bluuebunny/crossref_metadata_2025 huggingface dataset:
An Investigation Into Properties of Foam‐Mat‐Dried Spinach Powder and Physical Properties of Spinach Cube
Authors: Emami Maryam Sadat , Mohebbi Mohebbat , Khalilian-Movahhed Mohammad
Published: October 2024

Today, producing fruit or vegetable cubes with high solubility and dispersibility can attract consumers’ attention, diminish agricultural waste, and raise people’s access to important nutritional sources. In recent years, it seems that cube‐making preserves the appearance and marketability of food. In this study, the physicochemical properties of foam‐mat‐dried spinach powders with thicknesses of 3 and 5 mm produced at 40°C, 50°C, 60°C, and 70°C were investigated. The resulting powder was used to produce spinach cubes. The physicochemical properties of the powders were evaluated. With an increase in foam thickness, the water activity, moisture content, dissolution time, particle size, density, and porosity increased, whereas the hygroscopicity and glass transition temperature decreased. The lowest chlorophyll content (0.166 mg/g spinach) was observed in samples dried at 70°C and 5 mm. The samples dried at 70°C and 3 mm had the highest L∗ and higher stability (lower moisture content and water activity and higher Tg). The Heckel equation showed that the highest k value was related to the spinach powder produced at 70°C and 5 mm. The cubes with the greatest heights, the highest chlorophyll content (0.245 mg/g spinach), and the lowest dissolution time were obtained from powders produced at 40°C and 3 mm. The cube of the powder produced at 70°C and 5 mm had the longest dissolution time. All cubes had a non‐Fickian release in both dissolution media. The product of this research is dried spinach powder and its cubes. This product provides easy access to the valuable nutritional compounds of spinach in all conditions. This product is used as an additive in soup. It can also be used with yogurt and smoothies.
Experimental Analysis of Weather Condition Effects on Photovoltaic Systems’ Performance: A Jordan Case Study
Authors: Al-Smairan Mohammad , Odat Alhaj-Saleh , Olimat Melad , Khawaldeh Habes Ali
Published: October 2024

Energy generation and economic development are closely linked, with energy playing a pivotal role in wealth creation. However, the finite nature of fossil fuel resources and associated environmental challenges has emphasized the need for sustainable energy alternatives based on renewable sources. Among these alternatives, photovoltaic (PV) systems hold significant potential for fostering a sustainable energy system. Despite ongoing material research, achieving a major breakthrough in enhancing the conversion efficiency of commercial PV modules remains a challenge. To optimize PV system yield, installation must consider geographic location and design factors. PV modules often yield different results compared to manufacturer specifications due to factors such as high temperatures, dust, and arid to semiarid climates. Therefore, it is crucial to conduct outdoor testing and characterization of PV modules tailored to specific locations, such as Jordan, to maximize system performance. This research aims to examine the impact of ambient temperature, dust accumulation, and solar irradiance intensity on PV system performance in Jordan, providing valuable insights through monitoring power output variations. The findings will contribute to improving PV system efficiency in Jordan’s unique climate and aid manufacturers in developing innovative PV applications. The collected data includes solar radiation, temperature, voltage, current, and output power. The results confirm that the Azraq site is a better location when compared to Mafraq based on the possibility of generated more output power using a PV system.
Effects of Vitamin C on Growth and Non‐Specific Immune Response of Labeo gonius Fry in Density‐Dependent Biofloc Rearing
Authors: Devi Ng Chinglembi , Waikhom Gusheinzed , Singh Soibam Khogen , Das Pronob , Debbarma Sourabh , Debbarma Reshmi , Singh Lukram Sushil , Meinam Martina , Biswas Pradyut , Irungbam Surajkumar
Published: October 2024

Biofloc technology offers a viable choice for the rearing of fish seed by offering a safe and protected habitat for young fish. Nevertheless, it is crucial to establish a standardised stocking density and implement effective ameliorative steps in order to successfully utilise this technology. In this study, a 90‐day investigation was conducted to assess the effect of stocking density and dietary vitamin C (Vit C) levels on the growth and immunity of Labeo gonius fry (1.03 ± 0.01 g) reared in a biofloc system. Three stocking density groups (5, 10 and 15 fish per 50 L) were established, and each experimental group was supplemented with three levels of Vit C (0, 500 and 1000 mg kg−1). The highest survival rate was observed in the lowest density group (five fish per 50 L) fed with 1000 mg kg−1 Vit C. A better feed conversion ratio and significantly higher protein efficiency ratio were found in the moderate stocking density group (10 fish per 50 L) fed with 500 mg kg−1 Vit C. Total leukocyte count (TLC), haemoglobin, and packed cell volume improved in Vit C‐fed groups. The total erythrocyte count (TEC) increased in groups fed Vit C and stocked at lower densities (5 and 10 fish per 50 L). Total serum protein (TPP) content increased when Vit C was added at a rate of 500 mg kg−1. Serum glucose and cortisol levels were significantly reduced in Vit C‐supplemented groups. Supplementation of Vit C at 500 mg kg−1 resulted in a significantly lower value of malondialdehyde (MDA). Thus, the findings confirm that the incorporation of Vit C in the basal diet promotes the growth and health status of L. gonius fry reared in the biofloc system at high‐density rearing.
Supplementary Tables from Mutational Landscape and Patterns of Clonal Evolution in Relapsed Pediatric Acute Lymphoblastic Leukemia
Authors: Waanders Esmé , Gu Zhaohui , Dobson Stephanie M. , Antić Željko , Crawford Jeremy Chase , Ma Xiaotu , Edmonson Michael N. , Payne-Turner Debbie , van de Vorst Maartje , Jongmans Marjolijn C.J. , McGuire Irina , Zhou Xin , Wang Jian , Shi Lei , Pounds Stanley , Pei Deqing , Cheng Cheng , Song Guangchun , Fan Yiping , Shao Ying , Rusch Michael , McCastlain Kelly , Yu Jiangyan , van Boxtel Ruben , Blokzijl Francis , Iacobucci Ilaria , Roberts Kathryn G. , Wen Ji , Wu Gang , Ma Jing , Easton John , Neale Geoffrey , Olsen Scott R. , Nichols Kim E. , Pui Ching-Hon , Zhang Jinghui , Evans William E. , Relling Mary V. , Yang Jun J. , Thomas Paul G. , Dick John E. , Kuiper Roland P. , Mullighan Charles G.
Published: October 2024

