bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of an Optimized Savory Cluster on Glycemic and Insulinemic Responses in Healthy Individuals: a Randomized, Cross‐over Study
Authors: Wolever Thomas MS , Jenkins Alexandra L , Campbell Janice , Ezatagha Adish , Pan Yang , Nisbet Mark , Harkness Laura
Published: November 2023

Postprandial glycemia has been implicated in the development of chronic metabolic diseases such as obesity, type 2 diabetes mellitus and cardiovascular diseases. Foods with high contents of slowly digestible starch elicit lower glycemic responses have been proposed as better choices to help decrease postprandial glucose excursions and thus to improve glycemic control and health, For this reason, there is a growing interest in developing foods with slowly digestible starch, particularly for snacks. A low moisture, low temperature process was developed to produce a savory cluster snack (test‐cluster) containing nuts and grains and bound together with wheat starch and soluble fiber which is slowly digested in‐vitro. The objective of this study was to compare the glucose and insulin responses elicited by the test‐cluster to those of a control‐cluster made with ingredients commonly used in commercially available snacks or bars including oats, peanuts, puffed rice and corn syrup and an available‐carbohydrate matched portion of white‐bread in healthy subjects. Healthy men and women (n=25) were studied on 3 occasions using a randomized, cross‐over design. After 2 fasting finger‐prick blood samples, subjects consumed 1 serving (56g) of the test‐ or control‐cluster or 47g white‐bread and had blood glucose and serum insulin measured at intervals over the next 4 hr. Each serving of test‐cluster, control‐cluster and white‐bread, respectively, contained: 5, 5 and 4g protein; 12, 11 and 0.5g fat; 24, 33 and 24g available‐carbohydrate; and 10, 2 and 1g dietary fiber. The blood‐glucose peak‐rise and incremental area under the curve (AUC) after test‐cluster were significantly lower than those after both control‐cluster and white‐bread (mean±SEM; peak‐rise, 1.24±0.09 vs 2.27±0.13 and 2.27±0.16 mmol/L; AUC, 67±8 vs 117±10 and 114±9, respectively). The serum‐insulin peak‐rise and AUC after test‐cluster, 128±13 pmol/L and 6.10±0.73 nmol×min/L, were similar to those after white bread, 141±20 pmol/L and 6.47±1.11 nmol×min/L, but significantly less than those after the control‐cluster, 205±26 pmol/L and 9.60±1.31 nmol×min/L. It is concluded that a serving of test‐cluster elicits lower glucose and insulin responses than a serving of a similar control‐cluster. The results support the hypothesis that the carbohydrates in the test‐cluster are slowly digested and absorbed in‐vivo.

Support or Funding Information

This study is funded by PepsiCo, Inc.
Cardio‐metabolic risk and lifestyle behaviours among police officers in Trinidad and Tobago
Authors: Prout Patrice , Nichols Selby
Published: November 2023

Objectives

To evaluate cardio‐metabolic risk among police officers.

Design and Methodology

This cross‐sectional study was conducted among law enforcement officers of the Trinidad and Tobago Police Services. Four jurisdictions were randomly selected and officers invited to participate. Blood pressure and anthropometric measurements (height, weight, body mass index, waist circumference, lean muscle mass, visceral fat and percentage body fat) were obtained using standard procedures. Participation in the study was voluntary. Ethical approval was granted by the Senior Superintendent on behalf of the Commissioner of Police.

Results

Four hundred (Females n= 138; Males n= 262) officers participated in the study. Male officers were more likely to have elevated systolic blood pressures than female counterparts (p <0.001). In partial correlation analyses controlling for age, ethnicity, education level and marital status; obesity was associated with an increased likelihood of elevated systolic blood pressure (> 140mmHg) (Odds ratio (OR) =3.82, 95%(CI): 2.92, 5.49; p<0.01); elevated diastolic blood pressure (> 90 mmHg) (OR= 3.89; 95% CI 2.88, 5.88; p<0.01); vegetables <2 servings per day (OR = 1.9, 95% CI: 1.15, 3.14; p= 0.015); red meat intake > 4 times per week (OR= 2.0, 95% CI: 1.04, 2.36; p=0.04); soda intake > 48 ounces per week (OR = 1.9, 95% CI:1.26, 2.88: p=0.002); participation in non occupational physical activity < 3 times per week (OR =4.70, 95% CI: 3.00, 10.86; p = 0.001).

