bluuebunny/crossref_metadata_2025 huggingface dataset:
Low‐Dose Methotrexate Alters Cellular Folate Mediated One Carbon Metabolism
Authors: Lai Sou Nga , Wang Yi‐Cheng , Isabel Chiang En‐Pei
Published: November 2023

Impairments in folate‐dependent metabolism are associated with several human pathologies including neural tube defects, cancers, and vascular diseases. Folate mediated one carbon metabolism is an important target for cell proliferation diseases in human including rheumatic diseases. Low‐dose methotrexate (MTX) is one of the most common immunosuppressants used in rheumatic and other inflammatory conditions. MTX is a potent inhibitor of dihydrofolate reductase and thymidylate synthase, which can deplete folates and inhibit DNA synthesis. MTX also interferes with the enzymes methylenetetrahydrofolate reductase and methionine synthase, resulting in reduced cellular 5‐methylTHF levels and decreased homocysteine remethylation. Methionine S‐adenosyltransferase (MAT) catalyzes the only reaction that produces the major methyl donor in mammals. We reported that low‐dose MTX inhibits MAT in vitro and in vivo. The present study was conducted to further investigate how low‐dose MTX alter 1‐carbon metabolic fluxes in vitro and in vivo. Hepatoma and human liver derived cell lines were cultured under folate restriction or in low‐dose MTX with or without folate or methionine supplementation. Male C57BL/6J mice received regimens that reflected low‐dose clinical use of MTX in humans. Metabolic fluxes in 1‐carbon metabolism were investigated using stable isotopic tracers and GCMS. Concurrent folinate supplementation ameliorated MAT2A reduction and restored S‐adenosylmethionine in HepG2 cells. However, posttreatment folinate rescue failed to restore MAT2A reduction or S‐adenosylmethionine level in cells preexposed to MTX. Furthermore, the enrichments from isotopic tracers indicated that MTX inhibits endogenous formate production and alter the partitioning of 1‐carbon flow in our cell models. The impacts of low dose MTX on one carbon metbolic kinetics are under investigation. This study may provide new insights on the regulatory mechanisms by which DMARDs alter cellular one carbon metabolism and thus give potential therapeutic strategies in treating human rheumatic diseases.

Support or Funding Information

supported by NSC102‐2320‐B‐005‐006‐MY3;MOST104‐2320‐B‐005‐010‐MY3;and MOST104‐2911‐I005‐ 301
Long‐term efficacy of a refreshing beverage, fortified with selected micronutrients, to improve the micronutrient status of schoolchildren and women in the context of the nutritional situation in rural Guatemala
Authors: Oliva Raquel Campos , Soto‐Méndez María José , Solomons Noel W. , Armas Laura , Selhub Jacob , Paul Ligi , Kraemer Klaus
Published: November 2023

Background

Nutritive beverages can be important sources of micronutrients. There is a need and an opportunity to determine how beverages can play their most healthful and nutritionally‐relevant role in the diets of rural populations at risk of nutrient deficiencies.

Objective

To compare the evolution of biomarkers of nutritional status with respect to four vitamins (riboflavin, folate, vitamins B12 and D), across two rural samples (schoolchildren and women of fertile age), randomized to receive supervised consumption of a daily serving of a beverage.

Methods

A total of 236 women, and 156 schoolchildren of both sexes in 12 villages. All with informed consent, were enrolled in the baseline sampling of blood for circulating concentrations of riboflavin, folate, vitamins B12 and D in March, 2011. As of May, 2011, 10‐month, household‐level, stratified‐randomization intervention commenced, distributing 350 mL bottles of a fortified or non‐fortified beverage available to participants. Nutritional evaluation was repeated at the endline in March 2012 in the same individuals.

Results

Important differential (baseline‐to‐endline) increases, contrasting between fortification and non‐fortification groups, existed for children with riboflavin (+16.4 vs +5%), vitamin B12 (+33.9 vs +6.5%) and vitamin D (+23.3% vs +5.0%), but not for folate. The corresponding analysis for adult women revealed an effect for folate (+30.4 vs +11.5%), but not for the remaining vitamins. All children were folate adequate at all stages. Differentially greater endline adequacy of biomarker status was seen in the fortification vs non‐fortification groups for riboflavin (25.5% vs 6.5%), vitamin B12 (46.2% vs 6.2%) and vitamin D (41.0 vs 8.6%). For women and folate, the two treatment groups had 92.3 vs 43.7% reaching normal levels at endline. Among the other three vitamins, only up to a third of women had normal status at endline without a treatment‐wise effect.

Conclusion

Increases in circulating biomarkers occurred for most nutrients, variously among adult women and children, but complete resolution of initial low‐vitamin status was not seen for any nutrient.

Support or Funding Information

cbc, Guatemala; DSM Nutritional and Sight & Life, Basel, Switzerland
Impact of Vitamin C supplementation on Risk for Cardiovascular events: future scenario for the elderly US population
Authors: Vecchio Maria Gabriella , Minto Clara , Gregori Dario
Published: November 2023

Background

Micronutrients are an essential component in nutrition, ensuring the maintenance of a good state of health. Nowadays, among the global population, there is a high consumption of food supplementation in particular for what concern micronutrients. Several studies demonstrated that vitamins (i.e Folic acid, Vitamin C, β‐carotene), due to their intrinsic properties, have a proven efficacy toward a series of potential health targets. The aim of this study was to produce an estimation of the public health impact that vitamin supplementations have in terms of cardiovascular events risk reduction on the U.S. elderly population over the next 10 years.

