El‐Batti Salma , Viranta‐Kovanen Suvi , Keay Kevin , Kitahara Shuji , Martínez‐Abadías Neus , Esteban‐Torné Maria E. , Helms Jill , Sforza Chiarella , Gagliano Nicoletta , Norris Madeleine , Harmon Derek , Yasui Masato , Ichiko Midori , Lee Sammi , Reid Shaina , Lang Ariella , Kunzel Carol , Joseph Michael , Buehler Leo , Hardy Mark , Patel Snehal , Bernd Paulette , Kielstein Heike , Stewart William , Kellett Anne , Wu Anette , Noel Geoffroy
Published: May 2022

Hands‐on cadaveric dissection is often considered an important factor in shaping the emotional identity of medical and dental students as healthcare providers. This study explores how demographic and/or experiential factors affect the empathy of students in their pre‐clinical year(s) of medical or dental school. In the Summer of 2021 and Fall of 2021, a total of 530 students from 21 universities around the world participating in the International Collaboration and Exchange (ICE) Program, completed a validated questionnaire containing the Santa Clara Brief Compassion (SCBC) Scale and the Toronto Empathy Questionnaire (TEQ). Responses to the SCBC and TEQ were tested for variance and covariance against age group, sex, clinical experience, year of health professional school, format of anatomy education, hours of study on prosections and/or hours of hands‐on cadaveric dissection in their respective curricula; and whether their school provides an opportunity for reflection, information about the body donors, a memorial service, and/or addresses empathy in their curricula. Results show that having 40‐90 hours of hands‐on cadaveric dissection vs 0 hours yielded higher SCBC averages (p = 0.0206) and TEQ scores (p = 0.0031); and having 20‐40 hours of hands‐on cadaveric dissection vs 0 hours also resulted in higher TEQ scores (p = 0.0105). Comparisons of hours of study on prosections, format of anatomy education, clinical experience, and year of health professional school yielded no significant results in relation to empathy scores. Our study found that across different regions of the world, curricula emphasizing dissection are best at preparing students to become more empathetic healthcare providers. While none of the other curricular factors proved significant, this study confirms the merit of hands‐on cadaveric dissection in the emotional development of medical and dental students.
Glucocorticoid Hormones Regulate Pendrin Abundance and Subcellular Distribution
Authors: Abood Delaney , Pham Truyen Derek , Mullins Linda , Mullins John , Wall Susan
Published: May 2022

Introduction

Following a low salt diet or aldosterone administration, total and apical plasma membrane pendrin abundance increase through a mechanism thought to involve aldosterone binding to the mineralocorticoid receptor (MR). However, in other cells, aldosterone binding to the MR depends on 11 b Hydroxysteroid Dehydrogenase 2 (11 b HSD2) to oxidize and inactivate glucocorticoids, which are also MR ligands. Although 11 b HSD2 abundance is low or possibly absent in intercalated cells (ICs), it’s abundance might be sufficient to inactivate glucocorticoids, making aldosterone the preferred ligand. If so, 11 b HSD2 gene ablation should reduce pendrin expression. Alternatively, the IC MR might preferentially bind another ligand such as glucocorticoids. To test these possibilities, we examined the effect of 11 b HSD2 gene ablation as well as glucocorticoid administration on pendrin abundance and subcellular distribution.

Methods

11 b HSD2 KO rats and age‐, and sex matched wild type rats on the same background (controls) were given either a diet with a low or high NaCl content for 10 days. Wild type mice were adrenalectomized and given a diet with either zero, normal, or high (i.e. stress level) doses of corticosterone or dexamethasone for 10 days. Pendrin total protein abundance and subcellular distribution were determined in rats and mice from each group by immunoblot and immunohistochemistry.

