bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of polyphenolic‐rich dark chocolate and almonds on cardiovascular risk factors in overweight and obese adults
Authors: Lee Yujin , Berryman Claire , West Sheila , Chen C.‐Y. Oliver , Blumberg Jeffrey , Preston Amy , Lapsley Karen , Fleming Jennifer , Kris‐Etherton Penny
Published: November 2023

Chocolate is a source of flavanols and has been shown to reduce the risk of cardiovascular disease (CVD) via beneficial effects on vascular health. Almonds are high in unsaturated fat and bioactive compounds that have been shown to favorably affect multiple CVD risk factors. The objective of this study was to evaluate the individual, additive, and synergistic effects of dark chocolate/natural cocoa and almonds on CVD risk factors. Thirty‐one participants [age (mean ± SD): 46 ± 10 y; BMI (in kg/m2): 29.6 ± 2.8; 13 women; 18 men] were randomized to a 4 period crossover, controlled feeding study. All participants consumed each of the 4 diets for 4 weeks. The 4 diets were: Average American diet (AAD: 49% CHO, 17% PRO, 34% FAT, 13% SFA, 276 mg/d CHOL, no almonds or dark chocolate/cocoa), Almond diet (ALD: 49% CHO, 16% PRO, 35% FAT, 8% SFA, 153 mg/d CHOL, 1.5 oz. almonds/d), Chocolate/cocoa diet (CHOC: 53% CHO, 16% PRO, 32% FAT, 11% SFA, 157 mg/d CHOL, 43 g dark chocolate/d, 11 g natural cocoa/d), and Chocolate/cocoa + Almond diet (CHOC + ALD: 50% CHO, 16% PRO, 34% FAT, 8% SFA, 116 mg/d CHOL, 43 g dark chocolate/d, 11 g natural cocoa/d, 1.5 oz. almonds/d). Results from twenty‐six participants are reported; the study will be completed in December, 2015. Compared to baseline, the ALD and CHOC + ALD diets decreased total cholesterol (−15.6 ± 3.1 mg/dL, P < 0.001; −10.2 ± 3.1 mg/dL, P < 0.01, respectively) and LDL‐C (−11.9 ± 2.7 mg/dL, P < 0.001; −9.0 ± 2.7 mg/dL, P < 0.01, respectively). Compared with the AAD diet, the ALD diet demonstrated greater reductions in total cholesterol (−10.6 ± 2.9 mg/dL, P < 0.01) and LDL‐C (−11.3 ± 2.9 mg/dL, P < 0.01). Compared to baseline, CHOC and CHOC + ALD diets increased HDL cholesterol (2.20 ± 0.97 mg/dL, P = 0.03; 2.86 ± 0.97 mg/dL, P < 0.01, respectively). Compared with the AAD diet, the CHOC + ALD had a greater increase in HDL‐C (2.56 ± 0.97 mg/dL, P < 0.05). The ALD, CHOC, and CHOC + ALD diets improved fasting plasma insulin (−1.50 ± 0.58 mU/L, P = 0.01; −1.45 ± 0.58 mU/L, P = 0.02; −1.25 ± 0.58 mU/L, P = 0.04, respectively) and insulin resistance (via the homeostasis model assessment of insulin resistance: −0.38 ± 0.16, P = 0.02; −0.32 ± 0.16, P = 0.06; −0.30 ± 0.16, P = 0.07, respectively). The ALD diet significantly lowered fasting glucose (−1.69 ± 1.28 mg/dL) compared with the CHOC diet (2.12 ± 1.28 mg/dL, P < 0.01) and CHOC + ALD diet (1.50 ± 1.28 mg/dL, P < 0.05). Our results demonstrate that the individual and combined consumption of dark chocolate/cocoa and almonds improved multiple CVD risk factors including lipid profile and insulin resistance. This trial was registered at the ClinicalTrials.gov as NCT01882881.

Support or Funding Information

This study was supported by the Hershey Company and the Almond Board of California
Role of Hypoxia‐Inducible Factor (HIF) on colonic goblet cell differentiation and function during mucosal healing
Authors: Whitney Alyssa K , Schwisow Kalya D , Bayless Amanda J , Golden‐Mason Lucy , Mehta Gaurav , Kuhn Kristine A , Colgan Sean P , Campbell Eric L
Published: November 2023

Secreted mucus is essential for intestinal homeostasis, separating luminal bacteria from the colonic epithelium and preventing inappropriate inflammation. Depletion of the mucus‐secreting goblet cells has been shown in mice to lead to development of spontaneous colitis. Treatment of mice with a prolyl hydroxylase inhibitor (AKB‐4924) leads to stabilization of hypoxia inducible factor‐1 alpha (HIF‐1α) and ultimately regulation of hypoxia‐responsive genes that promote mucosal protection. Interestingly, goblet cell hyperplasia was observed following in vivo administration of AKB‐4924, which may account for the mucosal protection observed. Thus, we hypothesized epithelial stabilization of HIF‐1α leads to goblet cell differentiation and hyperplasia within the GI tract.

Utilizing a three‐dimensional murine intestinal stem cell spheroid culture system, we were able to analyze the influence of HIF‐1α stabilization on epithelial differentiation in a physiologically relevant context. Spheroids were treated with a goblet cell promoting factor (a gamma secretase inhibitor, DBZ), AKB‐4924 or hypoxia and analyzed by qPCR for changes in differentiation markers for secretory goblet cells (i.e. Atoh1, Muc2, and SPDEF) or absorptive enterocytes (i.e. Hes1, ALPI, and Fabp2). Additionally, DBZ and AKB‐4924 treated organoids were visualized using immunofluorescence for changes in the expression of Muc2, the predominant colonic mucin. Contrary to our hypothesis, neither AKB‐4924 nor hypoxia resulted in the induction of goblet cell differentiation factors. Furthermore, mice lacking HIF‐1α in the intestinal epithelium (HIF‐1αfl/fl‐VillinCRE) exhibited normal colonic goblet cell numbers. However, goblet cells in mice lacking epithelial HIF‐1α appeared less organized with mucin granules less uniform in size and more randomly dispersed, and have a significantly altered expression profile of both secreted and adherent mucins, indicating that epithelial HIF‐1α is involved in goblet cell function.