Methods

Relevant articles published until September 2014 were collected using electronic database PubMed. The search string was “vitamins”, limited to “clinical trials or cohort studies” and “humans”. 18091 records were retrieved and screened. Selected papers entered the meta‐analysis for effects of micronutrients. A random‐effect Der‐Simonian Laird model was estimated for the risk of having any cardiovascular event in cases of supplementation (irrespectively of dosage) or no supplementation. Dose‐response curve has been estimated using a over‐dispersed binomial meta‐regression model. Scenarios have been extrapolated to US via stochastic simulations (10000 Monte Carlo runs).

Results

No significant reduction of Cardiovascular Events (CE) risk was found for Folic Acid (OR 0.97, 95% CI 0.91 – 1.02), β‐Carotene (OR 1.01, 95% CI 0.96 – 1.05) and Vitamin C (OR 1.03, 95% CI 0.95 – 1.11), irrespectively from dose. Only for Vitamin C, reduction in risk is significantly associated with dose of supplementation higher than 400 mg/day.

Conclusion

In USA, more than 40 million people are older than 65 years. The figures increased from 2000 to 2010 census by 5276231 people. Assuming a steady growth also in the forthcoming years, a wide adoption of Vitamin C supplementation in adult and elderly population of 400mg/day could be associated in avoiding approximately additional 377670 (95% CI 256450 – 459998) CE in the forthcoming 10 years.

Cardiovascular events risk and vitamin supplementation

Vitamin
Dose (mg/day)
OR for CE
95% CI

Vitamin C
400
0.93
(0.88, 0.99)

β‐Carotene
50
0.28
(0.03, 3.00)

Folic Acid
2000
21.20
(0.01, 60.11)
Iron Status Modulates Circadian Rhythm and Cellular Metabolism in C2C12 Cells
Authors: Soh Traces P. , Fiddler Joanna L. , Wu Lei , Xin Gou , Lin Dingbo , Lucas Edralin A. , Smith Brenda J. , Clarke Stephen L.
Published: November 2023

In mammals iron deficiency (ID) can lead to hyperglycemia and dyslipidemia, though the factors responsible for these changes in glucose and lipid homeostasis remain unclear. Similar changes in glucose and lipid metabolism are also observed in response to abnormalities in circadian rhythm. Cellular oscillations in circadian rhythm are controlled through a network of negative feedback and feed‐forward loops that regulate the transcription of clock‐related and metabolic genes. The nuclear hormone receptors REV‐ERB and ROR are negative and positive regulators for Bmal1, respectively. BMAL1:CLOCK dimers function not only to increase Per and Cry gene expression, but also increase the expression of REV‐ERB and ROR mRNA thereby forming a regulatory circuit to maintain circadian oscillations. REV‐ERBa and REV‐ERBb are heme‐regulated transcription factors, suggesting that alterations in heme synthesis may affect circadian rhythm. In the presence of heme, REV‐ERBs repress transcription of Bmal1, G6pase, and Pepck in the liver and reduced heme levels leads to derepression of these genes. In the skeletal muscle, BMAL1 regulates cellular differentiation and mitochondrial function through the transcription of MyoD and Pgc1α/β. Dietary iron affects circadian signaling through REV‐ERB in the liver, though how iron status impacts REV‐ERB activity in skeletal muscle remains unknown. The aim of the study was to investigate the effect of iron status on the regulation of circadian rhythm and cellular metabolism in C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes and synchronized prior to treatment. Cells were treated with the iron chelator desferroxamine (DFO) or iron in the form of ferric ammonium citrate (FAC). After 16 hr, RNA was extracted for gene expression analyses and cytosolic protein extracts were prepared for measurement of iron regulatory protein (IRP) RNA binding activity. FAC increased Rev‐erbβ and Pgc1β mRNA abundance by 1.5 and 1.7‐fold respectively. In DFO treated cells, the expression of Rorα and Bmal1 mRNA increased by 3.1‐fold and 1.7‐fold, respectively. In contrast, DFO decreased Pgc1α and Pgc1β mRNA abundance by 50%. Both FAC and DFO treatment reduced MyoD mRNA abundance by 40%. IRP‐RNA binding activity increased in DFO treated cells and was reduced over 50% when treated with FAC. These results suggest that REV‐ERBs are sensitive to iron status in C2C12. Additional studies are on‐going to determine the extent to which iron status contributes to altered glucose and lipid metabolism through REV‐ERB‐dependent mechanisms in muscle.
Glucose‐lysine Maillard reaction products fortified with caffeic acid potentiate anti‐oxidant and anti‐inflammatory properties in macrophages
Authors: Kwak Eun Jung , Chung Soonkyu
Published: November 2023