Supplementary Tables
Table S4 from Tumor-Intrinsic Activity of Chromobox 2 Remodels the Tumor Microenvironment in High-grade Serous Carcinoma
Authors: Iwanaga Ritsuko , Yamamoto Tomomi M. , Gomez Karina , Nguyen Lily L. , Woodruff Elizabeth R. , Post Miriam D. , Mikeska Railey G. , Danis Etienne , Danhorn Thomas , Boorgula Meher P. , Mitra Siddhartha S. , Marjon Nicole A. , Bitler Benjamin G. , Brubaker Lindsay W.
Published: October 2024

TIMERv2
Table S2 from Tumor-Intrinsic Activity of Chromobox 2 Remodels the Tumor Microenvironment in High-grade Serous Carcinoma
Authors: Iwanaga Ritsuko , Yamamoto Tomomi M. , Gomez Karina , Nguyen Lily L. , Woodruff Elizabeth R. , Post Miriam D. , Mikeska Railey G. , Danis Etienne , Danhorn Thomas , Boorgula Meher P. , Mitra Siddhartha S. , Marjon Nicole A. , Bitler Benjamin G. , Brubaker Lindsay W.
Published: October 2024

Primers
Table S1 from Tumor-Intrinsic Activity of Chromobox 2 Remodels the Tumor Microenvironment in High-grade Serous Carcinoma
Authors: Iwanaga Ritsuko , Yamamoto Tomomi M. , Gomez Karina , Nguyen Lily L. , Woodruff Elizabeth R. , Post Miriam D. , Mikeska Railey G. , Danis Etienne , Danhorn Thomas , Boorgula Meher P. , Mitra Siddhartha S. , Marjon Nicole A. , Bitler Benjamin G. , Brubaker Lindsay W.
Published: October 2024

Antibodies
Figure S3 from Tumor-Intrinsic Activity of Chromobox 2 Remodels the Tumor Microenvironment in High-grade Serous Carcinoma
Authors: Iwanaga Ritsuko , Yamamoto Tomomi M. , Gomez Karina , Nguyen Lily L. , Woodruff Elizabeth R. , Post Miriam D. , Mikeska Railey G. , Danis Etienne , Danhorn Thomas , Boorgula Meher P. , Mitra Siddhartha S. , Marjon Nicole A. , Bitler Benjamin G. , Brubaker Lindsay W.
Published: October 2024

Modulation of CBX2 enhances monocyte infiltration
Is Intravenous and Oral Topotecan in Small-Cell Lung Cancer Truly Equal? A Case Report
Authors: Deraedt Davien , Verfaillie Saartje , Wynants Jokke , Cuppens Kristof
Published: October 2024

Introduction: Treatment with topotecan is standard-of-care therapy for relapsed small-cell lung cancer (SCLC). Both oral and intravenous administrations of topotecan have been extensively researched and are found to be equally effective with less adverse events in the oral group. Case Presentation: We report a case of a patient with SCLC, who had previously received oral topotecan, with radiological stable disease with no changes in tumor or metastasis diameter size after two administrations. Subsequently, this patient received intravenous topotecan instead of oral due to supply difficulties. After one administration of intravenous topotecan, we saw significant disease regression. Conclusion: This is to our knowledge the first reported case of better response of intravenous topotecan than oral topotecan. Multiple extrinsic (e.g., food, medication) factors were investigated but could not deliver an explanation.
Cognitive Disorders in Type 1 Diabetes: Role of Brain Glucose Variation, Insulin Activity, and Glucocorticoid Exposure
Authors: Brossaud Julie , Barat Pascal , Moisan Marie-Pierre
Published: October 2024

Background: The number of patients with type 2 diabetes (T2D) and type 1 diabetes (T1D) is on the rise, partly due to a global increase in new T1D cases among children. Beyond the well-documented microvascular and macrovascular complications, there is now substantial evidence indicating that diabetes also impacts the brain, leading to neuropsychological impairments. The risk of developing neuropsychiatric symptoms is notably higher in childhood due to the ongoing maturation of the brain, which makes it more susceptible to damage. Despite this awareness, the specific effects of diabetes on cognitive function remain poorly understood. Summary: This review synthesizes literature on the impact of diabetes on cognition and its relationship with brain structural changes. It presents data and hypotheses to explain how T1D contributes to cognitive dysfunction, with a particular focus on children and adolescents. The emphasis on the pediatric population is intentional, as young diabetic patients typically have fewer comorbidities, reducing confounding factors and simplifying the investigation of cognitive alterations. Key Message: We examine the roles of hypo- and hyperglycemia, as well as the emerging role of glucocorticoids in the development of neuropsychological disorders. When specific mechanisms related to T1D are available, they are highlighted; otherwise, data and hypotheses applicable to both T1D and T2D are discussed.
Acesso a medicamentos, o Sistema Único de Saúde e as injustiças interseccionais
Authors: Bastos João Luiz , Mujica Elba Marina Miotto , Boing Alexandra Crispim
Published: October 2024