Results

Both pendrin total protein abundance and pendrin’s relative abundance in the most apical region of the cell were greater in the 11 b HSD2 null than in control rats following either a NaCl‐replete or ‐restricted diet. Moreover, we observed a dose‐dependent increase in both total pendrin abundance and pendrin abundance in the most apical region of the cell following either dexamethasone or corticosterone administration, although the increase appeared greater in mice given corticosterone than dexamethasone.

Conclusions

Glucocorticoids increase pendrin total protein abundance and its relative abundance in the most apical region of the cell. These results might explain, in part, why pendrin is upregulated in the known absence of 11 b HSD2.
Aldosterone Regulates Vasopressin Escape through Changes in Water and Urea Transporters
Authors: Wang Yanhua , LaRocque Lauren M. , Sands Jeff M. , Klein Janet D.
Published: May 2022

Vasopressin escape is a process that prevents the continuous decrease in serum sodium even under conditions of sustained high vasopressin. Previous reports suggest that this process is regulated by aldosterone but the mechanism is not fully defined. In this study, aldosterone synthase (Cyp11b2) knockout and wild‐type mice were implanted with 14‐day osmotic mini‐pumps delivering 5 ng/h dDAVP (desmopressin, type 2 vasopressin receptor agonist) to promote distal nephron water reabsorption. Urine osmolality was measured before and then daily (24‐h urine collections) throughout the experiment. After 4 days of increased urine osmolality, mice were given a gel diet to force additional fluid consumption. Urine osmolality of the wild type mice was significantly lower (successful escape) than that of aldosterone synthase knockout mice. Protein analysis showed that both AQP2 and UT‐A1 protein abundances were higher in the aldosterone synthase knockout than wild‐type mice at the end of the escape period. To confirm the findings from the aldosterone synthase knockout mice, the mineralocorticoid receptor blocker spironolactone (20mg/kg/day, s.c.) or vehicle was given to rats that were receiving vasopressin by mini‐pump in a comparable vasopressin escape protocol to the mice. The spironolactone‐treated rats displayed a higher urine osmolality and lower serum sodium level versus untreated escaping rats. Phosphoserine 256‐AQP2 (PS256‐AQP2) was significantly increased in the spironolactone‐treated rats. Our results indicate that: vasopressin escape is prevented if aldosterone or its receptor are not available; and increased abundance of AQP2 or UT‐A1, or PS256‐AQP2, contributes to deficient vasopressin escape in the absence of aldosterone.
Loss of Biased Signaling Specificity of the Angiotensin II Type 1 Receptor in Overexpressed Systems
Authors: Li Angus , Liu Samuel , Huang Rennica , Ahn Seungkirl , Lefkowitz Robert J.
Published: May 2022

G protein‐coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β‐arrestins. [Sarcosine1Ile4Ile8]‐angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in multiple primary cell lines. Despite this, SII induces phosphorylation of the extracellular signal‐regulated kinases 1 and 2 (ERK1/2) through β‐arrestin‐2‐dependent mechanisms. However, studies reliant on exogenous overexpression of the AT1R indicate that SII is a partial agonist for G protein signaling and lacks β‐arrestin‐exclusive signaling specificity. We investigated this apparent discrepancy by profiling changes in signaling specificity at the AT­­1R with increasing levels of receptor expression. We hypothesized that overexpression of the AT1R causes loss of signaling pathway specificity, such that ligands capable of inducing only β‐arrestin‐mediated responses at lower physiologic receptor expression levels gain the ability to activate G proteins upon receptor overexpression. We established a tetracycline‐inducible cellular system for titratable expression of the AT1R to assess signaling responses at varying levels of receptor expression when stimulated with AngII, SII, and four other AngII analogs with different signaling biases. Unbiased and G protein‐biased ligands activated dose‐dependent calcium responses at all levels of AT1R expression. In contrast, β‐arrestin‐biased ligands induced dose‐dependent calcium signaling only at higher levels of receptor overexpression. Using inhibitors of different G proteins, we demonstrated that both Gi and Gq mediated this overexpression‐dependent calcium signaling by β‐arrestin‐biased ligands. All ligands induced ERK1/2 phosphorylation at receptor levels below those required for calcium signaling by β‐arrestin‐biased ligands. Thus, receptor overexpression causes loss of biased signaling specificity of AT1R ligands, highlighting the potential risks of using such systems to simulate GPCR ligand behavior in physiologically relevant contexts.
Mechanosensitive Responses of Resident Alveolar
Authors: Mthunzi Liberty , Gusarova Galina A. , Islam Mohammad N. , Bhattacharya Jahar
Published: May 2022