Conclusion

The results indicated that poor dietary practices and physical activity among police officers are associated with cardio‐metabolic risk as indexed by blood pressure and obesity.
A Method Comparison Study between Two Hemoglobinometer Models (Hemocue Hb 301 and Hb 201+) to Measure Hemoglobin Concentrations And Estimate Anemia Prevalence among Women in Preah Vihear, Cambodia
Authors: Rappaport Aviva I , Karakochuk Crystal D , Whitfield Kyly C , Kheang Khin Meng , Green Timothy J
Published: November 2023

Background

Anemia affects 1.62 billion people worldwide. Defined as a hemoglobin (Hb) concentration <120 g/L for non‐pregnant women, anemia can negatively impact pregnancy outcomes and reduce work capacity. Hb is often measured in global health and nutrition surveys using a portable hemoglobinomter such as the Hemocue Hb 201+ (Hemocue, Angelholm, Sweden). More recently, a newer model was released (Hemocue Hb 301) utilizing slightly different methods to measure Hb as compared to the older model.

Objective

To measure bias and concordance between Hb concentrations using the Hemocue Hb 301 and Hb 201+ models in a rural field setting.

Methods

Hb concentration was measured in 175 rural Cambodian women (18–49 y) from Preah Vihear province using three Hemocue Hb 301 and three Hemocue Hb 201+ machines and using the same finger prick of blood in alternating order. Half of the samples (n=94) were measured using the Hb 301 model first, and the remaining samples (n=81) with the Hb 201+ first. Bias (the difference in means) and concordance were determined.

Results

Overall, mean ± SD Hb concentration was 116 ± 13 g/L using the Hb 201+ and 118 ± 12 g/L using the Hb 301. Anemia prevalence (Hb <120 g/L) for the Hb 201+ and Hb 301 was 58.3% (n=102) and 57.7% (n=101), respectively. Bias ± SD between methods was 2.0 ± 10.5 g/L and concordance (95% CI) was 0.63 (0.54, 0.72).

Conclusions

Bias was low and concordance was high between the two models, suggesting that the new Hemocue Hb 301 measures anemia prevalence comparably to the previous Hemocue Hb 201+ model. Further, anemia prevalence was very similar among the two models (57.7 vs. 58.3). The comparability of Hb measurements among different Hemocue models has important implications for the accurate estimations of anemia prevalence across time and among different surveys.

Support or Funding Information

The funding for this study was provided by the Danish Red Cross (Copenhagen, Denmark) and the University of Guelph (Guelph, Canada).
Assessment of Vitamin D Knowledge/Awareness of Black College Students
Authors: Jerrod Tiffany , Peterson Catherine A
Published: November 2023

It is commonly accepted that African Americans are at great risk for vitamin D deficiency. African Americans have the highest prevalence of insufficiency among any other ethnic group in the United States (US); and poor vitamin D status may in part explain the health disparities between Black and non‐Blacks. However, little data are available on the knowledge of and attitudes towards vitamin D and health within the Black population living in the US. A researcher‐developed 27‐question survey was used to assess vitamin D awareness among Black college students. Participants were University of Missouri students, at least 18 years old, and identified as Black and/or African American. Of the 110 students who started the survey, 108 responses were included in analysis. Nearly 99% of participants had heard of vitamin D and the most common information source was the media at 60%. When asked if young healthy people get enough vitamin D, 30% responded correctly while, 30% answered incorrectly and 40% did not know. In response to the question “You can be vitamin D deficient and not know it,” students studying the Humanities/History or Business were the least likely to answer correctly at 27% respectively. Interestingly, 40% of Life/Health Science majors did not know if young healthy people had adequate vitamin D intake, while 29% believed that they did. Nearly one‐third indicated that they did not know the sources of vitamin D. Slightly less than half (42.3%) of respondents said that they knew the functions of vitamin D in the body. These findings show a lack of vitamin D knowledge among Black college students; thus, revealing a need for public health education to increase vitamin D awareness among this young, potentially vulnerable yet malleable, population.