The Maillard reaction (MR) occurs between reducing sugars and amino compounds during thermal processes of food. MR produces an array of MR products (MRPs) that promote food quality (i.e., color and flavor) and may increase anti‐oxidative, anti‐microbial, and anti‐hypertensive activities of food. Caffeic acid is one of the most widely consumed polyphenol abundant in fruits, vegetables, and coffee. It is well known that caffeic acid possesses strong antioxidant activity that may delay or prevent the prevalence of inflammatory diseases. However, no study has been conducted to determine whether MRPs fortified with caffeic acid (caffeic acid‐MRPs) possess higher anti‐oxidative and/or anti‐inflammatory potential than caffeic acid. The aim of this study is to compare anti‐oxidative and/or anti‐inflammatory properties of caffeic acid before and after MR using glucose and lysine model. Browning (A420), UV‐visible spectra, and antioxidant activities of caffeic acid and caffeic acid‐MRPs were evaluated while heating at 100°C for 0~12 h. Caffeic acid alone was unstable during heating compared to other cinnamic acid derivatives of cinnamic or coumaric acids. In contrast, caffeic acid‐MRPs enhanced browning and potentiated antioxidant activities of caffeic acid per se by DPPH and FRAP assay. More importantly, LPS‐mediated proinflammatory gene expression levels of IL‐6, IL‐8, and TNFα were significantly lower in caffeic acid‐MRPs treatment than caffeic acid alone in J774 macrophages. Consistently, caffeic acid‐MRPs incubation markedly attenuated the LPS‐induced ROS production and NF‐kβ activation in J774 cells. Moreover, pre‐incubation of caffeic acid‐MRPs significantly decreased caspase‐1 cleavage and IL‐1b secretion compared to caffeic acid in J774 macrophages. Collectively, these data suggest that caffeic acid‐MRPs not only promote browning but also potentiate anti‐inflammatory and anti‐oxidative properties of food. Fortification of caffeic acid during MR might be a useful approach to improve food qualities as well as nutritional values for food that requires thermal processing.
Effect of thymol on oxidative stress and inflammation‐related gene expression in high fat diet‐induced hyperlipidemic rabbits
Authors: Yu Ya‐Mei , Chao Tzu‐Yu , Chang Weng‐Cheng , Chang Margaret J. , Lee Ming‐Fen
Published: November 2023

Cardiovascular diseases contribute to one major cause of deaths worldwide, where atherosclerosis plays a key role in the development of the diseases and is often associated with oxidative stress and local inflammation. Thymol, a polyphenolic compound in thyme which has been widely used in cooking, food preservation, and folk medicine, exhibits both antioxidant and anti‐inflammatory properties. This study was aimed to investigate the effect of thymol in a high fat diet‐induced hyperlipidemic animal model. New Zealand white rabbits were fed with regular chow, high fat and cholesterol diet (HC), T3 or T6 (HC supplemented with thymol at 3 or 6 mg/kg/day), for 8 weeks. Aortic intimal lipid lesion, serum lipid profiles, inflammation‐related biomarkers including C‐reactive protein (CRP), interleukin‐1 (IL‐1)β, IL‐6, tumor necrosis factor (TNF)‐α, TNF‐β, and atherosclerosis‐associated indicators including vascular cell adhesion molecule‐1 (VCAM‐1), monocyte chemotactic protein‐1 (MCP)‐1, and matrix metalloproteinase (MMP)‐9, were significantly increased in the HC group but lessened upon thymol supplementation. Our data indicate that thymol not only displays strong antioxidant activity but also reduces aortic intimal thickness, lower serum lipids and oxidative stress, and alleviate inflammatory reactions, thereby may suppress the development of high fat diet‐induced hyperlipidemia and atherosclerosis.

Support or Funding Information

This study was supported by NSC102‐2320‐B‐309‐001‐MY3 to M‐F Lee and NSC99‐2320‐B‐309‐002 to Y‐M Yu.
The Effects Of Soluble Dietary Fiber On Blood Pressure: A Systematic Review And Meta Analysis Of Randomized Controlled Trials
Authors: Jovanovski Elena , Khan Kashif , Sievenpiper John , Ho Thanh , Zurbau Andreea , Au‐Young Rodney , Mejia Sonia Blanco , Vuksan Vladimir
Published: November 2023

Objective

Epidemiological evidence suggests an inverse relationship between soluble fiber intake and blood pressure, while evidence from randomized controlled trials remains largely controversial and poorly characterized. The present study aim is to conduct a systematic review and a meta‐analysis of randomly controlled trials to assess the effects of soluble fiber intake on blood pressure.

Study Design

Medline, EMBASE, CINAHL and Cochrane Central Register of Controlled Trials, through to January 2015 were searched. Randomized, controlled, clinical trials that were ≥30 days in duration testing soluble fiber supplementation against an adequate comparator were included. Two independent reviewers extracted the data and assessed study quality and risk of bias. Data were pooled using the generic inverse variance method with random effects model and were expressed as mean differences (MD) with 95% confidence intervals (CI). Study heterogeneity was estimated using Cochran Q‐statistic and quantified I2 statistic. Studies were also assessed for their quality using the Heyland Methodological Quality Score and for risk of bias using Cochrane's risk of bias tool.

Results

Fourteen studies (n=1112 participants) were included for analysis. Soluble fiber had no effect on systolic (MD=1.00 mmHg [95% CI 2.46 to 0.46]) or diastolic (MD=L0.23 mmHg [95% CI 1.17, 0.71]) blood pressures. Substantial and moderate unexplained heterogeneity were present for the systolic (I2=63%, P=0.18) and diastolic (I2=40%, P=0.63) blood pressure analyses.

Conclusion

Pooled analyses show that dietary supplementation of soluble fiber from all sources is not associated with systolic or diastolic blood pressure reduction. More studies will need to be included in future meta analyses to better understand the potential blood pressure reducing effects of soluble fiber, especially in patients with hypertension, and to delineate potential benefits of highly viscous soluble fibers.

Support or Funding Information

Supported by Internal research funding.
Enhancement the Antioxidant Activity, Total Phenolic and Flavonoid Contents by Optimizing the Germination Conditions of Desi Chickpea (Cicer arietinum L) Seeds
Authors: Domínguez Dulce , Cuevas Edith , Milan Jorge , Garzón Jose Antonio , Canizalez Adrian , Gutirerrez Roberto , Rochin Saraid Mora , Reyes Cuauhtemoc
Published: November 2023

Legumes have high nutritional value and play an important role in traditional diets throughout the world. Consumption of legumes has been linked to reduced risk of diabetes and obesity, coronary heart disease, colon cancer, prostate cancer, and gastrointestinal disorders. Some biotechnological processes such as germination are considered both simple and economical to improve the nutritive and functional value of legumes due to the increase in the bioactive compounds and related antioxidant acitivity. Chickpea (Cicer arietinum L.) is one of the most important grain‐legume crops in the world. Chickpeas contain high levels of proteins, carbohydrates, minerals, vitamins, unsaturated fatty as well as bioactive compounds.