Background

The extent of micromechanical coupling between resident alveolar macrophages (AMs) and the surrounding tissue niche remains unclear. Lung overexpansion due to therapeutic mechanical ventilation imposes distortional stresses on the alveolar epithelium (AE), causing acute lung injury (ALI). Sessile AMs, lying adjacent to the AE, are causally implicated in ALI. However, it is not clear whether micromechanical coupling with the AE induces proinflammatory distortional stresses in sessile AMs. We addressed this question in live mouse lungs by real‐time confocal microscopy (RCM).

Methods

For RCM, we inflated and blood‐perfused mouse lungs at physiological pressures. To detect cytosolic Ca2+ (cCa2+), we microinfused fluo‐4 by alveolar micropuncture. To detect sessile AMs, we microinfused fluorophore‐conjugated anti‐Siglec‐F and anti‐CD11c Abs. To induce transient lung overexpansion, we increased the alveolar pressure from 5 to 15 cmH2O for 15 seconds. We determined alveolar diameter in terms of fluo‐4 fluorescence, which marked the epithelial margins. We determined cell diameter of sessile AMs in terms of Siglec‐F fluorescence, which marked the cell boundary. We detected gap junctional communication (GJC) by fluorescence recovery after photobleaching (FRAP).

Results

Sessile AMs were exclusively located in non‐distending microniches of the alveolus and therefore, did not stretch during lung expansion. Nevertheless, a subset of AMs responded to lung overexpansion by transiently increasing cCa2+. The cCa2+ increase initiated in 5 min, reaching peak at two‐times baseline in 20 min, then subsiding to baseline after a further 20 min. Concomitantly, in AE adjacent to AMs, there was marked increase in cCa2+ oscillations, but no increase in mean cCa2+. In lungs with AM‐specific Cx43 deletion, the transient cCa2+ response was blocked in AMs, but not in adjoining AE. FRAP revealed GJC only in cCa2+‐responsive AMs. In paired comparisons, AMs lacking GJC failed to show expansion‐induced Ca2+ responses (p<0.05).

Conclusions

We report the unexpected finding that sessile AMs were exclusively located in immobile microniches of alveoli and therefore did not stretch during lung expansion. Nevertheless, following expansion there was Cx43‐mediated Ca2+communication with the AE, causing cCa2+ increases in sessile AMs. Although further studies are required to evaluate the potentially proinflammatory responses induced by this Ca2+ response, we propose Cx43 of sessile AMs might be a therapeutic target to mitigate lung expansion‐induced ALI.
Giα and β Proteins Associate in a Complex with Cannabinoid Receptor Interacting Protein 1a (CRIP1a)
Authors: Hughes Erin K. , England Andrew C. , Leone‐Kabler Sandra , Lowther W. T. , Howlett Allyn C.
Published: May 2022