OBJECTIVE: To estimate the prevalence of general and public access to prescription drugs in the Brazilian population aged 15 or older in 2019, and to identify inequities in access, according to intersections of gender, color/race, socioeconomic level, and territory. METHODS: We analyzed data from the 2019 National Health Survey with respondents aged 15 years or older who had been prescribed a medication in a healthcare service in the two weeks prior to the interview (n = 19,819). The outcome variable was access to medicines, subdivided into general access (public, private and mixed), public access (via the Unified Health System - SUS) for those treated by the SUS, and public access (via the SUS) for those not treated by the SUS. The study’s independent variables were used to represent axes of marginalization: gender, color/race, socioeconomic level, and territory. The prevalence of general and public access in the different groups analyzed was calculated and the association of the outcomes with the aforementioned axes was estimated with odds ratios (OR) using logistic regression models. RESULTS: There was a high prevalence of general access (84.9%), when all sources of access were considered, favoring more privileged segments of the population, such as men, white, and those of high socioeconomic status. When only the medicines prescribed in the SUS were considered, there was a low prevalence (30.4% access) that otherwise benefited marginalized population segments, such as women, black, and people from low socioeconomic backgrounds. CONCLUSIONS: Access to medicines through the SUS proves to be an instrument for combating intersectional inequities, lending credence to the idea that the SUS is an efficient public policy for promoting social justice.
Abstract 02: Time restricted feeding reduces kidney fibrosis in diet-induced obesity through limiting kidney CD8+ T cell infiltration
Authors: Edell Claudia , Walker Savannah , Erickson John , Colson Jackson , Molina Patrick , De Miguel Carmen , Pollock Jennifer
Published: October 2024

Obesity is a leading cause of chronic kidney disease (CKD). Our lab previously showed that a 20 week diet-induced obesity (DIO) protocol (45% high fat) significantly increased kidney medullary fibrosis in male mice. Further, we found that time restricted feeding (TRF, feeding during 12-hr dark phase only) during the final 2 weeks of the 20-week DIO protocol significantly reduced kidney fibrosis. DIO mice showed significantly greater CD8+ T cells, compared to mice on a normal diet (ND) only during the dark phase. TRF in the DIO mice normalized the CD8+ T cell to that similar to ND mice. We hypothesized that pro-fibrotic CD8+ T cells infiltrate the kidney and mediate damage in the DIO mice. We used anti (a)-CD8 treatment during the final 2 weeks of DIO to deplete all CD8+ T cells. Separate cohorts of ND and DIO mice received control a-IgG treatment during the final 2 weeks of the protocol. DIO mice treated with a-CD8 showed significantly reduced kidney fibrosis compared to DIO mice treated with a-IgG and similar to ND mice treated with a-IgG (assessed by blue trichrome and picrosirius red staining; one-way ANOVA, p=0.018; NDa-IgG vs DIOa-IgG: p=0.033; DIOa-IgG vs DIOa-CD8: p=0.032). We next investigated the intestinal mucosa (IM) as a source of the kidney-infiltrating CD8+ T cells in DIO mice since the IM is one of the primary non-lymphoid sites of T cells in the body. Previous studies have shown that migration of T cells from the IM to the kidney can drive acute kidney injury, but it is unclear if this occurs with chronic models such as DIO. We used the KikumeGreen (KikGr) mouse model to identify migrating cells into the kidney. When KikGR protein is exposed to 408 nm blue light it is photoconverted to a KikRed. We photoconverted the IM at the end of the ND, DIO, and DIO+TRF protocols. Kidneys were analyzed by flow cytometry at 2.5 days post-photoconversion. We detected significantly higher KikRed+ CD8+ T cells in the kidneys of DIO mice compared to ND mice indicating that CD8+ T cells migrate to the kidney from the IM. DIO+TRF mice showed reduced KikRed+ CD8+ T cells in the kidney compared to DIO mice (one-way ANOVA: p=0.006, ND vs DIO: p=0.008; DIO vs TRF: p=0.047). DIO did not have any significant effect on CD4+ T cell migration (one-way ANOVA: p=0.997). This study indicates that DIO induces migration and infiltration of pro-fibrotic CD8+ T cells from the IM to the kidney, while TRF specifically limits migration and infiltration of CD8+ T cells.
Abstract 04: Renal- and TAL-Specific Inhibition of Traf2 and NCK Interacting Kinase (TNIK) Decreases NKKC2 Phosphorylation and Blunts Salt-Sensitive Hypertension in Dahl Salt-Sensitive Rats
Authors: Granados Jessica , Ortiz Pablo
Published: October 2024