Support or Funding Information

McNair Scholars Program and Department of Nutrition & Exercise Physiology, University of Missouri.
Young soluble growth factors/cytokines improves wound healing in old rats
Authors: lluch Monica Caceres , Fernandez Christian , Saldias Maria Paz , Folli Vincenzo , Cerda Oscar
Published: November 2023

Aging is a natural process which is associated with the appearance of a number of diseases that contribute to progressive deterioration in the quality of life. Aging affect negatively gingival and skin wound healing. However, the mechanisms underlying this phenomenon remain unclear. To investigate the possible role of soluble growth factors/cytokines from the blood in this processes we performed in vitro experiments with serum derived from blood of young and old rats, primary skin fibroblast and in vivo wound skin healing. Animal procedures were conducted in accordance to the Administrative Panel on Laboratory Animal Care at Universidad de Chile.

We used skin fibroblast from old (18 months) and young (2 months) rats and serum derived from 1 ml of blood. Gel contraction was evaluated using a three‐dimensional cultures. Myofibroblast differentiation was evaluated by immunofluorescence and immunoblot. We evaluated α‐SMA after 2 days of treatment with young and old serum, we observed that young serum stimulates the expression of α‐SMA on young and old cells. However, we observed that α‐SMA does not incorporate into the stress fiber in old cells. Interestingly, these effects were reverted by the incubation of young serum. Old rats show a decrease in collagen gel contraction, suggesting the functional role of α‐SMA into the stress fiber. Skin fibroblast from old rats shows a decrease in collagen contraction in comparison with young skin

Full‐thickness 2,5 mm biopsy were made in the back of rats. Cross sections 5 um were stained with Masson trichrome, observed a delay in wound repair in old rats compared with young rats, this effect is partially reverted with serum derived from young rats after 7 days. We observed an improvement of cellular responses associated to wound healing in old rats topically treated with young serum such as increased in collagen expression and myofibroblast differentiation and diminished erythrocytes number into the wound.

These data demonstrate that young soluble growth/cytokines significantly accelerated wound healing in old rats. These effects might be related to the induction of differentiation to myofibroblast observed in young rats and old rats treated with young serum.

Support or Funding Information

Funding: FONDECYT 11140064, U‐Inicia
Continuous Efforts of the Vitamin Society of Japan to Increase Folate Intake in Japan
Authors: Amagase Harunobu , Ichinose Hiroshi , Kondo Atsuo , Abe Koichi , Watanabe Toshiaki , Sueki Kazuo , Shigeoka Shigeru
Published: November 2023

Successful implementation of Folate Fortification Program in 1998 led significant reduction in incidence of neural tube defects in the USA. Currently more than 70 countries have employed this fortification program. However, the Government of Japan (GOJ) has not mandated folate fortification at this moment, while Ministry of Health, Labor and Welfare (MHLW) of GOJ recommends expecting mother taking 400 μg of folate daily. In reality, average folate intake in Japanese expecting mother does not meet recommendation made by MHLW; it is even lower than 300 μg per day. The incidence of spina bifida, one of neural tube defects, in Japan has been gradually increasing, ranging from 2.2 per 10,000 births in 1980, 3.6 in 1990, 4.6 in 2000, to 5.9 in 2010. In another aspect, it has been suggested in many reports that supplementing folate, vitamin B6 and B12 reduces stroke incidence in the elderlies. Based upon these aspects, the Vitamin Society of Japan has proposed GOJ the following in 2015:

To enlighten child‐bearing age of women as to importance of taking recommended amount of folate to prevent a congenital disease.