Cannabis usage in recent years has increased due to the legalization of both recreational and medical use. The CB1cannabinoid receptor (CB1R) is a G‐protein coupled receptor (GPCR) found in the central nervous system and modulates neuroprogenitor development, neural commitment and migration, and neurotransmitter release. Cannabinoid receptor interacting protein 1a (CRIP1a) is a beta‐barrel protein that interacts with CB1R. Fluorescence polarization studies demonstrated that CRIP1a interacts directly with myristoylated Gαi (Booth et. al. 2021). We hypothesized that this occurs through the intercalation of the myristoyl moiety into the interior of the barrel through a gapped site that is bordered by the C‐terminal β‐strand. We tested this hypothesis by immunoprecipitation of CRIP1a with antibodies developed to two different epitopes of CRIP1a: to the loop‐helix‐loop motif that is located at a distance from the proposed myristoylated peptide access site, and to the C‐terminal β‐strand of CRIP1a which is directly adjacent to this region. We immunoprecipitated CRIP1a from N18TG2 mouse neuroblastoma cell homogenates without the addition of detergent for solubilization. We then probed for CRIP1a and determined if the either the Gαi subunit or the Gβγ dimer were associated with CRIP1a. Immunoprecipitation using the antibody to CRIP1a directed at the loop‐helix‐loop motif revealed complexes with Gai1 and Gai3 and not Gβγ. In contrast, immunoprecipitation of CRIP1a using the antibody directed at the C‐terminal β‐strand co‐immunoprecipitated Gβ and not Gαi1 or Gαi3. Collectively, these data suggest CRIP1a is a cargo‐carrying protein capable of carrying the G‐protein α‐subunit, but also exhibits an interaction with the β‐subunit.
Investigating the interaction of E6/E6AP heterodimer with p53‐DBD by fluorescence resonance energy transfer
Authors: Qu Shanna , Huang Yongqi
Published: May 2022

Human papilloma viruses (HPV) are small DNA viruses, among which the type 16 is the most prevalent and responsible for about 50% of human cervical carcinomas. The E6 oncoprotein of HPV‐16 interacts with numerous host proteins by recognizing their LXXLL motifs. One of the HPV‐16 E6 cellular binding targets is the E3 ubiquitin ligase E6AP. Once binding, the E6/E6AP heterodimer recruits and ubiquitinates p53, leading to subsequent degradation of p53. Importantly, neither E6 nor E6AP interacts with p53 alone. Therefore, blocking the interaction between E6 and E6AP could stabilize p53. Indeed, a LQELL peptide has been found capable to interact with E6 and suppress degradation of p53. Interaction of ligands with p53 may also reduce or inhibit binding of p53 with E6. While the crystal structure of E6/E6AP/p53 has been determined, efficient ligands targeting E6/E6AP/p53 are still missing. In this work, we studied the interactions between E6/E6AP fusion protein and p53 DNA binding domain using fluorescence resonance energy transfer (FRET). E6/E6AP fused to YFP and p53 fused to CFP were expressed from E. coli and purified using Ni affinity chromatography. We characterized the complex stability and obtained a dissociation constant consistent with previously determined by isothermal titration calorimetry. We further obtained the rate constant for dissociation of p53 from E6/E6AP using unlabeled p53 protein. Since the intensity of FRET signal is related to the amount of E6/E6AP/p53 complex, we further investigated the effect of p28, a p53 binding peptide, on the stability of E6/E6AP/p53 complex. Our preliminary results showed that p28 interferes with the binding of p53 to E6/E6AP.
HDAC3 Inhibition Improved Urinary Concentrating Defect in Hypokalemia by Promoting AQP2 Transcription
Authors: Xu Long , Xie Haixia , Hu Shan , Zhao Xiaoduo , Han Mengke , Liu Qiaojuan , Feng Pinning , Wang Weidong , Li Chunling
Published: May 2022