The aim of this study was to optimize the germination conditions of desi chickpea seeds that would maximize the antioxidant activity (AoxA), total phenolic (TPC), and flavonoid (TFC) contents. To optimize the germination bioprocess, response surface methodology was applied over three response variables (AoxA, TPC, TFC). A central composite rotable experimental design with two factors [germination temperature (GT), 20–35ºC; germination time (Gt), 10–240 h] in five levels was used; 13 treatments were generated. The sprouts from each treatment were lyophilized, tempered, and ground to obtain germinated chickpea flours (GCF). The best combination of germination bioprocess variables for producing optimized GCF with the highest AoxA [ABTS: 14,435 □mol Trolox equivalents (TE) / 100 g simple, dw; ORAC: 15,143 □mol TE/100 g sample, dw], TPC [217.69 mg Gallic acid equivalents (GAE) / 100 g sample, dw], and TFC [122.61 mg Quercetin equivalents (QE)/100 g sample, dw] was GT= 34ºC/Gt=171 h. The germination bioprocess increased AoxA, TPC, and TFC in 146–178, 98, and 111 %, respectively. The germination is an effective strategy to increase the TPC and TFC of desi chickpea seeds for enhancing functionality with improved antioxidant activity. The chickpea germinated flour or extract might be recommended for use as a source of natural antioxidants in functional foods.

Domínguez‐Arispuro DM1, Cuevas‐Rodríguez EO1,2, Milán‐Carrillo JM1,2, Garzón‐Tiznado JA1, Canizalez‐Roman VA1, Gutiérrez‐Dorado R1,2, Reyes‐Moreno C1,2*

1 Programa Regional de Posgrado en Biotecnología, Facultad de Ciencias Químico Biológicas (FCQB), Universidad Autónoma de Sinaloa (UAS), Ciudad Universitaria, AP 1354, CP 80,000 Culiacán, Sinaloa, México.

2 Programa de Posgrado en Ciencia y Tecnología de Alimentos, FCQB‐UAS, Cd Universitaria, AP 1354, CP 80,000 Culiacán, Sinaloa, México.

(*) C Reyes‐Moreno, Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Calle Lichis Ote 1986, Col La Campiña, CP 80,060 Culiacán, Sinaloa, México, e mail: creyes@uas.edu.mx
Cytotoxic effect of proanthocyanidins from a Mexican endemic blackberry (Rubus liebmannii) on the synthesis of ROS on two murine glioma cell lines
Authors: Cuevas‐Rodríguez Edith‐Oliva , Sanchez‐Velazquez Oscar‐Abel , Rangel‐Lopez Edgar , Saraid Mora‐Rochin , Reyes‐Moreno Cuauhtemoc , Milan‐Carrillo Jorge
Published: November 2023

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion, and it indicates possible cellular damage. In tumor cells the accelerated metabolism produces a high number of ROS. Brain tumors represent the third cause of death in cancer patient's woldwide. Gliomas are the most frequents tumors of the neuroepitelial tissue, and they are graded on a scale from I to IV based on their malignancy. The alternative medicine offers some options of natural origin as possible therapeutic agents against neoplastic cells, such as proanthocyanidins extracted from berries and other fruits and grains. Here, the objective was to determine the cytotoxic effect of proanthocyanidins from a wild blackberry (R. liebmanii) from Mexico on two murine glioma cell lines (C6 and RG2), and determinate the formation of ROS. After 22 and 46 hours of incubation (1 × 105 cells/mL) at 37°C, the viability was determined with two concentrations of proanthocyanidins (0.5 y 100 μg/mL) extracts by 24 and 48 h, respectively, in 96 well plates. ROS concentration was determined with spectrophometric method using 2′,7′‐DCFH‐diacetate (DCFH‐DA). Our results show that the neoplastic cells has a dose dependent cytotoxic response and the major effect was at 48 hours of exposition at 100 μg/mL in RG2 (>80%) vs the control. The results obtained will allow us to suggest suitable compounds for the development of alternative treatment against tumors of glial origin.

Support or Funding Information

PROFAPI2014, Universidad Autonoma de Sinaloa, Mexico
Preparation technology of feruloylated fatty acyl structured lipids and its stabilities in high temperature
Authors: Wang Qiang , Long Jie , Zhang Yubin
Published: November 2023

Ferulic acid has obvious antioxidant, scavenging free radicals, anti infection and neuroprotective effects. However, the poor solubility of ferulic acid, which limits its application in the oil based food, medicine, and cosmetics. Several studies have found that the natural antioxidant composition of the modified structure of lipid can significantly improve the application of natural antioxidant components. To model and optimize the reaction conditions of enzymatic esterification of ferulic acid with maize germ oil in solvent system by response surface metodology (RSM) based commercial lipases Lipozyme RM IM. Central composite design was used to design the experiments. The reaction factors investigated were the substrate molar ratio of maize germ oil and ferulic acid (Sr=4:1~10:1), temperature (T=60~80°C) and time (t=20~30h). The yield of of eruloylated fatty acyl structured lipids was selected as response value. All factors investigated positively affected the yield of of eruloylated fatty acyl structured lipids. The optimal reaction conditions were: Sr =8.9, T=76.0°C and t=28.1h, and yield of of eruloylated fatty acyl structured lipids was 59.1% under these conditions. The actual value was in reasonable agreement with the predicted value of 58.8%. The above prepared structured lipids produces low polymer in the frying experiment, and it has high storage stability. RM IM Lipozyme is an effective and specific technique for the synthesis of ferulic acyl fatty acyl group. It can be used in the produced process of feruloylated fatty acyl structured lipids.