To promote and support research to examine impact of vitamins and bio‐factors for health maintenance and disease prevention.
Immunomodulatory Effects of high‐purity β‐glucan on macrophage, natural killer (NK) cells, and T cell mediated factors
Authors: Kwon Han Ol , Lee Minhee , Ha Yejin , Kim Hye Sook , Shim Hyejin , Lee Jeongmin
Published: November 2023

The present study investigated the immunomodulatory effects of high‐purity β‐glucan on macrophage, natural killer (NK) cells, and T cell mediated factors. High‐purity β‐glucan on cytotoxicity in macrophage was investigated. Using macrophage, cytotoxicity of high‐purity β‐glucan was evaluated by MTT assay. We treated high‐purity β‐glucan at concentrations of 50, 100, 150, 200, and 250 μg/mL in macrophage. High‐purity β‐glucan did not affect the macrophage viability. Phagocytic activity was used zymosan. High‐purity β‐glucan cell was significantly increased as compared with the zymosan. We treated high‐purity β‐glucan to murine NK cells co‐incubated with YAC‐1 cells. High‐purity β‐glucan resulted in a significantly increased activity of Natural killer cell as compared with the control. In addition, treatment of macrophage with high‐purity β‐glucan resulted in a significantly increased activity of T cell mediated cytokines (IL‐2, IL‐12, IFN‐γ, and TNF‐α) levels as compared with the control. In conclusion, high‐purity β‐glucan could enhance immune response through activation of macrophage, NK cell, and T cell mediated factors.

Support or Funding Information

This reserch was financially supported by Korea Institute for Advancement of Technology(KIAT, 20151810) through the research and development for regional industry.
Anti‐Infective and Histogenic Bioscaffold for Bone Defect Repair
Authors: Luo Yangyang , Mills David
Published: November 2023

The objective of this study was to develop a stable drug delivery platform that is biocompatible, biodegradable and would provide sustained combinatorial drug release for the control of bone infection while simultaneously supporting bone tissue regeneration. Chitosan has been widely used in tissue engineering and in a variety of biomedical applications due to its eco‐friendly and biodegradable properties and it possesses a physiochemistry similar to the extracellular matrix of bone. However, the application of chitosan has been limited by its inherent mechanical weakness. Halloysite nanotubes (HNTs) are a naturally‐occurring aluminosilicate clay with a hollow tubular structure. HNTs are widely used as a bulk filler to materially reinforce a variety of polymer materials from bone cement to rubber. They have also been shown to be a potent nanocontainer for the sustained release of varied bioactive factors. In this study, chitosan and HNTs and chitosan and HNTs, doped with gentamicin and BMP‐2, were combined in different wt./wt. ratios and cross‐linked with tripolyphosphate. The biodegradability, mechanical properties, and surface structure of the hybrid nanocomposites were tested. The ability of the nanocomposites to control bacterial growth while supporting bone tissue formation was also evaluated. Gentamicin was selected as a model drug to evaluate the drug release capability of the chitosan‐HNTs nanocomposites and with release, its effectiveness against E. coli was analyzed through growth inhibition studies. Results indicate that the addition of HNTs to the chitosan hydrogel significantly improved the gels’ mechanical properties, permitting an extended period of drug release, and without a negative affect on drug efficacy. The coatings were capable of inhibiting bacterial growth and osteoblasts cultured on the hydrogel surfaces showed enhanced growth and functionality. Our doped clay/chitosan nanocomposite may overcome the limitations of traditional anti‐bacterial hydrogels and its ability to support bone tissue formation may enable its use in bone defect repair results from osteomyelitis.

Support or Funding Information

Funding for this project was provided by the Louisiana Governor's Biotechnology Initiative and Louisiana Board of Regents OPT‐IN program.
Breastfeeding and maternal physical and mental health among food insecure families with infants in Canada
Authors: Orr Sarah Kathleen , Tarasuk Valerie
Published: November 2023