Up to 50% of hypertensive patients are salt-sensitive. Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) in the kidney contributes to the development of salt-sensitivity. NaCl reabsorption in the TAL is entirely mediated by the apical Na/K/2Cl cotransporter NKCC2. We showed that NKCC2 phosphorylation at Thr-96,101 is increased in TALs from Dahl salt-sensitive (DSS) rats. We discovered a new kinase, TNIK (Traf2-and-NCK-interacting kinase), that binds the amino-terminus of NKCC2 and phosphorylates NKCC2 at Thr-96,101. TNIK is abundantly expressed in TALs and other nephron segments. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 phosphorylation in TALs from DSS rats. First, we measured TNIK expression in isolated TALs. TNIK expression was 8±4-fold higher in TALs from DSS compared to SD rats (n=5, p<0.05). Then, we used a pharmacological specific inhibitor of TNIK, NCB-0846. We implanted renal medullary catheters connected to minipumps to directly infuse NCB-0846 or vehicle into the left kidney of uninephrectomized DSS rats. After recovery rats were fed 4% NaCl diet and measured systolic blood pressure (SBP) via tail-cuff. NCB-0846 decreased SBP by 21±6 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs vehicle). To specifically decrease TNIK gene expression in medullary TALs, we used Cas9-mediated gene editing. We injected the renal medulla of the left kidney with AAV-gRNA-TNIK simultaneously with AV-NKCC2promoter-Cas9 (drives Cas9 expression in TALs) or AV-NKCC2promoter-Cas9 alone (AV-control) in DSS rats. After 10 days, rats were fed a 4% NaCl diet and SBP was measured. Rats transduced with AAV-gRNA-TNIK had a 18±5 mmHg lower BP compared to AV-control group (AV-control:147 ± 4.7 vs AAV-gRNA-TNIK:129 ± 5.6 mmHg n=5, p<0.03 vs control) which remained lower after 14 days (AV-control:163 ± 4.8 vs AAV-gRNA-TNIK:140 ± 4.9 mmHg, n=5, p<0.01). After 14 days on high salt, TNIK expression was decreased by 30 ± 4% (n=4, p<0.05), and pThr-96,101-NKCC2 decreased by 43 ± 11% in AAV-gRNA-TNIK transduced TALs compared to AV-control transduced TALs (n=4, p<0.02). We conclude that hypertension and enhanced NKCC2 phosphorylation in the DSS rat is partly due to higher TNIK expression in TALs and that inhibition of this novel kinase could be a target for salt-sensitive hypertension.
Abstract 07: Ambulatory Blood Pressure Variability, Progression of Kidney Disease, and Cardiovascular Outcomes in the Chronic Renal Insufficiency Cohort
Authors: Byfield Rushelle , Cohen Debbie , Townsend Raymond , Zhang Rachel , Hossain Alavi , Shimbo Daichi , Cohen Jordana
Published: October 2024

Background:
Visit-to-visit blood pressure variability is strongly associated with adverse kidney and cardiovascular outcomes in a wide range of patient populations. Patients with chronic kidney disease (CKD) have abnormal circadian blood pressure patterns which may affect 24-hour ambulatory blood pressure variability (ABPV).

Methods:
We used ambulatory blood pressure monitoring (ABPM) data from participants in the Chronic Renal Insufficiency Cohort (CRIC). Blood pressures were obtained every 30 minutes during day and night. We assessed 24-hour, daytime and nighttime systolic ABPV using average real time variability (ARV); the sum of the absolute differences between consecutive BP readings divided by the total number of readings minus one. Cox proportional hazards models were used to evaluate the association of ARV with the development of 50% reduction in eGFR or end stage kidney disease or adverse cardiovascular (CV) events (myocardial infarction, stroke, peripheral artery disease, new atrial fibrillation, and heart failure).

Results:
There were 1502 CRIC participants included in the analysis of whom 44% were female and mean age was 63 years (standard deviation 10). Anthropometric and clinical factors associated with higher tertile of ARV included older age, higher BMI, higher urine protein to creatinine ratio, lower estimated glomerular filtration rate, diabetes, hypertension, dyslipidemia and atherosclerotic heart disease. In unadjusted Cox proportional hazard models, 24-hour systolic ARV was associated with both adverse kidney (Hazard Ratio [HR] 1.16 95% confidence interval [CI] 1.06-1.27) and CV events (HR 1.35 95%CI 1.22-1.50). However, in adjusted models that included clinical and sociodemographic factors, there were no statistically significant associations of 24-hour systolic ARV with either adverse kidney (HR 1.02, 95%CI 0.91-1.14) or CV events (HR 0.93, 95%CI 0.84 -1.04). Similarly, daytime and nighttime ARV were not statistically significantly associated with kidney and CV events in adjusted analyses.

Conclusions:
In a large cohort of patients with CKD, ABPV was not associated with adverse kidney and CV outcomes in adjusted models. Thus, in the context of the many cardiometabolic risk factors present in patients with CKD, ABPV is not an independent mechanism of adverse clinical outcomes in this high-risk population.
Abstract 20: Orthostatic Hypotension in Patients with Heart Failure taking Carvedilol or Metoprolol
Authors: Carranza Leon Daniel , Samarakoon Ryan , Shaffer Lauren Lee , Wells Quinn , Okamoto Luis , Biaggioni Italo
Published: October 2024

Background:
Metoprolol succinate and carvedilol are widely prescribed beta-blockers for the treatment of heart failure (HF). Both can reduce mortality, but unlike the β-1 selective metoprolol, carvedilol has α-1 blocking effects. Since orthostatic hypotension (OH) is a common comorbidity in HF patients, we conducted a case-control study to assess if the vasodilatory effect of carvedilol, compared to metoprolol, is associated with a higher risk for newly diagnosed OH.

Methods:
A deidentified retrospective database of all HF patients seen at the Vanderbilt University Medical Center who were taking metoprolol or carvedilol (n=272,019) was used. Cases of newly diagnosed OH were defined as those who had a new diagnosis of OH after the first mention of either drug (n=3,006) and controls as those who did not have a diagnosis of OH (n=269,013). Demographic information, presence of other comorbidities, and concomitant use of other drugs known to cause hypotension were obtained. One-way analysis of variance (ANOVA) and Pearson’s chi-square tests were used to compare continuous and categorical variables between both groups, respectively.

Results:
OH was diagnosed in 1% of all patients and metoprolol was more commonly prescribed than carvedilol (81% vs. 19%). Compared to controls, OH patients were older (58 vs. 66 yr) and had a higher rate of hypertension (22% vs. 96%), Parkinson disease (1% vs. 3%) and concomitant use of diuretics, calcium channel blockers, other α-1 blockers and other drugs associated with OH (Table). The prevalence of OH cases was higher in the carvedilol group compared to the metoprolol group (1.96% vs. 0.91%), but only age differed between the OH cases in both groups (metoprolol: 64 vs. carvedilol: 71 yr). We found that patients were more likely to be diagnosed with OH after taking carvedilol vs. metoprolol (unadjusted odds ratio 2.2078, 95% CI 2.0448 – 2.3838, p < 0.001).