Support or Funding Information

This research was supported by the Natural Science Foundations of China (No. 31401559)
Genome‐Wide Association Analysis Identified Candidate Variants Associated With Responsiveness to a Blood Pressure Reduction Intervention in Rural North Carolina
Authors: Lenhart Kaitlin Christine , Robasky Kimberly , Jones Wendell , Halladay Jacqueline , Ammerman Alice , Patterson Cam , Schisler Jonathan Cummings
Published: November 2023

Lenoir County, North Carolina, is among the poorest in the state with significantly elevated incidences of heart disease, stroke, and obesity relative to state and national levels. We developed the Heart Healthy Lenoir project (an NHLBI‐funded Center for Population Health and Health Disparities) to create long‐term, sustainable approaches to reducing cardiovascular disease risk disparities in Lenoir County, with a focus on controlling hypertension and promoting healthy lifestyle changes. Complementing our clinical interventions, we genotyped 347 study participants utilizing genome‐wide technologies to identify genetic‐based factors that contribute to increased cardiovascular disease (CVD) risk, with a particular focus on hypertension and obesity. Among the 124 Caucasian (CAU) and 193 African American (AA) study participants, we observed a marked reduction in systolic blood pressure (SBP) one year following initiation of the intervention (−6.28 mmHg ± 1.25 and −4.52 mmHg ± 1.16 in CAU and AA groups, respectively). We also observed a modest reduction in body weight (−1.09 kg ± 0.36 and −0.91 kg ± 0.29 in CAU and AA groups, respectively). A workflow was established to identify problematic single nucleotide polymorphisms (SNPs), potential problematic samples, and subsequent optimization of genotyping calls based on the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium that resulted in 585,865 SNPs used in our analyses. We developed a linear model accounting for age, gender and smoking status in the African American cohort: ΔSBP12‐month ~ Age + Gender + Smoking + Gender*Age + Smoked*Age + SNP + SNP*Age. The resulting Q‐Q plot identified a skewed region that we filtered for p‐values < 0.0001 resulting in the identification of 46 loci with low p‐values for both the homozygous SNP term and the interaction term associated with SBP reduction at one year post‐intervention. Interestingly, we found 7 SNPs mapped to 3 separate loci linked to 6 candidate genes (CELF2, SFTA1P, CHST9, AQP4, CPVL, and CHN2) that may be involved in CVD risk and inflammation. It is important to note that our study is limited by cohort size power and further investigation is required to delineate these associations and responsiveness to hypertension interventions, however, our data identified novel genetic loci associated with positive intervention implementation in a rural North Carolinian cohort.
Salt Exacerbates Development of Aortic Dissection and Rupture in LNK/SH2B3 Deficient Mice
Authors: LAROUMANIE Fanny , Saleh Mohamed A. , McMaster William , Kirabo Annet , Madhur Meenakshi s.
Published: November 2023

Background

Inflammation plays a key role in the development of aortic dissection. In particular, the Th1 cytokine IFNγ has been detected in patients with aortic dissection. A polymorphism in the gene SH2B3 encoding the lymphocyte adaptor protein, LNK, is associated with hypertension and other cardiovascular and autoimmune diseases in human. We previously showed that mice deficient in LNK have exacerbated hypertension, enhanced T cell production of IFNγ and increased aortic inflammation. Salt has been shown to modulate T cell polarization. We hypothesized that LNK deficiency promotes aortic dissection/rupture at least in part through enhanced production of IFNγ, and that this is exacerbated by salt.

Methods

LNK−/− mice or wild type (WT) controls were infused with angiotensin II (1000 ng/kg/min) for 14 days with or without an initial intraperitoneal injection of saline equal to 10% of their body weight. Kaplan‐Meier survival curves were generated and flow cytometry, histology, and quantitative RT‐PCR were employed to evaluate aortic inflammation, remodeling, and gene expression.

Results

LNK−/− mice infused with angiotensin II exhibited an accelerated rate of suprarenal aortic dissection or rupture compared to WT mice, and this was exacerbated by salt injection (Figure). Aortas from LNK−/− mice treated with salt plus angiotensin II expressed higher mRNA levels of IFNγ (2.8 fold increase, p<0.05) and the Th1 transcription factor T‐bet (6.2 fold increase, p<0.05) compared to aortas from WT mice that underwent the same treatment. Moreover, the aortic expression of the metalloproteinase MMP‐9 was upregulated compared to WT mice. In mice that developed aortic rupture, loss of LNK was associated with a decrease in the aortic expression of both pro collagens type 1 and 3.

Conclusion

Loss of LNK combined with salt exacerbates the development of aortic dissection and rupture. Targeting LNK and reducing salt intake may be a potential therapeutic strategy for the management of aortic dissections.