Therefore, we conclude that AKB‐4924 does not act directly on the epithelium to promote epithelial goblet cell differentiation, however, epithelial HIF‐1α does alter goblet cell function, potentially regulating intestinal mucus layer, which may contribute to the mucosal‐protective role for stabilized HIF.

Support or Funding Information

R01 DK103639 from NIDDK, CDA from Crohn's and Colitis Foundation of America to E.L.C.
Pharmacological Inhibition of p38/MAPK Improves Cardiac Function in Cardiac‐specific Bag3‐P209L Transgenic Mice
Authors: Eaton Samuel C. , Takayama Shinuchi , Sidorova Tatiana N. , Murray Katherine T. , Willis Monte S.
Published: November 2023

Bag3 (Bcl‐2‐associated athanogene‐3) is a small heat shock protein expressed predominantly in cardiac and skeletal muscle that regulates proteasomal and autophagosomal protein elimination pathways. Several mutations in Bag3, including a P209L mutation, result in childhood myopathy leading to heart failure. However, the mechanisms by which Bag3 contribute to the pathophysiology of heart failure remain unclear. We recently created cardiac‐specific (alphaMHC) Bag3‐P209L transgenic mice and identified, by conscious echocardiography, a progressive systolic and diastolic heart failure phenotype starting at 8 months of age. We hypothesized that proteotoxicity resulting from misfolded Bag3‐P209L protein contributes to this progressive heart failure phenotype. We first identified increased levels of pre‐amyloid oligomers in Bag3‐P209L mice by immunohistochemistry at one year. Since p38/MAPK signaling is a known regulator of proteotoxicity in neurodegenerative disorders, such as Alzheimer's disease, we next investigated p38 activation in Bag3‐P209L cardiac tissue by immunoblot. At one year, Bag3‐P209L hearts have increased activation of p38 compared to age matched wildtype hearts(p<0.05). To test p38 as a clinically relevant target in Bag3‐P209L heart failure, we treated Bag3‐P209L mice daily with the p38 inhibitor SB203580 (1 mg/kg bw). Conscious echocardiography of treated Bag3‐P209L (n=4) and wildtype (n=4) mice revealed a reversal of systolic heart failure (ejection fraction) by 10 days post‐injection, indicating a role of p38 signaling in Bag3‐P209L cardiomyopathy. Future studies will focus on the biochemical effects of SB203580 treatment to elucidate the therapeutic mechanism of p38 inhibition in Bag3‐P209L mice. This study provides a novel therapeutic target for human Bag3‐mutation related cardiomyopathy where no therapy currently exists.
Serial Femtosecond Crystallography Opens a New Era in Membrane Protein Structural Biology
Authors: Fromme Petra
Published: November 2023

Serial Femtosecond Crystallography (SFX) provides a novel concept for structure determination, where X‐ray diffraction “snapshots” are collected from a fully hydrated stream of nanocrystals, using femtosecond pulses from the world's first high energy X‐ray free‐electron laser, the Linac Coherent Light Source. The large membrane protein complex Photosystem I was used as the model system for the first serial femtosecond crystallography experiments that showed the proof of concept that diffraction of nanocrystals can be observed using femtosecond pulses that are 1012 stronger than 3rd generation synchrotron sources and destroy any material that is placed in its focus [1],[2]. By using femtosecond pulses briefer than the time‐scale of most damage processes, femtosecond nanocrystallography overcomes the problem of X‐ray damage in crystallography [3] and extends to atomic resolution [4],[5]. It is also applied to membrane proteins crystallized in lipidic environments [6],[7], and has recently led to the structure determination of the Angiotensin receptor which regulates the blood sugar level and is one of the major targets for drugs fighting hypertension [8] and the structure of the rhodopsin‐arrestin complex [9]. Femtosecond crystallography also opens a new avenue for determination of protein dynamics. First experiments on the proof of principle for time resolved serial femtosecond nanocrystallography have been performed on Photosystem I‐ferredoxin [10] and Photosystem II nano‐crystals [11] and conformational changes of the Mn4Ca cluster and its protein environment were observed for the first time in the transition from the dark to the double excited state [12]. Very recently we were able to show that TR‐SFX studies extend to atomic resolution using the photoactive yellow protein as a model system [13]. This pioneering work paves the way for the determination of molecular movies of the dynamics of membrane proteins “at work” in the future including the determination of molecular movies of water splitting.
Hunger and satiety responses to saturated fat‐rich meals before and after a high PUFA diet
Authors: Cooper Jamie A , Stevenson Jada L , Paton Chad M
Published: November 2023

Objective

High‐fat meals rich in poly‐unsaturated fats (PUFAs) have been shown to induce greater satiety than meals rich in saturated (SFA) or mono‐unsaturated (MUFA) fats in an acute setting. It remains unknown, however, what the long‐term diet effects of PUFAs are on hunger and satiety.

Purpose

To determine hunger and satiety responses to high SFA test meals before and after a 7‐day high PUFA diet.