Conclusions:
The prevalence of OH among patients with heart failure was higher in patients taking carvedilol compared to metoprolol. Given that both drugs have similar beneficial effects on mortality, metoprolol appears to be preferable in the treatment of heart failure in patients at risk of developing OH. Studies to further assess the relationship between heart failure classification and orthostatic hypotension are needed.
Abstract 35: The Dual Endothelin-1 Antagonist Aprocitentan Attenuates Mitochondrial Oxidative Stress in Human Cardiac Fibroblasts
Authors: Varzideh Fahimeh , Jankauskas Stanislovas , Densu Agyapong Esther , Kansakar Urna , Santulli Gaetano
Published: October 2024

Endothelin-1 (ET-1) is a peptide hormone primarily produced by endothelial cells that functions as a potent vasoconstrictor through two receptors, ETA and ETB. While most research on ET-1 has focused on endothelial dysfunction and hypertension, increasing evidence shows that ET-1 is also involved in other physiological processes such as cell proliferation, inflammation, and tissue remodeling. Dysregulation of ET-1 has been implicated in the pathogenesis of various diseases, including heart failure, atherosclerosis, fibrosis, systemic sclerosis, and cancer. Aprocitentan (also known as ACT-132577) is a relatively new dual ET-1 receptor antagonist that prevents ET-1 from binding to both endothelin receptors and has been shown to significantly reduce blood pressure in patients with resistant hypertension. Our hypothesis was that aprocitentan could reduce the proliferation rate and mitochondrial ROS production induced by TGF-β in human cardiac fibroblasts (HCFs). HCFs were isolated from hearts from fully de-identified cadaveric donors and cultured in cardiac fibroblast growth medium. HCFs were treated with aprocitentan (4 μM) and/or TGF-β1 (10 ng/ml) for 72 hours (doses and times identified in preliminary dose-response and time-course assays). Aprocitentan reduced the TGF-β1-induced proliferation rate in HCFs by approximately 23% (
Figure 1A
). Our study also aimed to determine if aprocitentan could mitigate mitochondrial dysfunction. Since the mitochondrion is a major source of reactive oxygen species (ROS), which can promote myofibroblast differentiation, mitochondrial properties in HCFs were assessed using MitoTracker Green and MitoSOX staining. TGF-β1 treatment increased mitochondrial ROS production, which was significantly reduced by aprocitentan (
Figure 1BC
); furthermore, aprocitentan reduced mitochondrial fragmentation induced by TGF-β1 treatment (
Figure 1D
). To the best of our knowledge, we are the first to demonstrate that aprocitentan can reduce the increased production of mitochondrial ROS, mitochondrial fragmentation, and cell proliferation rate induced by TGF-β1 in HCFs. This discovery could pave the way for new research and therapeutic strategies for treating fibrosis in cardiovascular disorders.
Abstract 45: Deletion of a 4-bp noncoding genomic segment orthologous to human rs1882961 results in sex-divergent changes in salt-induced hypertension in Dahl salt-sensitive rats
Authors: Xue Hong , Grzybowski Michael , Pandey Rajan , Liu Yong , Liu Pengyuan , Therani Bhavika , Vanden Avond Mark , Cowley Allen , Geurts Aron , Liang Mingyu
Published: October 2024

Most single nucleotide polymorphisms (SNPs) associated with human blood pressure (BP) are in noncoding regions of the genome and have small effect sizes in the general population. Functional studies in a controlled model system are essential to ascertain the effect of these noncoding loci on blood pressure. Such direct testing is challenging and largely absent, contributing to a critical gap between genetic discoveries and physiological understanding. We reasoned that physiological studies in genetically engineered and phenotypically permissive animal models would help to address this critical gap. We tested the hypothesis by studying the noncoding SNP rs1882961. The T allele of rs1882961, which has a frequency of approximately 30% in the European population, is associated with 0.23 mmHg higher systolic blood pressure in humans. We mapped the human rs1882961 SNP to the rat genome and used CRISPR/Cas9 to delete a 4-bp segment encompassed the rs1882961 orthologous site in the genome of the Dahl salt-sensitive (SS) rat (SS-Δrs1882961). Male SS-Δrs1882961
-/-
rats exhibited significantly higher mean arterial pressure (MAP) and systolic and diastolic blood pressures (SBP and DBP) than wild-type littermates after the rats were fed a high-salt (4% NaCl) diet for 14 days (156.6 ± 5.9 vs 149.3 ± 5.1 mmHg for MAP, 179.4 ± 7.8 vs 171.9 ± 6.0 mmHg for SBP, and 133.2 ± 4.6 vs 125.1 ± 4.5 mmHg for DBP; n=7 for SS-Δrs1882961
-/-
and 9 for wild-type, p<0.05). In contrast, MAP, SBP, DBP, and pulse pressure of female SS-Δrs1882961
-/-
rats were significantly lower than wild-type littermates on a high-salt diet (139.8 ± 1.8 vs 149.5 ± 4.7 mmHg for MAP, 159.3 ± 2.5 vs 172.3 ± 6.0 mmHg for SBP, 116.8 ± 1.5 vs 124.5 ± 3.7 mmHg for DBP, and 42.5 ± 1.6 vs 47.7 ± 2.3 mmHg for pulse pressure; n=8 for SS-Δrs1882961
-/-
and 7 for wild-type, p<0.05). The difference in systolic blood pressure reached more than 10 mmHg in female rats. Urinary albumin and protein levels of SS-Δrs1882961
-/-
rats after 14 days on a high-salt diet tended to be higher in male and lower in female compared with wild-type littermates.