Support or Funding Information

The Fondation pour la Recherche Medicale (FRM)
Intronic regulation of human GLI1 DNA by cis DNA elements and epigenetic marks
Authors: Iannaccone Philip , Taylor Robert , Long Jun , Childs Ronnie , Yoon JoonWon , Sylvestersen Kathrine , Nielsen Michael Lund , Walterhouse David O. , Robbins David
Published: November 2023

In humans, a large cancer burden is associated with dysregulation of the Hedgehog/Patched/GLI (HH/PCTH/GLI) pathway. The canonical Hedgehog signal (including Sonic Hedgehog [SHH]) is mediated by the GLI family of transcription factors (GLI1, GLI2 and GLI3). GLI1 plays an important role in normal development and differentiation and is a human oncogene that is believed to be active in up to one‐third of all cancers. GLI1 gene targets sustain proliferation, inhibit apoptosis, and promote angiogenesis and tumor cell migration. Wild‐type p53 competes with GLI1 for the co‐activator TAF9, inhibiting GLI1's oncogenic activity. GLI1 and GLI2 form a positive‐feedback loop that drives their expression. Mechanisms that regulate this positive‐feedback loop remain unknown. We identified 6 GLI binding sites (GBS) in the large first intron of the human GLI1 gene that are highly conserved among mammals and are associated with histone H3K27Ac marks and DNase hypersensitivity clusters. GLI1 binds all of the GBS and GLI2 binds 2 of them, shown using gel mobility shift assays. Elimination of some of the GBS attenuates transcriptional activation. Removal of all GBS eliminates reporter gene activation. Preliminary evidence suggests GLI1 multimerizes and can bind the pioneer protein CBP. ChIP qPCR shows SHH signal dependent (Smoothened agonist) occupancy of the 1st intron near the GBS by GLI2, and increased occupancy by the histones H3K4me3 and H3K27Ac and the histone bromodomain reader protein BRD4. BRD4 can be driven off the intron and transcription start site region with a small inhibitory molecule, I‐BET151, providing a possible route to disrupting GLI1 expression. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 could auto‐regulate GLI1 expression at GBS in the 1st intron of GLI1 through protein‐protein interactions involving complexes of the transcription factors, histone variants, and reader proteins. Given the widespread involvement of GLI1 and GLI2 in human cancers including medulloblastoma and rhabdomyosarcoma, it is important to try and understand key elements of the regulation of their expression.

Support or Funding Information

Supported in part by PHS grant ES1054, Illinois Excellence in Academic Medicine award, Illinois Regenerative Medicine Institute and the Eisenberg Foundation for Charities.
Use of Hepatic Gene Expression Analysis to Investigate Iron Accumulation in Rats Treated with an RGMc Antagonist Monoclonal Antibody
Authors: Liguori Michael J. , Boeser Preethne , Fan Leimin , Huang Lili , Seemann Dietmar , Blomme Eric A.G. , Popp Andreas , Mueller Bernhard K.
Published: November 2023

As the development processes mature for large molecule biologics, additional technologies to assess potential safety issues would be favorable, especially as related to target engagement. ABT‐207 is a humanized antibody that inhibits repulsive guidance molecule C (RGMc). The antibody has possible application to treat anemia of chronic disease. In this study, we used an affinity maturated variant of ABT‐207 (PR‐1285929; h5F9‐AM8) to examine the toxicological effects of RGMc inhibition. We applied hepatic whole genome transcriptomic profiling using the Affymetrix platform to investigate the effects of PR‐1285929 and to examine whether there were toxicologically relevant pathway perturbations. Female Sprague Dawley rats were treated for 3 weeks (i.v.) with PR‐1285929 at doses of 0.02, 0.20, 2.0, or 20.0 mg/kg/week. At the end of the dosing period, the rats were sacrificed, and liver was collected for histology and gene expression analysis (rat RAE230 2.0 array). Histological examination showed mild to moderate dose dependent accumulation of hepatic iron as determined by Prussian blue staining. This effect was histopathologically assessed as non‐adverse since no other effects (e.g. inflammation or degeneration) were observed. Global gene expression profiling revealed a relatively minor impact on the hepatic transcriptome. Levels of hepatic hepcidin (Hamp) mRNA, the main regulator of serum iron homeostasis, declined in excess of 20‐fold in a dose dependent manner. This finding was also reflected in liver Hamp mRNA expression as measured using RT‐PCR and decreased serum hepcidin protein levels. Interestingly, RGMc is a main regulator of hepcidin, and thus, its inhibition is likely associated with the reduced Hamp. At the transcript level, a minor oxidative stress response induction, mostly related to glutathione metabolism, was apparent at the highest dose (20 mg/kg), but lower doses revealed few consistent changes in these genes. Only minor modulations were apparent with respect to select BMPs and ferroportin. No other toxicologically relevant pathways were consistently up‐ or down‐regulated. This experimental example shows the potential utility of transcriptomic profiling to investigate plausible mechanisms of cellular changes mediated by biologic therapies and can serve as a method for risk mitigation in the development of protein pharmaceuticals.

Support or Funding Information

Author Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Regulome‐Seq: Searching for Single Nucleotide Variants (SNVs) Associated with Disease Beyond Protein‐Coding Regions
Authors: Pinsach‐Abuin Mel·lina , Mates Jesus , del Olmo Bernat , Allegue Catarina , Brugada Ramon , Garcia‐Bassets Ivan , Pagans Sara
Published: November 2023