Hypokalemia is a common electrolyte disturbance frequently encountered in clinical medicine. Animals with hypokalemia had a significant arginine vasopressin (AVP)‐resistant polyuria with reduced expression of aquaporin 2 (AQP2) levels in the collecting ducts of the kidneys. The precise molecular mechanisms of hypokalemia‐induced nephrogenic diabetes insipidus remain unresolved. AVP induced a marked increase in histone H3K27 acetylation (H3K27ac) across the body of the AQP2 gene, in the promoter and in upstream of the AQP2 transcriptional start site in mouse mpkCCD cell line (PMID: 29572403). The purpose of the present study was to investigate whether enhanced histone acetylation prevented decreased AQP2 expression and improved urinary concentrating defect in rats with hypokalemia. Male Wistar rats were fed potassium‐free diet with or without 4‐phenylbutyric acid (4‐PBA) or a selective inhibitor of histone deacetylase 3 (HDAC3) RGFP966 for 4 days. 4‐PBA or RGFP966 markedly increased AQP2 mRNA and protein levels in kidney inner medulla of rats with hypokalemia, which was associated with decreased urine output and increased urinary osmolality. Protein expression of H3K27ac was markedly decreased in kidney inner medulla of hypokalemic rats, which was significantly prevented by 4‐PBA or RGFP966. Expression of H3K27ac was decreased in primary cultured inner medullary collecting duct cells (IMCD) or mpkCCD cells cultured in potassium‐deprivation media, in particular, H3K27ac in the promoter region of AQP2 was markedly decreased, which was associated with reduced mRNA levels of AQP2. Protein expression of HDAC3 was significantly upregulated in both mpkCCD and IMCD cells in response to potassium deprivation; the binding of HDAC3 to the promoter of AQP2 was also dramatically increased. RGFP966 significantly increased expression of H3K27ac and AQP2 protein and enhanced the binding of H3K27ac to AQP2 in mpkCCD cells. In conclusion, HDAC3 inhibition prevented downregulation of AQP2 and improved urinary concentrating defect induced by potassium deprivation, likely through enhancing acetylation of H3K27 and transcription of AQP2.
The Impact of Sorghum Polyphenols on DNA Methylation and Signaling Pathways in Colon Cancer
Authors: Kamagate Zeguela , Smolensky Dmitriy , Tsuji Petra
Published: May 2022

Colorectal cancer (CRC) is a major public health issue, accounting for nearly 150,000 cases annually. Because CRC correlates with inflammation and oxidative stress, exploiting the anti‐oxidative and anti‐inflammatory nature of bioactive components in our diet, such as polyphenols, could potentially contribute to the inhibition of cancer initiation. Polyphenols are an immense family of naturally occurring secondary plant metabolites found in fruits, vegetables, and cereal grains, and have been demonstrated across many in vitro and in vivo studies to protect against carcinogenesis. Not only are polyphenols implicated to impact cancer development but they may also be involved in epigenetic regulation. Abnormalities in epigenetic modifications can lead to aberrant regulation of gene expression, and depending on the genes in question, this may contribute to the cell’s ability to acquire hallmarks of cancer. New varietals of the grain Sorghum bicolor provided by the USDA contain high amounts of polyphenols, which may contribute to CRC prevention due to their strong antioxidative and anti‐carcinogenic properties. The expression of genes involved in the promotion or prevention of CRC, like the Wnt/β‐catenin signaling pathway, may be regulated via epigenetic mechanisms. We hypothesize that the high polyphenol sorghum extracts may decrease DNA methylation, thus impacting the expression genes involved in the Wnt/β‐catenin signaling pathway. For our current study, we treated HCT116 and SW480 human colon cancer cells with novel high‐polyphenol S. bicolor extracts (HP and SC) or an extract of low‐polyphenol Macia‐Sorghum for up to 48 hours. Controls included cells treated with solvent or 5 µM epigallocatechin gallate as a benchmark. Total RNA was harvested, and the mRNA expression of selected genes was quantitated via qPCR. Relative to solvent control, the high‐polyphenol S. bicolorextract HP increased mRNA expression of DNA methyltransferase 1 (DNMT1), the key enzyme that upholds methylation patterns after DNA synthesis. Furthermore, mRNA expression of DNMT3A, the enzyme responsible for de novo DNA methylation, was also increased. The SC extract significantly decreased the expression of β‐catenin while increasing expression of transcription factor 4 (TCF4), a gene that mediates the expression of pro‐tumorigenic Wnt target genes such as VEGF and cMYC. Encouraged by these preliminary findings, we will investigate mRNA and protein expression of genes that are involved in epigenetic mechanisms, as well as those of downstream signaling pathways. Our study may help elucidate the question, whether consumption of high‐polyphenol S. bicolor as part of the human diet may lead to beneficial changes in DNA methylation. Subsequently, the expression of specific genes involved in the promotion or prevention of colorectal cancer may be modified, providing a dietary intervention to CRC.
Integrated single cell RNA‐seq and ATAC‐seq of iPSC‐derived kidney organoids reveals the complexity of TGFβ signalling to chromatin during the origin of fibroblasts
Authors: Davis Jessica L. , Kennedy Ciaran , Clerkin Shane , Treacy Niall , Andrews Darrell , Dodd Thomas , Murphy Alison , Moss Catherine , Brazil Derek , Brougham Dermot , Cagney Gerard , Finlay Darren , Crean John
Published: May 2022