Methods

Eighteen, normal weight, sedentary adults (BMI=21.4±2.3kg/m2; age=22.7±4.1y) were randomly assigned to either a PUFA or control diet. Following a 3‐day lead‐in diet, participants reported for the baseline visit where anthropometrics and a fasting blood sample were collected. Participants then consumed two high‐fat meals (breakfast and lunch) that were rich in SFAs (47% of total energy as SFA). Postprandial blood draws were collected approximately every 30 minutes for 4 hour after each HF meal, for a total of 8 hours. Participants then consumed a high PUFA diet (50% carbohydrate, 15% protein, 35% fat, of which 21% of total energy was PUFA,) or control diet (50% carbohydrate, 15% protein, 35% fat, of which 7% of total energy was PUFA) for the next 7 days. All food and drink were provided by research personnel, and diets were designed to meet each participants estimated daily energy needs. Following the 7‐day diet, participants completed the post‐visit which included same procedures as the baseline visit (2 high SFA meals and 8 hours of blood draws). Blood samples were analyzed for ghrelin (hunger hormone) and Peptide YY (PYY), insulin, and leptin (satiety hormones).

Results

For fasting concentrations, the PUFA group showed a significant decrease in ghrelin (1147.1±101.4 vs. 967.0±85.9pg/mL, p<0.05) and significant increase in PYY (89.2±9.3 vs. 113.5±11.8pg/mL, p<0.05) from pre‐to post‐diet. There were no change in fasting insulin or leptin concentrations, or in the control group for any hormone. In response to the high SFA meal challenges, there was greater postprandial PYY after the 7‐day high PUFA diet (postprandial average of 140.8±10.4 vs. 150.6±8.8pg/mL for pre‐ to post‐PUFA diet, respectively; p=0.05). Conversely, there were no significant differences in the postprandial response for ghrelin, insulin, leptin, or VAS measures from pre‐ to post‐diet in either the PUFA or control groups (ns).

Conclusion

A high PUFA diet consumed for seven days favorably altered physiological markers of hunger and satiety in the fasted and fed state. This study is the first to show that the types of fat consumed on a daily basis can alter long‐term appetite hormone profiles.

Support or Funding Information

This study was funded by the California Walnut Commission.
Application of the Evidence Review Cycle Model to Review Food and Health Evidence to Inform Canadian Dietary Guidance Decision‐Making
Authors: Lowell Hélène , Ellis Ann , Colapinto Cynthia K
Published: November 2023

Canada's population‐level dietary guidance policies and education tools are used by health professionals, policy makers and academics to support nutrition policies, programs and research. While Health Canada has always reviewed the evidence base, the growing volume of nutrition related research stimulated Health Canada to develop the Evidence Review Cycle (ERC) for dietary guidance, a model that formalizes the evidence review process. A Health Canada review of reports on food topics in relation to nutrition‐related chronic diseases and conditions will be described to illustrate the implementation of the ERC model in Canada to support decision‐making for dietary guidance policy.

Reports published between 2006 and July 2015 by leading scientific organizations or Federal agencies, as well as health claims assessments from Health Canada, were reviewed. Reports were included if the organization involved an expert committee, systematically reviewed evidence and an evidence grade supported the conclusions. Excluded conclusions were specific to populations less than 2 years of age or pregnant, were not original to the report or were commissioned by industry.

A total of 693 conclusions were retained from 28 reports and 58 of these conclusions were graded as convincing (strong), which included those related to sodium and increased risk of high blood pressure, trans fatty acids and increased risk for cardiovascular disease, and dietary patterns—characterized by higher consumption of vegetables, fruits, whole grains, low‐fat dairy, and seafood, and lower consumption of red and processed meats, and lower intakes of refined grains, and sugar‐sweetened foods and beverages—and positive cardiovascular disease outcomes. Convincing evidence was also found on the relationships for isocaloric replacement of saturated fatty acids and cardiovascular disease outcomes, red and processed meat and colorectal cancer, and soy protein and lowered blood cholesterol.

Food and health relationships are one important input in the broad range of considerations that assist Health Canada in developing population‐level dietary guidance. Guidance is also informed by evidence on stakeholder and Canadians use dietary guidance and by national‐level data on what Canadians are eating.
Effect of Dietary Pulses in a Low Glycemic Index Diet on Renal Function in Participants with Type 2 Diabetes Mellitus
Authors: Mejia Sonia Blanco , Ireland Christopher , Augustin Livia S.A. , Vidgen Edward , Kendall Cyril W.C. , Mitchell Sandra , Sahye‐Pudaruth Sandhya , Chiavaroli Laura , Mirrahimi Arash , Bashyam Balachandran , Coveney Judy , de Souza Russell J. , Sievenpiper John L. , Leiter Lawrence A. , Josse Robert G. , Wolever Thomas M.S. , Vuksan Vladimir V.V. , Pencharz Paul B. , Jenkins David J.A.
Published: November 2023

Background

There is uncertainty in the effects of dietary pulses so far tested. Reducing the glycemic (GI) of the diet using Acarbose, the α‐glucosidase inhibitor, has been shown to be associated with a lower incidence of cardiovascular disease and hypertension in patients with impaired glucose tolerance. Similar dietary maneuvers to lower the glycemic index of the diet using plant protein (e.g. pulses) may be beneficial in people with diabetes.

Objective

To determine the effect of a low GI diet through increase pulse consumption, on renal function in study participants with type 2 diabetes mellitus.

Methods

We conducted a secondary analysis of a 12‐week randomized controlled trial in participants with type 2 diabetes mellitus. The intervention was a low GI diet with emphasis on pulses (LGI‐pulse diet, ~190 g/day) versus a high fiber control diet with emphasis on wheat products (HF‐wheat diet). Markers of renal function (urea, creatinine, albumin, albumin/creatinine ratio, blood urea nitrogen, estimated glomerular filtration rate, creatinine clearance, sodium, phosphorus, and potassium) were assessed in those who completed the study and provided 24h urine collections.