Heart disease is a leading cause of morbidity and mortality in children and adults and it largely depends on genetic factors. Genome‐wide association studies suggest that single nucleotide variants (SNVs) in cis‐regulatory regions increase the susceptibility to cardiac disease. Here we describe the development of an efficient strategy for selective capturing and sequencing of cis‐regulatory regions from human patients (Regulome‐seq) and the application of this strategy for the comprehensive evaluation of cis‐regulatory regions in patients clinically diagnosed with Brugada Syndrome (BrS). BrS is an electrical disease in the heart with high susceptibility to ventricular arrhythmia and sudden cardiac death (SCD). The main genes involved in BrS pathogenesis are SCN5A (which encodes for the cardiac sodium channel and represents 20% of the cases), SCN2B, SCN3B, CACNA1C, CACNA2D1 and CACNB2. SNVs in exonic regions of BrS‐associated genes account for 25–30% of BrS cases. We hypothesize that SNVs at cis‐regulatory regions of BrS‐associated genes could be the cause of a number of BrS cases that currently cannot be explained by exonic SNVs (orphan BrS cases). We identified approximately 1,500 cis‐regulatory regions potentially relevant to BrS by combining information of topological organization in the human genome, chromatin accessibility, histone marks, and binding of transcriptional regulators over six BrS‐associated genes in cardiac cells. Then, we prepared DNA libraries, enriched with the 1,500 cis‐regulatory regions, from genomic DNA of 72 BrS orphan cases using Nextera Rapid Capture (NRC). DNA libraries were sequenced on a HiSeq 2500. After computational integration of the sequencing data, we identified a total of 114,937 variants (>2,500 variants per patient). From those variants identified, 79% represent single nucleotide polymorphisms (SNPs), while 20% represent insertions and deletions (INDELs). The highest percentage (46.15%) of these variants is located within the topological domains of beta subunit genes (SCN2B and SCN3B). Beta subunits are known to modulate the cardiac sodium channel function. In summary, our Regulome‐seq approach gives the possibility to analyze a massive number of human samples at the same time because it simplifies and reduces the cost of the analysis of genetic variants and it could be used for the future diagnosis of patients. Regulome‐seq could potentially provide novel therapeutic targets for pharmacological intervention in the treatment of genetic diseases.

Support or Funding Information

Predoctoral fellowship, Generalitat de Catalunya, Spain (MP).

Marie‐Curie International Reintegration Grant, IRG07‐GA‐2010‐268395 (SP).
Oxime Reactivation Mechanisms for Organophosphate Conjugated Human Acetylcholinesterase
Authors: Hou William C. , Radic Zoran , Sharpless K. Barry , Taylor Palmer
Published: November 2023

Organophosphates (OP) interacting with acetylcholinesterase (AChE) carry narrowly prescribed therapeutic benefits and are effective, biodegradable pesticides when judiciously used. However, certain volatile OP's have been employed insidiously in terrorism. To develop efficacious OP antidotes and scavenging agents, we have adopted a new chemistry with zwitterionic oximes in order for the antidote to cross the blood‐brain barrier and enable reactivation of AChE in the central nervous system. Such compounds also exhibit appropriate oral bioavailability, eliminating parenteral administration in field situations. Studies of oxime catalysis of low molecular weight OP esters show that the oximate is the general base, nucleophile in solution. To investigate whether a similar mechanism occurs in the active center of AChE, we examined the pH dependency of oxime reactivation on the OP conjugated human AChE. Quaternary pyridinium aldoximes, such as pralidoxime (2‐PAM), and a series of zwitterionic oxime species, such as RS 41A and RS194B, were compared at various oxime concentrations to deconstruct the overall kinetic constant for reactivation, kov, into an apparent dissociation constant, Kox and a maximal rate, k2, of reactivation. General base catalysis predicts that, since the anionic oximate is the attacking species, rate constants would be expected to increase with pH, but oximolysis of the conjugated OP in the human AChE shows a broad pH dependence with a peak reactivation rate constant at pH values around 7.5 at both partial and full saturation of the active center. These findings suggest the possibility of transient electric fields and hydrogen bonding allow for proton tunneling of the oxime hydrogen for oximate formation in the active center gorge of AChE.

Support or Funding Information

Supported by USPHS grants GM 18360 and NS 058046.
Directly binding to HNE and up‐regulating α1‐AT mediate the anti‐migration effects of EGCG on neutrophil elastase‐induced migration in A549 cells
Authors: LI Xuejun , Xiaokaiti Yilixiati
Published: November 2023

EGCG has been extensively investigated as a multi‐targeting anti‐tumor and anti‐inflammatory compound. Lung cancer is a complex process that occurs in unregulated inflammatory environment. In this study, we demonstrated a novel mechanism by which EGCG inhibits the neutrophil elastase‐induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1‐antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1‐antitrypsin was up‐regulated after EGCG treatment in neutrophil elastase treated A549 cells. We preliminarily discovered that the EGCG‐mediated induction of α1‐antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase‐induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1‐antitrypsin by regulating the PI3K/AKT pathway.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China (No. 91129727, 81473235 and 81020108031) and the Research Fund from the Ministry of Education of China (111 Project, No. B07001).
Salubrinal inhibits glioma growth and sensitizes malignant gliomas to ER stress‐ and Temozolomide‐induced death
Authors: Li Zhaozhong , Bezalel Spencer , Chen Jin , Belal Cherine , Al'Khafaji Aziz , Ameli Neema J , Baharani Akanksha , Chigurupati Srinivasulu , Mughal Mohamed , Wersto Robert R , Cheng Zixi Jack , Chan Sic L
Published: November 2023