One in six children in Canada live in food insecure households, but we know very little about the early‐life conditions facing infants in these households. Data from the US finds infants living in food insecure households face higher risk of poor health, hospitalization, and psychosocial and developmental problems. Also in the US, food insecurity has been shown to increase risk of maternal depression and reduce odds of following recommended infant feeding practices. Using nationally representative data available from the 2007–2012 Canadian Community Health Survey, we examined the relationship between food insecurity, breastfeeding practices, and maternal self‐reported general and mental health among mothers of infants 0–24 months of age (n = 6,470). When controlling for sociodemographics and maternal mental health, mothers living in food insecure households, compared to those living in food secure households, were as likely to initiate breastfeeding (aOR: 1.11, 95% CI 0.70–1.76), but once initiated, they were more likely to stop exclusive breastfeeding earlier than recommended (<4 months (aOR: 1.59, 95% CI 1.17–2.17), <6 months (aOR: 1.51, 95% CI 1.05–2.17)). In multivariate analyses controlling for maternal‐and household‐level sociodemographics, household food insecurity independently predicted mothers of infants reporting self‐perceived poor general health (aOR: 3.43, 95% CI 1.88–6.26), poor mental health (aOR: 4.48, 95% CI 2.81–7.14), and high life stress (aOR: 2.21, 95% CI 1.73–2.82). This study suggests that food insecurity interferes with a mother's ability to optimally care for her infant, although these relationships are cross‐sectional. More research is needed to understand the impact of food insecurity on infant health in Canada.
Low‐grade Fibromyxoid Sarcoma of the Sigmoid Presenting as Uterine Fibroid
Authors: MENDOZA ALEJANDRO S. , Peng Shi‐Kaung , French Samuel W.
Published: November 2023

BACKGROUND

Low‐grade fibromyxoid sarcoma(LGFMS) is a rare soft tissue tumor with a slight male predominance. The tumor has a tendency to arise from deep soft tissue of the trunk and lower extremities. Rare cases are reported to arise from the mediastinal and retroperitoneal areas. Its deceptively bland histologic appearance makes this tumor difficult to diagnose. We report a case of low‐grade fibromyxoid sarcoma presenting as uterine fibroid.

CASE REPORT

The patient is a 48‐year‐old obese female with a history of hypertension and menometrorrhagia who presented complaining of persistent lower abdominal pain and vaginal bleeding. A transvaginal ultrasound revealed an 8.7 cm uterine fibroid and MRI of the pelvis revealed a 10.6 × 6.9 × 8.1 cm lobulated mass within the mid pelvis. The patient was taken to the operating room for a total abdominal hysterectomy with bilateral salpingo‐oophorectomy for a mass of presumed gynecologic origin. Intraoperatively, a large 12 cm mass located in the pelvis was seen to be arising from the sigmoid colon. A shave biopsy was obtained, abdomen closed, and further staging of the colonic mass ensued. Further tests were negative of metastatic disease. Patient consequently underwent emergent sigmoidectomy due to bowel obstruction. Histologically, the tumor showed a swirling whorled growth pattern with alternating fibrous and myxoid areas. The myxoid area had a low to moderate cellularity composed of bland spindle cells with scant cytoplasm, uniform elongated nuclei, and small inconspicuous nucleoli. The tumor cells stained negative for most muscle, neural and mesenchymal stains but positive for CD99, BCL2 and MUC4 stains.

CONCLUSION

LGFMS has a deceptively bland histologic appearance making this tumor difficult to diagnose. CD99 and BCL2 stains will yield 90% positivity. MUC4 stain is highly specific and 100% sensitive for low grade fibromyxoid sarcoma. Molecular study will be 90% positive for t(7;16)(q32–34;p11) FUS‐CREB3L2 fusion gene.

Support or Funding Information

We declare no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
Thermal Manipulation during Mid‐term Broiler Chicken Embryogenesis: Effect on Muscle Growth Factors and Muscle Marker Genes
Authors: Al‐Zghoul Mohammad borhan
Published: November 2023