Results

We included 109 participants with type 2 diabetes mellitus who completed the study and provided 24 hr. urine collections, 52 in the LGI‐pulse diet, and 57 in the HF‐wheat control diet. Dietary protein intake (DPI) was not significantly different between diets. The change in urinary urea was positively correlated with the change of DPI (r=0.23, p=0.01) and animal protein (r=0.22, p=0.02), but not with plant protein, No significant changes within and between treatments were seen in markers of renal function. There was a lack of effect seen on markers of renal function despite a significant relation between dietary protein and urinary urea.

Conclusions

Increase in plant protein through increased dietary pulses consumption as part of a low GI diet did not affect renal function in participants with type 2 diabetes mellitus.

Trial Registration

NCT01063361.

Support or Funding Information

This work was supported by ABIP through the PURENet and the Saskatchewan Pulse Growers.
Impact of Folic‐acid Intervention and MTHFRC677T Gene Polymorphism on All‐cause Mortality Associated with Elevated Serum Homocysteine Levels in Chinese Adults with Hypertension
Authors: Xu Benjamin , Kong Xiangyi , Xu Richard , Zhao Min , Song Yun , Zhang Chunyan , Yu Tao , Liu Lishun , Fan Fangfang , Zhang Yan , Qin Xianhui , Tang Genfu , Wang Binyan , Hou Fan Fan , Huo Yong , Li Jianping
Published: November 2023

Background

Mortality is one of the most fundamental metrics of population health. The role of nutrition in reducing mortality is of great public health interest. High levels of homocysteine (HHcy, in part, due to low folate intake) are known to increase the risk of cardiovascular diseases. However, data are limited on the longitudinal association of HHcy levels with all‐cause mortality in low folate regions such as China.

Objective

In a population with relatively low folate intake and high rate of HHcy, we aimed to test the hypothesis that HHcy is associated with increased mortality and folic acid therapy can reduce the risk of all‐cause mortality associated with HHcy. We also examined whether the effect may be affected by individual MTHFR (a key enzyme involved in Hcy metabolism) gene C677T polymorphism.

Methods

We analyzed the data of 20,702 hypertensive adults (age: 59.6 ± 7.5 years) enrolled in the China Stroke Primary Prevention Trial (CSPPT), with measurements for individual baseline serum total Hcy (tHcy) levels and MTHFR C677T genotypes. These participants were randomized to receive 5‐year, double‐blind daily treatment with 10mg enalapril and 0.8mg folic acid (n=10,348) or 10mg enalapril alone (n=10,354). Kaplan‐Meier survival analysis and Cox proportionate hazard model were used to estimate the effect of tHcy levels on all‐cause mortality and to test effect modification by folic acid intervention and MTHFR C677T genotype.

Results

There were 622 (3%) all‐cause deaths during a mean treatment period of 4.5 years. There was a dose‐response relationship between tHcy quartiles and all‐cause mortality (P for trend<0.001) after adjusting for pertinent covariables. This relationship was stronger among participants with CC or CT genotypes than among those with TT genotype. Compared to the enalapril alone group, enalapril plus folic acid therapy further lowered the risk of all‐cause mortality associated with increased tHcy levels. The risk reduction was more pronounced in participants with CC or CT genotypes.

Conclusions

In this large‐scale study of population residing in a low folate region, we found that baseline tHcy levels were associated with all‐cause mortality in a dose‐response fashion; and folic acid therapy can lower the all‐cause mortality risk associated with HHcy. Our data suggest that tHcy and MTHFR genotypes may serve as biomarkers for risk stratification and folic acid therapy may offer a simple, inexpensive and safe intervention strategy to reduce mortality among population characterized by low folate intake and high rate of HHcy.
ATF3 as an Important Factor of the Acute Phase Lung Inflammatory Response in an Animal Model
Authors: Caruso Carla Romina , Cabello Noe , Sinha Utkarshna , Ekpa Ndifreke , DiAngelo Susan , Chroneos Zissis , Silveyra Patricia
Published: November 2023

Background

Ground level ozone, an oxidative photochemical air pollutant, is highly corrosive and once inhaled triggers acute lung inflammation, leading to damage of bronchioles and alveoli, and compromising the innate pulmonary immunity. Our previous research has indicated that the expression of lung pro‐inflammatory chemokines and cytokines, such as Interleukin‐6 (IL‐6), are increased after ozone exposure. Since the activating transcription factor 3 (ATF3) is a known regulator of IL‐6 in other models, in this project we sought to determine whether ATF3, via IL‐6, affects and/or modify the lung acute inflammatory response in an experimental animal model.

Methods

Male wild type (WT, n=6) and ATF3 knockout (ATF3‐KO, n=5) mice were exposed to 2ppm ozone or filtered air (FA, control) for 3 hours. Lung tissue was collected 4 hours after exposure, and bronchoalveolar lavage fluid (BAL) was obtained 24h post exposure. Total RNA form lung tissue was extracted and retro‐transcribed to measure mRNA levels of IL‐6 by Real Time PCR. Changes in cell counts were evaluated by BAL cytospin analysis, and the expression of the lung injury marker lipocalin‐2 was determined by ELISA in BAL.

Results

The expression of IL‐6 mRNA was significantly increased by ozone in both WT and KO mice (p<0.05). The lungs of ATF3‐KO mice expressed higher levels of IL‐6 than WT mice, when exposed to FA (10‐fold) or ozone (4‐fold). Moreover, in response to ozone, ATF3‐KO mice expressed significantly higher IL‐6 levels than WT mice (4‐fold, p<0.05). In the BAL, we found an increase in lipocalin‐2 expression in response to ozone, as well as a highly elevated neutrophilic count in ATF3‐KO mice exposed to both FA (23% of total BAL cells) and ozone (40% of total cells) vs. the WT groups (0.2% in FA, 6% in ozone).