Glioblastoma multiforme represents a particularly difficult‐to‐treat type of cancer with a dismal prognosis. Recent studies showed that human malignant gliomas adapt to their local cellular environments by triggering the endoplasmic reticulum (ER) stress response. This response not only activates critical adaptive pathways for cell survival but also switches to apoptosis when ER stress signaling is prolonged or excessive. Therefore, approaches that either down‐regulate the protective ER stress response or enhance ER stress signaling in malignant gliomas may significantly improve treatment outcome. Here we report that Salubrinal (Sal), an inhibitor of phospho‐eukaryotic translation initiation factor (p‐eIF2α) dephosphorylation, impedes glioma proliferation and invasiveness. The anti‐tumorigenic properties of Sal are associated with impaired receptor‐mediated calcium signaling resulting from the down‐regulation of the ER‐resident calcium release channel protein, inositol triphosphate receptor (IP3R). We also found that Sal elevates ER stress signaling and sensitizes gliomas to apoptosis in the presence of ER stress‐inducing agents. Furthermore, Sal sensitizes gliomas to apoptosis induced by Temozolomide (TMZ), a methylating drug widely used to treat malignant gliomas. In contrast, Sal protected primary astrocytes and neurons against ER stressors suggesting that this differential cell type‐specific response to Sal could be harnessed to selectively sensitize malignant gliomas. Collectively, our findings indicate that Sal should be evaluated alone or in combination, as a safe and effective anti‐cancer therapy.

Support or Funding Information

Florida Center for Brain Tumor Research to Sic L Chan and Zixi (Jack) Cheng
AXL Knockdown For Therapy of Uterine Cancer
Authors: Palisoul Marguerite , Nguyen Mai , Pan Hua , Lohrey Anne , Desai Riva , Powell Matthew , Mutch David , Wickline Samuel , Fuh Katherine
Published: November 2023

Objective

To validate that siAXL‐p5RHH can be a potential therapeutic inhibitor in uterine cancer.

Methods

A novel serum‐stable, cell‐penetrating, endosomolytic amphipathic peptide delivery system (p5RHH) was used for genetic knock‐down of a receptor tyrosine kinase, AXL. p5RHH peptide was dissolved in 10mM in DNase‐, RNAse‐, and protease‐free sterile purified water. The p5RHH siRNA nanoparticles were prepared by mixing equal volumes of the p5RHH peptide and siRNA at a peptide/siRNA ratio of 1:20 in HBSS with Ca2+ and Mg2+. This preparation results in a nanoparticle size of 55nm after applying a stabilizing albumin coating. Total RNA and total cell lysates were recovered from p5RHH‐siAXL nanoparticle treated uterine cancer tumor cells to evaluate for RNA and protein expression knockdown. AXL knockdown by siRNA was compared with either a traditional transfection reagent (DharmaFECT) or with the novel serum‐stable, cell‐penetrating, endosomolytic amphipathic peptide delivery system (p5RHH). Invasion assays were performed using Boyden chambers coated with Matrigel, and migration assays were performed using Boyden chambers.

Results

Western blotting confirmed over‐expression of AXL in uterine cancer cell lines. The novel delivery system, p5RHH, was loaded with siAXL, and shown to inactivate AXL in ARK1 uterine cancer cells at the RNA and protein level to a similar extent as traditional transfection technique (8.4‐fold vs 8.7‐fold decrease for RNA, respectively; 67% vs 83% knockdown at 48h and 84% vs 97% knockdown at 96 hours for protein, respectively). Uterine cancer cells with genetic inactivation of AXL (through shRNA to AXL) showed less invasive cells than those with AXL (shRNA to scrambled Control) of 32 vs 117 cells/high power field (hpf), p=0.0001 as well as less migratory cells (10 vs 35 cells/hpf, p<0.001).

Conclusions

AXL expression is associated with metastatic uterine cancer cell lines. AXL plays a role in uterine cancer invasion and migration. p5RHH‐siAXL is able to inactivate AXL and can be a potential therapeutic inhibitor in uterine cancer.
Organophosphorus Pesticides Promote Cytokine Production in Human Macrophages
Authors: Grodzki Cristina , Fryer Allison , Lein Pamela
Published: November 2023

Macrophages are sentinel immune cells that reside in most if not all tissues and play a critical role in innate immunity. We previously established that exposure to organophosphorus pesticides (OPs) increases airway hyperreactivity in animal models. OPs are neurotoxicants that cause acute cholinergic toxicity via inhibition of the enzyme acetylcholinesterase (AChE); however, our studies demonstrated that the OP parathion causes airway hyperreactivity independent of inhibition of AChE activity. Rather, parathion‐induced airway hyperreactivity is mediated by dysfunction of autoinhibitory muscarinic M2 receptors expressed on the parasympathetic nerves that innervate airway smooth muscle. Recently, we reported that the airway hyperreactivity triggered by parathion requires macrophages and immune cytokines. Here, we test the hypothesis that OPs interact directly with macrophages to stimulate them to express cytokines that modulate neuronal M2 receptors. Using the human THP‐1 monocyte line, we evaluated cytokine expression, NFkB activation through a secreted embryonic alkaline phosphatase (SEAP) reporter expression and nuclear localization of the NFkB p65 subunit and intracellular calcium mobilization in PMA‐differentiated THP‐1 cells exposed to different OPs and their metabolites. Preliminary experiments indicate that in differentiated THP‐1 macrophages, OPs significantly increase the mRNA expression of the pro‐inflammatory cytokines TNFα and IL‐1β. OPs also significantly increased NFkB activation but did not increase intracellular calcium levels. Therefore, our in vitro data strongly indicate that OPs interact directly with macrophages to provoke release of pro‐inflammatory mediators via activation of the NFkB signaling pathway. Ongoing studies are exploring the action of carbamates and pyrethrins on macrophages and further evaluating the mechanism by which these pesticides stimulate macrophages.

Support or Funding Information

This work was supported by funding from the National Institutes of Health grant R01 ES017592.