The remarkable effects on BP as well as the sex-divergent changes resulted from a 4-bp noncoding segment deletion around rs1882961 orthologous site indicate that noncoding genomic segment may have profound impact on phenotypes on permissive genomic background and with informative stressors.
Abstract 49: Activator protein 1 (AP-1) Complex in Antigen Presenting Cells contributes to Salt-Sensitive Blood Pressure in Humans
Authors: Ahmad Taseer , Saleem Mohammad , Mutchler Ashley , Ertuglu Lale , Albritton Claude , Haynes Alexandria , Demirci Mert , Khan Mohd , Kirabo Annet
Published: October 2024

Salt-sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure is complex and not fully understood. We previously found that sodium entry into antigen-presenting cells (APCs) via the epithelial sodium channel (ENaC) activates proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. The activator protein 1 (AP-1) transcriptional factors (FOS/JUN) have been implicated in activating the pro-inflammatory pathway, but its role in SSBP is unknown. We hypothesized that high salt activates the AP-1 signaling pathway and inflammation in APCs and contributes to SSBP. Our bulk RNA-sequencing data in human monocytes, demonstrated that high salt increases the expression of the AP-1 gene family, FOS (2,378.1 ± 480.7 vs 6,494.0 ± 945.5, p= 0.0009), and JUN (7,313.9 ± 984.9 vs11,370.0 ± 1,286.3, p= 0.0015) compared to normal salt-treated monocytes. In additional experiments, we enrolled patients with hypertension and phenotype them for SSBP using an established inpatient protocol of salt-loading/depletion and performed single-cell transcriptomic analyses on peripheral blood mononuclear cells (PBMCs). We observed expression of the e FOS (r= 0.5041, p= 0.0464) and JUN (r= 0.7083, p= 0.0021) genes changes in concert with blood pressure in salt-sensitive (SS) but not in salt-resistant (SR) patients. To confirm whether genes expression translates to protein expression, we cultured total splenocytes from salt-sensitive SV/129 mice for 24 hours in either normal (150 mM) or high salt media (190 mM) and performed flow cytometry. Compared to normal salt, high salt-induced a significant increase in the expression of FOS-JUN genes in monocytes of the spleen. We also adoptively transferred PBMCs from salt-sensitive (SS) and salt-resistant (SR) hypertensive individuals into immunodeficient NSG mice, treated with the high salt diet for three weeks and performed flow cytometry on immune cells of the kidney, aorta, and spleen. We found that immune cells from SS people demonstrated a higher propensity to infiltrate mouse tissues with increased expression of FOS-JUN genes than immune cells from SR people. These preliminary findings disclose the role of the AP-1 gene family in salt-sensitive hypertension and may provide a potential therapeutic target for the treatment and diagnosis of SSBP.
Abstract 66: Early-Life Sodium Restriction Programs Salt-Sensitivity of Autonomics and Blood Pressure in Adult C57BL/6J Mice
Authors: Lawton Samuel , Ziegler Alisha , Grobe Connie , Wagner Valerie , Brozoski Daniel , Fekete Eva , Sigmund Curt , Nakagawa Pablo , Grobe Justin , Segar Jeffrey
Published: October 2024

Extremely premature birth is a risk factor for sodium (Na) depletion due to renal immaturity and urine Na losses. We have proposed that Na depletion in preterm infants mechanistically contributes to their increased risk for cardiovascular and metabolic disease observed in adulthood, potentially due to changes during hypothalamic development. In humans, the hypothalamus matures between 20-25 weeks of gestation; in mice, this developmental window occurs between postnatal day (PD) 21 and PD42. Previously, we demonstrated in mice that dietary Na restriction from PD21 to PD42 induced long-lasting increases in energy expenditure via exaggerated autonomic activity. Therefore, we hypothesized that restricting dietary Na from PD21 to PD42 would similarly induce programmed changes in cardiovascular control via changes in autonomic and neurohypophyseal functions. Male C57BL/6J mice were provided a customized low (0.04%, n=7) or supplemented (0.30%, n=8) Na version of the soy protein-free Teklad 2920x diet from PD21 to PD42, after which they were returned to the standard (0.15% Na) 2920x diet. Radiotelemetric blood pressure (BP) transducers were implanted at 7-8 weeks of age. At 10 weeks of age, no differences in BP or heart rate (HR) were noted between groups. All animals were switched to a high (1%) Na diet at 12 weeks of age. At 16 weeks of age, systolic BP was not altered in the 0.30% Na group but significantly increased in the 0.04% Na group compared to the week 10 baseline (24h avg: +6±2 mmHg, p<0.05). Ganglionic block with chlorisondamine or β-adrenergic block with propranolol reduced heart rate (HR) to a greater extent (each p<0.05) in the 0.04% group. Conversely, muscarinic block with atropine resulted in a smaller increase in HR in this group (p<0.05). Urine elimination rates of aldosterone (0.30%: 1.96±0.44 vs 0.04%: 2.92±0.064 ng/d) and copeptin (0.30%: 0.16±0.03 vs. 0.04%: 0.14±0.02 ng/d) were similar between groups. Finally, at 18 weeks of age, all mice received the angiotensin AT
1
R antagonist losartan in drinking water. This caused a greater BP reduction in the 0.04% group at 20 weeks (24h avg: -12±2 vs -19±2 mmHg, p<0.05). Together, these data indicate that dietary Na restriction during hypothalamic development programs increased salt sensitivity of BP via a mechanism involving AT
1
R and characterized by increased sympathetic yet decreased parasympathetic activity.
Abstract 74: The rs1173771 Haplotype Associated with Blood Pressure Influences Chromatin Interactions Involving the Natriuretic Peptide Receptor 3 Gene Promoter
Authors: Liu Yong , Mishra Manoj , Pandey Rajan , Geurts Aron , Liang Mingyu
Published: October 2024