Conclusion

Our preliminary results indicate that ATF3 is an important factor that mediates the lung inflammatory response to acute ozone exposure. This is reflected by the increased levels of acute inflammatory phase cytokines (IL‐6) and cells (neutrophils). Future studies will evaluate the specific mechanisms involved in this response, as well as potential sex differences when including female mice.

Support or Funding Information

BIRCWH K12HD055882
Genistein alleviates testicular ischemia and reperfusion injury‐induced spermatogenic damage and oxidative stress by suppressing abnormal testicular matrix metalloproteinase system.
Authors: Al‐Maghrebi May
Published: November 2023

The aim of the study is to examine the role of Matrix metalloproteinases (MMPs) and their inhibitors (TIMP) during testicular ischemia/reperfusion (t I/R), the involvement of the Notch pathway, and their modulation by the antioxidant, Genistein. Three groups of male Sprague– Dawley rats were used: sham rats, tI/R rats, and genistein‐treated rats (10mg/Kg). The t I/R rat model underwent testicular artery occlusion of the left testis and was subjected to 60 min ischemia followed by 4 h reperfusion. Protein expression of MMP‐2, MMP‐9, TIMP‐1, and TIMP‐2 were measured in testicular tissue. Histological examination was performed to assess spermatogenesis. Protein levels of Notch 2, Jagged 1, and hairy/enhancer of split 1 (hes‐1) was quantified. The degree of testicular oxidative stress, DNA damage and germ cell apoptosis were also evaluated. t I/R induced severe tubular damage, a significant increase in MMP‐2 and MMP‐9 expression and decreased expression TIMP‐1 and TIMP‐2. Genistein treatment normalized the MMP‐TIMP imbalance. Rats subjected to t I/R had low total antioxidant capacity of the testis, decreased superoxide dismutase activity, and increased oxidative DNA damage. Enhanced activities of caspase 8, caspase 3 and PARP were also observed during t I/R. Genistein suppressed the t I/R‐induced activity of the Notch 2/Jagged 1/hes‐1 pathway. Genistein was also able to salvage the testicular structure and function through restoring the MMP‐TIMP anti‐proteolytic balance, suppressing spermatogenic damage, alleviating oxidative stress and apoptosis. The Notch pathway is partly involved in inhibiting the t I/R‐induced testicular impairment.

Support or Funding Information

This study was supported by Kuwait University Research Grant SRU02/13.
Amphiregulin Promotes Fibroblast Activation in Pulmonary Fibrosis
Authors: Liu Tianju , De Los Santos Francina Gonzalez , Ding Lin , Wu Zhe , Phan Sem H
Published: November 2023

Amphiregulin (AREG), an epidermal growth factor receptor ligand, is implicated in tissue repair and fibrosis by recent studies. Group 2 innate lymphoid cells derived AREG is reported to be important for the epithelial integrity, and involved in repair of airway epithelium following injury. However AREG is essential for pulmonary fibrosis induced by TGFβ overexpression, and its deficiency significantly impairs bleomycin‐induced pulmonary fibrosis. AREG is significantly induced in BLM‐induced fibrotic lung tissues primarily in bone marrow‐derived CD11c+ cells that play a significant role in promoting lung fibrosis. Moreover recombinant AREG stimulates mouse lung fibroblast proliferation accompanied by the induction of telomerase reverse transcriptase (TERT) expression. To further elucidate the possible mechanism by which AREG activates fibroblasts during the development of pulmonary fibrosis, its direct role in fibroblast proliferation and motility was investigated. The results showed that AREG significantly increased human lung fibroblast proliferation. The response to AREG was significantly enhanced in the telomerase expressing lung fibroblasts from an IPF patient compared to telomerase negative cells from control subjects. To assess the importance of induced TERT expression in the AREG‐induced cell proliferation, the effect of TERT overexpression was investigated in the human BJ fibroblast cell line. In response to AREG treatment, BJ5ta cells with stably transfected hTERT exhibited significantly greater proliferation relative to control BJ cells. Conversely, significant reduction of AREG‐induced cell proliferation was noted in TERT deficient fibroblasts. Thus induction of TERT by AREG significantly enhanced fibroblast proliferation. Moreover AREG caused significantly increased lung fibroblast motility in an in vitro scratch assay, which was associated with increased expression of α6 integrin. Finally lung α6 integrin mRNA was highly induced in bleomycin‐induced pulmonary fibrosis, which was significantly diminished in AREG deficient mice. Thus AREG‐induced cell motility might be associated with the induction of α6 integrin expression. Taken together, these findings indicated that AREG‐induced fibroblast activation, motility and invasiveness may contribute to the increased number of fibroblasts in pulmonary fibrosis.

Support or Funding Information

This study is supported by NIH grants HL052285, HL091775 and HL112880.
Epigenetic signatures of alcohol abuse in hepatocellular carcinoma
Authors: Puszyk William Matthew , Hlady Ryan , Robertson Keith , Cabrera Roniel , Liu Chen
Published: November 2023

There is a paucity of human in vitro models available to study the effects of alcohol in hepatocellular carcinoma (HCC). HCC cell lines are often deficient in either alcohol dehydrogenase (ADH) or cytochrome P450 2E1 (CYP2E1). VL‐17a is currently the most widely studied model available. VL‐17a is derived from a HepG2 cell line engineered to overexpress mouse ADH and human CYP2E1. LH86 is a novel HCC cell line previously developed in our laboratory.