Animal productivity for meat consumption is directly related to the cellular and molecular mechanisms regulating skeletal muscle growth and development. Thermal manipulation (TM) during mid and late broiler chicken embryogenesis has been shown to promote muscle development and growth. However, the molecular bases of promoting muscle development and growth are not fully understood. The aim of this study was to investigate the molecular bases of muscle growth and development in broiler chicken subjected to thermal manipulation (39° and 65% for 9, 12 or 18 h) daily during ED 12–18. This included the investigating the changes in mRNA expression levels of muscle marker genes (MyoD, Myogenin, Pax7 and PCNA), and muscle growth factors (IGF‐1, Myostatin and GH) during embryogenesis and at posthatch days 10 and 28. Fertile Cobb strain eggs (n=1500) were divided into four groups. Control group was incubated at 37.8°C and 56% relative humidity (RH), the second group (TM1) was subjected to 39°C daily during (ED 12–18) and 65% relative humidity (RH) for 9 hours. The third group (TM2) was subjected to 39°C daily during (ED 12–18) and 65% (RH) for 12 hours. The fourth group (TM3) was subjected to 39°C daily during (ED 12–18) and 65% (RH) for 18 hours. The body weight (BW) during embryogenesis and at posthatch days (1, 3, 5, 7, 14, 21, 28 and 35) was recorded. mRNA expression levels of muscle marker genes (MyoD, Myogenin, Pax7 and PCNA) and muscle growth factor genes (IGF‐1, Myostatin and GH) during embryonic days 12, 14, 16 and 18 and at the posthatch days 10 and 28 were analyzed using the real time RT‐ PCR. TM resulted in significant increase in the mRNA expression of muscle marker genes and muscle growth factors, which were accompanied by the improvement of muscle mass and body weight at posthatch days 28 and 35 of age.

Support or Funding Information

This project was funded by National Plan for Science, Technology and Innovation (MAARIFAH)‐King Abdulaziz city for Science and Technology‐The king of Saudi Arabia, award number (11‐B10 1805‐06).
Fluorinated Caffeic Acid Phenethyl Ester: a Novel Anti‐osteogenic molecule to Attenuate Excessive Bone Damage during Autoimmune Arthritis
Authors: Holland Lauren , Mula Ramanjaneya VR , Machiah Deepa , Wang Xinyu , Shashidharamurthy Rangaiah
Published: November 2023

Caffeic Acid Phenethyl Ester (CAPE), an antioxidant flavonoid isolated from hives of honeybee, is reported to exert its anti‐osteogenic function by inhibiting the NF‐kB activation. It also disrupts the microtubule network created by osteoclasts, which would also aid in the attenuation of bone resorption. However, commercially available parental CAPE molecules are highly insoluble and have low stability. Recent studies have shown that a new fluorinated derivative of CAPE (F‐CAPE) exhibited improved NF‐kB inhibition and stability. Herein we have investigated the anti‐osteoclastogenic activity of F‐CAPE using an in vitro osteoclastogenesis model with mouse monocytic cell line (RAW264.7 cells). We observed an increased dose dependent inhibition of receptor activator of nuclear factor kappa‐B ligand (RANKL)‐induced multinuclear tartarate resistant acid phosphatase (TRAP) positive osteoclast formation by F‐CAPE compared to the parental compound. F‐CAPE at 150nM concentration has shown more than 2 fold higher anti‐osteoclastogenic activity compared to CAPE. Western blot analysis revealed that F‐CAPE decreased the expression of c‐Fos and nuclear factor of activated T cells c1 (NFATc1), which are the master regulators during osteoclastogenesis. In addition, F‐CAPE inhibits the NF‐κB signaling pathway, as confirmed by the decreased degradation of IκBα. Our data suggests that F‐CAPE is a highly potent anti‐osteogenic compound and could be a novel therapeutic molecule to attenuate excessive bone degradation during autoimmune arthritis

Support or Funding Information

This work was supported by the Biomedical sciences graduate research program at Philadelphia College of Osteopathic Medicine – GA Campus and American Heart Association Award (11SDG5710004)
The development and characterization of Cardiolipin‐specific fluorescent protein markers to assess and understand the localization and behavior of Cardiolipin within mitochondrial membranes of yeast cells
Authors: Amouzgar Meelad , Labbé Katherine , Nunnari Jodi , Sarsam Reta , Gonser William
Published: November 2023