Determining the mechanisms of action for human blood pressure (BP) variants in the noncoding regions of genome, particularly large haplotypes containing multiple variants, is both critical and challenging. Human BP-associated rs1173771 locus includes a haplotype containing 11 single nucleotide polymorphisms (SNPs) spanning 17.4 kbp. The Linkage Disequilibrium (LD) region is approximately 126 kbp from the transcription start site of the closest protein-coding gene
NPR3
(natriuretic peptide receptor 3). We used genome editing and region-capture Micro-C to examine the effect of the rs1173771 haplotype on the expression of neighboring genes and the chromatin mechanisms involved. The BP-elevating and -lowering haplotypes of rs1173771 locus were precisely reconstituted in human induced pluripotent stem cells (iPSCs) using an efficient, two-step genome editing technique. We then differentiated the edited iPSCs to endothelial (iEC) and vascular smooth muscle cells (iVSMC). The BP-elevating haplotype significantly (p ≤ 0.05) decreased
NPR3
expression in iEC and iVSMC. Using region capture Micro-C targeting the rs1173771 haplotype region and the promoter regions of neighboring genes, we found in both iECs and iVSMCs, the BP-elevating rs1173771 haplotype increased chromatin contacts between the rs1173771 haplotype region and NPR3 promoter region, compared to the BP-lowering haplotype. Statistical comparison of chromatin interaction using HiCCompare showed the interactions of a genomic segment overlapping the 5’ end of the rs1173771 haplotype region with the NPR3 promoter region and a region just upstream of the promoter were 10 and 6 fold stronger (p.adj ≤ 0.001) in iECs with homozygous BP-elevating haplotype compared to BP-lowering haplotype. Similarly, in iVSMCs, the interaction of a genomic segment overlapping the rs1173771 haplotype region with a region just upstream of the NPR3 promoter was 4 fold stronger (p.adj = 0.02) for homozygous BP-elevating haplotype compared to BP-lowering haplotype. These results suggest that the BP-elevating rs1173771 haplotype interacts with the NPR3 promoter more strongly to suppress NPR3 expression, possibly through the recruitment of inhibitory protein complexes.
Abstract MP04: Brain miR-410-3p Expression Sex-dependently Reduces Ang-II-induced Hypertension
Authors: Xia Huijing , Tarhriz Vahideh , Lazartigues Eric
Published: October 2024

Dysregulation of microRNA (miRNA) is associated with hypertension and other diseases. We previously observed direct interaction of miR-410-3p with AT
1
R and sex-dependent downregulation of miR-410-3p in the hypothalamus of a mouse cardiometabolic disease (CMD) model, suggesting a possible involvement in the development of CMD through modulation of the renin-angiotensin system. To further investigate the role of hypothalamic miR-410-3p expression as a therapeutic target for hypertension, acute Ang-II injection and chronic infusion experiments were performed. In acute experiments, C57BL/6J mice (males and females) were injected with Lenti-miR-410-3p or a control virus (icv, 2 µl, n=5-6). After 3 weeks, mice were anesthetized and a catheter was inserted to the carotid artery for blood pressure (BP) monitoring, followed by a bolus icv injection of Ang-II (200 ng/200 nl). Changes in systolic BP (ΔSBP) were recorded. In chronic experiments, mice were fed with a high calorie diet for 2 months before icv virus injection (2 µl). Telemetry probes (DSI) were implanted for conscious BP monitoring. Three weeks following virus injection, all mice were infused with Ang-II using osmotic minipumps (sc, 600 ng/kg/min, 14 days) to induce CMD. BP was recorded weekly and 24 h average of mean arterial pressure (MAP) was calculated. Acute Ang-II injection-induced pressor responses were similar in males (ΔSBP; 22.2±4.7 mmHg) and females (20.6±6.5 mmHg) transfected with the control virus. In lenti-miR-410-3p transfected mice, the pressor responses were blunted in both males (6.7±4.8 mmHg,
P
<0.001
vs.
control) and females (10.9±4.7 mmHg, P<0.05
vs.
control), with a greater reduction observed in males (69.8%
vs.
47%). In the HCD model, baseline MAP was not different between male control and Lenti-miR-410, or female control and Lenti-miR-410 group. Following Ang-II infusion, MAP was increased in both sexes treated with control virus. Interestingly, this increase was blunted by Lenti-miR-410-3p expression in males (137.3 ± 9.4
vs.
160.7 ± 4.3 mmHg,
P
=0.05) but not in females (140.4±4.6
vs.
140.5 ± 4.2 mmHg). These data are aligned with our previous findings that male mice had higher expression of hypothalamic AT
1
R than in females in CMD. In summary, miR-410-3p inhibits both acute and chronic Ang-II induced increases in BP in a sex-dependent manner, with more profound effects in males. miR-410-3p could be a new strategy for the treatment of hypertension in CMD.
Abstract MP05: A Preliminary 10X Genomics Xenium Profiler Spatial Transcriptomics Map of Renin-Angiotensin System Genes in the Mouse Subfornical Organ
Authors: Hantke Guixa Ana , Vazirabad Ibrahim , Mathieu Natalia , Muskus Veitia Patricia , Brozoski Daniel , Grobe Justin , Nakagawa Pablo , Sigmund Curt
Published: October 2024