Differential methylation patterns and changes in post‐translational histone modifications are observed in patients with or without progressive diabetic kidney disease (DKD). TGFβ1 plays a central regulatory role in the determination of renal cell fate and the progression of DKD. Using ChIP‐seq, we identified an association between SMAD3 and the histone methyltransferase, EZH2, in iPSCs and in iPSC derived nephron progenitors. Using the 10X Genomics platform, we performed single cell RNA‐seq and ‐ATAC‐seq on human iPSC‐derived kidney organoids treated with the EZH2 specific inhibitor, GSK343, prior to treatment with TGFβ1. Integration of single cell RNAseq and ‐ATACseq identified de novo ACTA2+ve/POSTN+ve myofibroblasts in TGFβ1‐treated kidney organoids, characterised by increased SMAD3‐dependent cis chromatin accessibility and the expression of genes associated with fibroblast activation, which are similarly overexpressed in DKD patients. This fibrosis‐associated regulon is characterised by enrichment of SMAD3 and AP‐1, corresponding to FOS/JUN motifs at accessible loci identified by scATACseq. Treatment with GSK343 blocked SMAD3‐dependent cis co‐accessibility and inhibited myofibroblast activation. We propose that that the enzymatic function of the polycomb repressive complex is necessary for TGFβ1 induced increase in chromatin accessibility and its subsequent gene regulatory functions. Manipulation of the association between SMAD3 and EZH2 may be a useful therapeutic strategy for the resolution of renal fibrosis.
Functional Annotation and Cross‐study Comparison of DNA Methylation Regions uniquely associated with 24‐hour Blood Pressure Phenotypes in African Americans
Authors: Roberts Michelle L. , Kotchen Theodore , Pan Xiaoqing , Li Yingchuan , Yang Chun , Liu Pengyuan , Wang Tao , Laud Purushottam , Chelius Thomas H. , Munyura Yannick , Mattson David L. , Liu Yong , Cowley Allen W. , Kidambi Srividya , Liang Mingyu
Published: May 2022

Epigenetics may reflect the interactions between DNA and environment and lifestyle and DNA methylation marks have been implicated in the development and progression of hypertension. In an African American (AA) cohort of hypertensives and normotensives (n = 281), we have recently discovered up to 72 methylation regions (MRs) in whole blood DNA that were significantly associated with 24‐h BP phenotypes (False Discovery Rate (FDR) < 0.05) via reduced representation bisulfite sequencing (RRBS). One‐time clinic BP measurements were not associated with MRs. Upon adjustments for covariates age, sex, and body mass index, 13 MRs remained associated with 24‐h BP phenotypes (FDR < 0.05), each explaining 6.5% ‐ 9.4% of BP variance. Analysis of the chr19 MR in an independent cohort (n=117) using Bisulfite ULtrapLEx Targeted Sequencing (BULLET‐Seq), a new method that we developed, validated its association with 24‐h avg BP phenotypes (FDR < 0.05).