Mitochondria are double‐membrane organelles that generate most of the energy used by cells and play key roles in cellular growth, death and differentiation. Continuous remodeling and redistribution of the mitochondrial membrane structure are critical for integrating organelle physiology with cellular needs. Cardiolipin (CL) is a diphosphotidyl lipid found almost exclusively in bacteria and in the mitochondrial inner membrane of eukaryotes. CL distribution within mitochondrial membranes is important in determining membrane structure, regulating bioenergetic processes, and recruiting factors involved in cell death and mitochondrial dynamics to the outer mitochondrial membrane. However, CL's exact distribution and functional localization within mitochondria is poorly understood because there exists no means for detecting and visualizing CL in live cells. We have developed a CL sensor based on the CL‐binding properties of the “InsertB” domain of Mgm‐1, a yeast mitochondrial inner membrane fusion protein. In in vitro assays, Insert B specifically binds CL‐containing liposomes. In live yeast and mammalian cells, a fluorescent‐tagged Insert B protein forms distinct foci within mitochondria depending on the presence of CL. By making a series of site‐directed mutations in highly conserved regions of Insert B, we are further characterizing the marker's affinity and specificity for CL. We are now using this tool to better understand the localization and behavior of CL‐enriched regions within mitochondrial membranes and its association with mitochondrial function.

Support or Funding Information

UC Davis Molecular and Cellular Biology Summer Fellowship for Undergraduate Research. Special thanks to Dr. Katherine Labbé and Dr. Jodi Nunnari.
Gene Expression Profiling of Prion‐Infected Brains: a Novel Disease Signature for Neurodegeneration in Humans
Authors: Vanni Silvia , Barbisin Maura , Moda Fabio , Giaccone Giorgio , Tagliavini Fabrizio , Haïk Stéphane , Deslys Jean‐Philippe , Zanusso Gianluigi , Legname Giuseppe
Published: November 2023

Objectives

In a previous study (Barbisin et al. 2014), we identified a five‐gene signature able to distinguish BSE‐infected macaques (Macaca fascicularis) from healthy ones: in brief, hemoglobin beta (HBB) and alpha2 (HBA2) were down‐regulated in infected monkeys, whereas transthyretin (TTR), apolipoprotein C1 (APOC1) and serpin peptidase inhibitor clade A (SERPINA3) were up‐regulated. The aim of the present work is to investigate whether the gene dysregulation pattern observed in macaques is also a specific gene signature for Creutzfeldt– Jakob disease (CJD) in humans compared to both healthy subjects and other neurodegenerative diseases.

Methods

We collected about 120 samples from frontal cortex of frozen brain tissue, obtained from prion‐infected patients (variant (v)CJD, sporadic (s)CJD, iatrogenic (i)CJD), patients with non‐CJD neurodegenerative disorders (Alzheimer's Disease, Parkinson's Disease, tauopathies) and age‐matched controls). RNA was extracted using TRIzol with PureLink® RNA Mini Kit and on‐column DNase I digestion. Quantity, purity and integrity were checked and only samples with a RIN≥5 were included in the study. Reverse transcription was carried out using Superscript III and qPCR was performed for the selected transcripts, with ACTB, RPL19, GAPDH and B2M as reference genes. In addition, erythrocyte markers expression analysis was performed in order to exclude any relevant blood contamination.

Results

Gene expression trend observed in macaques was confirmed in humans, with similar FC values, for four genes. HBA1/2 was down‐regulated in sCJD cases and also in patients affected by non‐CJD neurodegenerative diseases, while APOC1, TTR and SERPINA3 were up‐regulated in CJD patients, but not in patients affected by non‐CJD neurodegenerative diseases. IHC and WB analyses on related proteins are ongoing. We also found a consistent up‐regulation of SERPINA3, in both RML‐infected ScGT1 and ScN2a cell lines.

Conclusions

Besides providing important hints on some prion‐specific alterations in CJD, these results suggest that various pathways, such as oxygen and iron homeostasis (HBB, HBA2) and in particular cholesterol and lipids metabolism (APOC1, SERPINA3, TTR) have to be taken into consideration to identifying potential biomarkers and drug targets for prion diseases and other neurodegenerative disorders.