The subfornical organ (SFO) is a brain region rich in angiotensin-II (ANG) AT1 receptors (AT1R) which regulates water and salt intake and communicates with other hypothalamic nuclei to regulate blood pressure (BP). Activation of the classical G protein-mediated AT1R signaling in the brain induces pressor and natri-dipsogenic responses. TRV027, a synthetic AT1R β-arrestin-biased agonist, decreases BP and increases saline avoidance. We examined the spatial transcriptomic makeup of the SFO to balanced or biased AT1R activation using 10X Genomics Xenium. We treated C57BL/6J mice with either aCSF, ANG (0.64 µg/h), or TRV027 (0.56 µg/h) through continuous ICV infusion for 11-days. Water intake was monitored on days 6-11. ANG caused an increase in water intake compared to aCSF or TRV027 groups (7.0±2.0, 3.0±0.2, and 1.9±0.6 mL/day, respectively, n=4, p<0.05). On day 11, brains were perfused, collected, and fixed. Subsequently, 5 µm paraffin-embedded coronal sections were mounted on Xenium slides and tested with the 10X mouse brain set of 247 genes plus 100 custom add-on genes including all RAS genes. A third of the cells expressed AT1R, and the pattern of AT1R-expressing cells Xenium-detected was qualitatively similar to NZ44 mice, which express GFP under the control of the AT1R locus. Expression of the prorenin receptor (
Atp6ap2
) was abundant throughout the SFO. A few cells in the SFO and more prominently in the adjacent blood vessels were angiotensinogen positive. Single cells were identified that expressed the ANG type 2 receptors (AT2R). Expression of Mas receptor was undetectable. Most of the AT1R-expressing cells were excitatory glutamatergic neurons (expressing
Slc17a6
, VGLUT2) but a few were inhibitory GABAergic neurons co-expressing
Slc32a1
(VGAT). Each section had cells classified as astrocytes, neurons, ependymal cells, and immune cells (e.g., microglia or macrophages). Increased numbers of immune cells were detected in the SFO of ANG-treated mice. Interestingly, although there was no evidence for expression of renin in the SFO, renin-expressing cells were identified in the choroid plexus which is consistent with highly sensitive in situ RNA hybridization data. Additional qualitative and quantitative analyses are ongoing to compare gene expression patterns in AT1R-positive cell types. Future studies will identify gene expression patterns in SFO neurons with connectivity to other brain regions controlling water/salt intake and BP.
Abstract MP13: Hemolysis-mediated Prolyl Endo-Peptidase plasma release – a natural protective mechanism against extreme blood pressure elevations?
Authors: Wysocki Jan , Ye Minghao , Forster Peter , Batlle Daniel
Published: October 2024

During hypertensive crises (BP >180/120 mm Hg), turbulent passage of red blood cells through severely constricted blood vessels can cause blood cell fragmentation and intravascular hemolysis. Consistent with this, in mice we found that a rapid and large increase in BP (by ~60-70 mm Hg) induced by an Ang II-infusion (0.2 ug/g BW) resulted in hemolysis. Prolyl Endo-Peptidase (PEP) is an intracellular enzyme that converts Ang II to Ang-(1-7), thereby shifting from a powerful vasopressor peptide to a counter regulatory peptide. The Ang II-induced acute BP increase that caused a visible hemolysis was associated with a ~4-fold increase in PEP activity (Figure, Panel A). A massive hemolysis induced by freeze-thawing of blood (cryo-hemolysis), moreover, was associated with a ~20x increase in PEP activity as compared to native plasma (Panel B). Of note, mouse cryo-hemolyzed plasma (HP)
ex vivo
was able to produce substantially more Ang-(1-7) peptide from spiked Ang II than equivalent amount of non-hemolyzed plasma (P). In addition, when PEP inhibitor, S-17092, was added to cryo-hemolyzed plasma (HP inh), Ang-(1-7) formation from Ang II was greatly diminished (Panel C). This suggests that blood corpuscles acting via intracellular PEP are endowed with a larger capacity than native plasma to catabolize the vasoconstrictor peptide Ang II to the vasodilator peptide Ang-(1-7).

In conclusion, a large and rapid increase in BP after Ang II infusion causes hemolysis which is associated with the release of intracellular PEP enzyme from blood cells. We propose that the release of PEP confers a protective mechanism against severe BP elevations during hypertensive crisis by granting higher plasma Ang II-degrading capability thereby lowering Ang II and BP. This occurs at the expense of temporary hemolysis in the setting of hypertensive crisis as seen with malignant hypertension.
Abstract MP15: Macrophage PPARα is essential to increased immune function induced by angiotensin converting enzyme (ACE).
Authors: Bernstein Kenneth , Saito Suguru , Cao Duo-Yao , Bernstein Ellen , Nishi Erika , Shibata Tomohiro , Khan Zakir
Published: October 2024

Mice with increased macrophage expression of angiotensin converting enzyme (ACE), called ACE 10/10 mice, have a profound increase of macrophage function in response to diverse immune challenges including tumors, infection, atherosclerosis, and Alzheimer’s disease. ACE 10/10 macrophages are highly efficient in oxidative ATP generation, and they express increased amounts of peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor that controls lipid metabolism. To pinpoint the role of PPARα in increasing macrophage immune function, we used LysM-Cre-LoxP system to generate ACE 10/10 mice selectively lacking macrophage PPARα (A10-PPARα-Cre mice). This reduced macrophage fatty acid metabolism, reduced oxidative metabolism, and lowered macrophage intracellular ATP. Functionally, A10-PPARα-Cre macrophage are impaired in both adaptive and innate immunity. A10-PPARα-Cre mice have larger tumor growth of B16-F10 melanoma, less intratumor macrophages, reduced anti-tumor cytokines, and reduced generation of tumor antigen-specific CD8
+
T cells. The mice also have a very attenuated anti-bacterial response to methicillin-resistant
Staphylococcus aureus
(MRSA) infection, with more severe MRSA tissue colonization
in vivo
. In addition, increasing ACE expression or adding a PPARα agonist to the human monocytic cell line THP-1 enhances tumor cell cytotoxicity, phagocytosis of bacteria, and bacterial killing. Thus, we find that elevated macrophage PPARα is absolutely required for the increased immunity of ACE 10/10 mice. This may indicate a pathway to increasing human immunity.