Here we compare the LH86 cell line with the current model to identify a novel tool to study the effects of alcohol in HCC.

The aim of this study is to evaluate the LH86 cell line as a novel human in vitro model to study the effects of alcohol in HCC. The cell lines were cultured with alcohol and gene expression and epigenomic changes were identified.

The LH86 cell line was observed to have unique functional properties indicating that it is a potential novel tool to study the effects of alcohol in HCC.

Support or Funding Information

R01 AA19976 TO KR AND CL
Androstenetriol enhances lung macrophage host defense against bacterial pneumonia in mice
Authors: Kobzik Lester , Yang Zhiping , Lu Peng , Mo Fengfeng , Loria Roger
Published: November 2023

The active metabolite of dihydroepiandrosterone, 17beta‐androstenetriol (AET), has potent immunomodulatory effects but its potential to improve host defense against the common problem of bacterial pneumonia has not been studied. Mice (C57BL/6, male) were pre‐treated with AET (0.5 mg, s.c.) or vehicle before infection with Strep. pneumoniae (serotype 3, 100K CFU, i.n.). Evaluation showed 8‐fold improved bacterial clearance at 24 h (p <.001, n>20/group) accompanied by decreased inflammation (5‐fold fewer neutrophils in lung lavage samples at 24 h, p < .001). Secondary bacterial pneumonia after influenza remains a major clinical problem. AET treatment of mice on day 7 after non‐lethal influenza infection produced resulted in 15‐fold improved bacterial clearance (p <.001, n >5/group). Survival analysis showed 37% survival in AET‐treated vs. 0% survival in vehicle‐treated mice in this model of secondary pneumococcal pneumonia. In vitro, AET improved macrophage killing of bacteria in vitro (e.g. % bacteria alive after ingestion, vehicle vs AET‐treated: 54 ± 8 vs. 36 ± 7, n>5). To explore mechanisms, we analyzed the role of MARCO, a macrophage scavenger receptor with important functions in host defense against bacteria. Western blot analysis showed that in vitro AET increased MARCO expression in both murine and human macrophages. Comparison of bacterial clearance after vehicle or AET pre‐treatment showed marked improvement in wild‐type mice, but no benefit in MARCO −/− mice. We conclude that AET enhances innate resistance to primary and secondary pneumococcal pneumonia, in part through increased expression of the scavenger receptor MARCO, and is a potential immunomodulator for improving lung host defense against bacterial pneumonia.

Support or Funding Information

NIH ES00002 ES11008
Hepatic stellate cells play an essential role in acetaminophen‐induced hepatocyte injury with or without prior endotoxemia
Authors: Rani Richa , Tandon Ashish , Kumar Sudhir , Gandhi Chandrashekhar R
Published: November 2023

Acetaminophen (APAP)‐induced liver injury is augmented by gram‐negative bacterial endotoxin (lipopolysaccharide: LPS). However, the understanding of the mechanisms of this clinically significant problem and the early predictors of cellular stress/injury is inadequate. LPS‐stimulated perisinusoidal hepatic stellate cells (HSCs) produce an array of cytokines that can affect survival of hepatocytes. The aim of this study was to investigate the role of LPS‐stimulated HSCs in augmenting APAP‐induced liver injury. Rats (n=6 each) were administered 5 mg/kg LPS or vehicle (PBS) (i.p.), followed by 200 mg/kg APAP or PBS (i.p.) 1h later, and sacrificed at 6h. Control rats received PBS at both time points. LPS and APAP alone induced mild liver injury, endoplasmic reticulum (ER) stress and autophagy as demonstrated by histologic examination, electron microscopy, increased CHOP expression and LC3‐II formation, and caspase‐3 activation. Augmentation of liver injury occurred when the rats were administered LPS and APAP. While CHOP expression and caspase‐3 activation increased further with LPS+APAP than with LPS or APAP, autophagy did not show significant change over that due to APAP alone. Notably, the liver injury was accompanied by proportionate increases in the serum levels of hepatocyte survival factor “augmenter of liver regeneration (ALR)”. In vitro, addition of APAP to hepatocytes incubated in LPS/HSC‐conditioned medium inhibited DNA and protein synthesis, and increased autophagy (LC3 II expression) and apoptosis (TUNEL staining). LPS‐stimulated HSCs expressed interferon‐β (IFNβ), and LPS/HSC ± APAP‐induced hepatocyte apoptosis was prevented by anti‐IFNβ Ab. Furthermore, consistent with the in vivo effects, LPS and APAP both stimulated secretion of ALR by hepatocytes, which increased strongly when hepatocytes incubated in LPS/HSC‐conditioned medium were challenged with APAP. Finally, HSC‐depleted mice were found to be protected from excessive liver injury due to APAP (600 mg/kg) treatment for 6h as well as LPS (5 mg/kg overnight) followed by 600 mg/kg APAP administration. These results suggest that HSCs play a critical role in causing liver injury due to APAP alone and in the presence of endotoxemia, and thus contribute to acute liver failure. The data also suggest that serum ALR can be a reliable diagnostic marker for hepatocyte stress or injury.