In the current study, we functionally annotated the MRs that we identified as uniquely associated with 24‐h BP phenotypes and examined CpG sites previously reported to be associated with BP. Two MRs were near chr1 linkage disequilibrium (LD) regions within genes, PRDM16 and CASZ1, containing BP‐associated sentinel single nucleotide polymorphisms (SNPs). Although no MRs were found to directly overlap with reported BP‐relevant expression or methylation quantitative trait loci (eQTLs or meQTLs), many overlapped with enhancer regions, CTCF‐binding sites, or genes related to hypertension and stroke. To demonstrate consistency with prior studies, we found associations between reported clinic BP‐associated methylation array chip CpGs and the 24‐h BP phenotypes in our study. Of the 16 BP‐associated CpGs from two reports that were detected in both the methylation chip and our RRBS data, several CpGs were significantly associated with 24‐h and clinic BP phenotypes in our cohort (p < 0.05) but not after adjustment for both covariates and FDR. A chr7 CpG (cg06340364) showed significant negative associations with all studied BP phenotypes except nighttime and clinic DBPs (p < 0.05, unadjusted). These investigations highlight the value of regional DNA methylation and the ability to capture the overall effect of environmental and genetic factors that influence BPs over a 24‐h period which have functional epigenomic relevance.
Modulatory effect of Luteolin on potassium dichromate‐induced nephrotoxicity, cardiotoxicity and genotoxicity via Kim‐1/Nrf2 signaling pathways
Authors: Hassan Fasilat O. , Awoyomi Omolola V. , Oyagbemi Ademola A. , Falayi Olufunke O. , Ogunpolu Blessing O. , Omobowale Temidayo O. , Adedapo Adeolu A.
Published: May 2022

Environmental and occupational exposure to chromium compounds has become a potential aetiologic agent for kidney disease through excessive generation of free radicals, induction of apoptosis and exaggerated inflammatory responses. These are some pathophysiologic mechanisms of potassium dichromate (K2Cr2O7) toxicity with attendant nephrotoxicity and cardiotoxicity. The cardioprotective and nephroprotective effects of Luteolin, a known potent antioxidant were evaluated in this study as described; Group A (normal saline), Groups B (30 mg/kg K2Cr2O7), Group C (Luteolin 100 mg/kg and K2Cr2O7 30 mg/kg), and Group D (Luteolin 200 mg/kg and K2Cr2O7 30 mg/kg), respectively.

Markers of antioxidant defense system, oxidative stress, blood pressure and micronucleated polychromatic erythrocytes (MnPEs) were determined. Immunohistochemistry of Kidney Injury molecule (Kim‐1), nuclear factor erythroid 2‐related factor 2 (Nrf2), and cardiac troponin I (CTnI) were also determined.

Our results showed that administration of K2Cr2O7increased systolic, diastolic and mean arterial blood pressures, markers of oxidative stress, the frequency of micronucleated polychromatic erythrocytes together with a significant reduction in serum nitric oxide bioavailability.

Immunohistochemical staining revealed higher expression of renal Kim‐1 and cardiac troponin I in rats intoxicated with K2Cr2O7. However, lower expression of renal and cardiac Nrf2 were also observed in Luteolin co‐administered rats. Furthermore, co‐treatment with Luteolin restored blood pressure parameters, with concomitant reduction in oxidative stress indicators, augmented in vivo antioxidant status and improved serum NO levels. Lowered expressions of Kim‐1, CTnI and up‐regulation of both cardiac and renal Nrf2, and reduced frequency of micronucleated polychromatic. Taken together, this study demonstrates for the first time the antihypertensive, cardioprotective, nephroprotective and antigenotoxic effects of Luteolin against potassium dichromate induced‐toxicity through antioxidant and radical scavenging mechanisms.