Support or Funding Information

VA Merit Review grant 1IO1BX001174‐01A1
Cell‐specific Wnts regulate liver zonation and regeneration
Authors: Preziosi Morgan , Yang Jing , Okabe Hirohisa , Diegel Cassandra , Williams Bart , Monga Satdarshan
Published: November 2023

Our lab has previously shown that β‐catenin regulates liver regeneration in a partial hepatectomy (Phx) model, under the control of Wnt and the canonical signaling pathway. This is true at early timepoints up to 40–72 hours. Using cell‐specific Wntless (Wls) knockout mouse models, we have previously determined epithelial cells are not the source of these Wnts. However, macrophage‐specific Wls knockouts (MP‐KO) have a regeneration deficit at 40 hours that is not seen earlier, suggesting they are a contributing source of Wnts in a temporal manner. We sought to further elucidate the source, identity, and initiation of Wnts using a Tamoxifen‐inducible endothelial cell‐specific Wls knockout model (EC‐KO). At basline, EC‐KO showed decrease in pericentral b‐catenin target genes such as GS, Cyp2e1, and notably Cyclin D1, suggesting a contribution to zonation and potentially regeneration. Since b‐catenin activation occurs early during regeneration, we also wanted to investigate the most proximal event that could be stimulating Wnt expression and secretion from relevant cells after hepatectomy. We hypothesized that shear stress immediately after Phx may initiate Wnt expression and secretion. Using an in vitro system, a preliminary analysis demonstrates that shear stress can initiate Wnt expression in EC, but not in MP or hepatocytes. In vivo, MP and EC demonstrate high levels of Wnt2 and 9b at baseline, which significantly increases after Phx. Taken together, we predict shear stress initiates secretion of Wnt2 and Wnt9b from EC at early timepoints, and MP secrete Wnt2 and Wnt9b at later timepoints, thus temporally regulating b‐catenin and contributing to liver regeneration.

Support or Funding Information

5T32HL094295‐05
Spatial Abilities and Pictures of Objects Recognized from Haptic Perception: Effect of Short Term Memory
Authors: Langlois Jean , Dagenais Yvan , Martin Marcel , Lemieux Renald , Lecourtois Marc , Yetisir Elizabeth , Bellemare Christian , Bergeron Germain , Hamstra Stanley J. , Wells George A.
Published: November 2023

Objective

As a basis for an application in anatomy education, vision‐based spatial abilities tests have been correlated with pictures of objects recognized from haptic perception. The objective of the current study was to determine the effect of short term memory on the recognition of pictures of objects from haptic perception. The hypothesis is that the use of short term memory has a negative effect on the performance.

Methods

A cohort of 48 medical graduates was enrolled in a prospective study. Ethics committee approval and written informed concent were obtained. Spatial abilities were measured with a redrawn Vandenberg and Kuse Mental Rotations Tests in two (MRTA) and three (MRTC) dimensions and a Surface Development Test (SDT). In a one‐month rotation preparing for residency, the experiment was done within a one‐week drawing course before a one‐week applied anatomy course. Twenty‐five objects constructed from various shaped parts glued together were identified on a picture by participants after haptic perception. In the first exercise, participants could touch the object for up to two minutes while identifying the corresponding picture. In the second exercise, 30 seconds were allowed for haptic perception of the object, 15 seconds to memorize, and up to 75 seconds to identify the corresponding picture without any further haptic access to the object. The maximum score was 24 for each of MRTA and MRTC, 60 for SDT, and 25 for the picture score. Descriptive statistics included median and lower and upper quartiles. Spearman's correlation coefficient was used to correlate the picture score to MRTA, MRTC and SDT scores. Wilcoxon signed‐rank test was used to compare the picture score in the first and second exercise.

Results

The picture score in the first exercise [18 (12, 21)] was correlated with MRTA [14 (9, 17)], MRTC [9.5 (6.5, 12)] and SDT [44.5 (36, 53)] scores with a correlation of 0.427 (p = 0.0025), 0.539 (p < 0.0001) and 0.429 (p = 0.0024), respectively. Similarly, the picture score in the second exercise [10 (7, 13)] was correlated with MRTA, MRTC, and SDT scores with a correlation of 0.444 (p = 0.0014), 0.384 (p = 0.0064) and 0.323 (p = 0.0236), respectively. The picture score in the first and second exercise was different (p < 0.0001).

Conclusion

Haptics is involved in the handling of anatomical structures. Vision‐based spatial abilities tests were correlated with pictures of objects recognized from haptic perception. The use of short term memory was found to have a negative effect on the performance. These findings on haptic perception and short term memory have promising avenues for education in the anatomy laboratory.

Support or Funding Information

This study was supported by an internal grant from the Department of Surgery, Université de Sherbrooke, Sherbrooke, QC, Canada.
Spatial Organization and Learning
Authors: Alazzeh Lena
Published: November 2023

Spatial organization is essential in the study of gross anatomy because it plays a central role in understanding the different anatomical organization differences in the body. However, undergraduate students consistently struggle with this concept resulting in poor performance on spatial organizing conceptual problems. The focus of this study was to understand where the root of the misconceptions originates. Over several semesters students have consistently missed a higher‐level multiple‐choice question regarding spatial organization of the transverse plane that crosses the 3 main body cavities. To understand the student's logic in answering the question we gave them the problem again and told them to respond to each option and why it would be scientifically correct or incorrect. The results showed the main misconceptions to be how they think of planes, mixing up the planes, and how body cavities are divided. These results give some insight into the concepts of spatial organization that students consistently struggle with and can thus help instructors better target the sources of confusion and figure out ways to better teach the material effectively.
Utilizing the CRISPR‐Cas System to Excise and Study the Insulin Enhancer
Authors: Yang Sophia , Rose Robert
Published: November 2023

The discovery of the CRISPR‐Cas system as an adaptive immune system for bacteria and archaea has stimulated much excitement in the scientific press. In type two CRISPR‐Cas systems, Cas9 is the RNA‐mediated endonuclease. Using a FLAG tagged Cas9, we propose to utilize the CRISPR‐Cas9 system to excise and pull down the insulin promoter both in vitro and in vivo to study the proteins associated with the enhancer. Guide sequences needed to target the insulin enhancer have been generated and we are currently in the process of expressing and purifying Cas9. In order to pull down the excised enhancer, we must determine how Cas9 releases products. Cas9 is a unique enzyme that has single turnover and must undergo significant conformational changes during catalysis. In vitro studies indicate Cas9 does not release cleaved DNA after cutting. We will crystallize the product form of the Cas9/DNA complex to characterize the structural rearrangement. Knowing the structure of the product complex will improve our understanding of how Cas9 associates and releases DNA post‐cleavage during genome editing.

Support or Funding Information

NCSU CALS Dean's Enrichment Grant – Wynne Innovation Category 07/01/14–06/30/16

Enhancer Excision based on CRISPR technology: a tool for deciphering gene regulation.
DNA Breaks and Damage Response Signaling are Coupled with RNA Polymerase II Promoter‐Proximal Pause Release and Required for Effective Transcriptional Elongation
Authors: Bunch Heeyoun
Published: November 2023

We have discovered a novel function of the factor tripartite motif‐containing 28 (TRIM28) to stabilize RNA polymerase II (Pol II) promoter‐proximal pausing at many stimulus‐inducible protein‐coding genes in mammalian cells during the un‐induced state. Upon transcriptional activation that releases paused Pol II, TRIM28 is phosphorylated at residue S824, dependent on phosphoinositide‐3 kinases such as DNA‐PK and ATM. Importantly, we have recently identified that the involvement of these DNA repair enzymes indeed indicates a coupling of DNA breaks and DNA damage response (DDR) signaling with processive elongation of Pol II. This was evidenced by enrichment of γH2AX in the gene bodies of transcriptionally activated genes and by global DNA‐PK co‐localization with Ser2 phosphorylated Pol II at C‐terminal domain (S2 Pol II). DNA breaks induced by transcriptional activation were also visualized in the cellular level and at a representative paused gene, HSPA1B through comet assay and primer extension analysis, respectively. Strikingly, DNA breaks and DDR signaling not only coincide with activated transcription but also are required for effective Pol II pause release and subsequent elongation. Inhibition of DNA‐PK catalytic subunit reduced the occupancy of Pol II, S2 Pol II, and γH2AX in the gene bodies of transcriptionally activated genes. We have also shown that the DNA breaks were the resultant of transcriptional activation because interfering P‐TEFb using a small molecule reduced γH2AX accumulation. In addition, we have revealed the important function of topoisomerase II‐mediated DNA double strand breaks in Pol II pause release and processive elongation in stimulus‐inducible genes. A chemical inhibitor for the catalytic activity of topoisomerase II led to retention of Pol II in the transcription start site, interfered with Pol II pause release, and reduced the level of γH2AX on the activated genes. Our findings suggest DNA breaks mediated by topoisomerase II and DDR signaling involving TRIM28 and DNA‐PK as a novel transcriptional mechanism to regulate the expression of a number of stimulus‐inducible protein‐coding genes in humans.
IDENTIFICATION OF ALTERNATIVELY SPLICED TRANSCRIPT OF BAX GENE IN MOUSE
Authors: Husain Mohammed Amir , Tabish Mohammad
Published: November 2023

Bax, a pro‐apoptotic member of Bcl‐2 family regulates apoptosis through homodimerization/heterodimerization with Bcl‐2. Bax‐α is the only product of the Bax gene that has been extensively studied. Bax‐α exists in inactive form and several conformational changes are required during apoptosis to activate it. Here, we have identified a novel transcript variant of Bax gene which contains alternatively spliced new first exon that is different from the first exon of previously reported transcript. Conceptual translation of new transcript encodes a protein (Bax‐α1), having different N‐terminus. The existence of the new transcript variant was confirmed by RT‐PCR, semi‐nested PCR using primers designed specific for the newly predicted transcript variant. The identity of PCR product obtained after semi‐nested PCR was confirmed by DNA sequencing. Relative expression of newly identified transcript variant with respect to reported transcript was also studied with the help of real time PCR. The existence of novel transcript variant was further supported by the presence of clusters of overlapping cDNA from the EST database. The novel variant was found to have different transcription factor binding sites with a distinct promoter. Bax‐α1 display heterogeneous properties as observed by post translational modification analysis tools. The differences in post translational modifications might play important roles in divergent function of the novel isoform. The three dimensional structure was generated by homology modelling to visualize the differences at N termini of known and newly identified variant.
The RS/RGG Domain of the SR‐like Protein Npl3 is Required for Efficient RNA Splicing in Yeast
Authors: Kress Tracy L , Gala Kinisha , Lobby Jenna , Moazamian Aron , Palvannan Prashanth
Published: November 2023

Npl3 is an SR‐like protein found in Saccharomyces cerevisiae that functions in co‐transcriptional pre‐mRNA splicing by promoting the recruitment of splicing factors to the pre‐mRNA during transcription. It contains two RNA recognition motifs (RRMs), an RS/RGG domain rich in arginine and serine repeats, and an uncharacterized N‐terminus. While the RRM domains are known to be required for Npl3 binding to RNA, the role of each of these functional domains in splicing is currently unknown. Here we have generated a panel of flag‐tagged Npl3 proteins lacking one or more of the protein domains. We identified genetic interactions between Npl3 mutations lacking the RS/RGG domain and mutations in genes that encode RNA splicing factors, suggesting that the RS/RGG domain is important for RNA splicing. Indeed, we show that deletion of the RS/RGG domain of Npl3 causes a significant decrease in RNA splicing efficiency of Npl3‐dependent pre‐mRNAs. In addition, deletion of the RS/RGG domain abolishes the ability of Npl3 to interact with splicing factors and proteins important for transcription, which strongly suggest that the RS domain is required to recruit the splicing machinery during transcription. This work demonstrates for the first time that the RS/RGG domain is required for the activity of Npl3 in RNA splicing.