Support or Funding Information

FIRB Accordi di programma 2011 “genomica funzionale delle malattie neurodegenerative” (Prot. RBAP11FRE9_001)
Optimizing Human Tissue Fixation for High‐Quality Downstream Analysis Using Real‐Time Fixation Monitoring
Authors: Lerch Melissa , Bauer Daniel R , Chafin David , Theiss Abbey , Otter Michael , Baird Geoffrey S.
Published: November 2023

Molecular markers are invaluable to characterizing and treating disease. The pathologist routinely uses immunohistochemistry of formalin‐fixed paraffin embedded tissues to determine diagnosis or molecular subtypes in cancer and other diseases. Most markers that have been developed to date are robustly detected after varied fixation conditions. A greater challenge will be to develop diagnostics for more difficult markers, such as phosphoproteins, that are highly labile yet are critical “companion diagnostic” biomarkers that indicate sensitivity to selected chemotherapeutics.. Meeting this challenge with formalin fixation, the standard clinical tissue preservation process, will be difficult because of the overall poor understanding of formalin's ability to preserve all tissue biomarkers. We have demonstrated that labile phosphoproteins are better preserved using a rapid fixation protocol of 2 hours cold formalin followed by 2 hours hot formalin, which allows the cold formalin to penetrate tissue specimens completely while minimizing other enzymatic activity and the subsequent hot formalin step to crosslink the tissue proteins rapidly and thoroughly. To aid in studying this problem as well as to provide a potential metric for assessing fixation, we have invented a real‐time ultrasound time‐of‐flight (TOF) instrument that is capable of monitoring and imaging the critical step in formalin fixation, diffusion of the fixative into tissue, thereby creating a quantifiable quality metric for tissue fixation. This technology has been applied to most human tissues types and tissue from multiple disease states to demonstrate a broad range of diffusion, and thus fixation, behaviors. Our measurements can be converted into tissue diffusivity constants that correlate with the apparent diffusion constant calculated using MRI, despite the differences in the approaches, indicating that our approach is biophysically plausible. Using data collected from TOF analysis of many tissues, we have therefore developed a novel rapid fixation program that will ensure high quality downstream analysis for a broad range of tissues that correspond to the thickness of a full histology cassette (6 mm).

Support or Funding Information

Funding was provided by Ventana Medical Systems, Inc. to support this research.
Inhibition of ABC‐Transporters by in silico Identified Small Molecules
Authors: Vogel Pia D , Follit Courtney A. , Nanajakkara Amila K. , Wise John G.
Published: November 2023

Multidrug resistance (MDR) is a complex phenotype characterized by simultaneous resistance to structurally and functionally dissimilar drugs. MDR is frequently associated with overexpression of transmembrane efflux proteins belonging to the ATP‐binding cassette (ABC) transporter superfamily including P‐glycoprotein (P‐gp), multidrug resistance associated protein (MRP1), and breast cancer resistance protein (BCRP). Normally expressed at tissue barriers, these proteins protect vulnerable cells from xenobiotic attack by pumping a wide range of toxins out of the cell. Unfortunately, this broad substrate range, characteristic of these transporters, poses a major obstacle to the successful chemotherapeutic treatment of cancers as the majority of clinically relevant chemotherapeutic drugs can be effluxed by P‐gp, MRP1, and BCRP. Treatment and prevention of MDR have been studied extensively for decades, and yet, there are still no FDA‐approved treatments available.

Using targeted molecular dynamics and massively parallel drug docking studies, we have screened millions of drug‐like molecules and identified thousands novel compounds predicted to inhibit P‐gp by specifically interacting with its ATP binding domain. We have previously shown that of four in silico identified compounds inhibited P‐gp ATP hydrolysis (Brewer, F. K., Follit, C. A., Vogel, P. D., & Wise, J. G. (2014) Molecular Pharmacology, 86(6), 716–726). When tested in cell culture, three of these compounds restored chemotherapeutic sensitivity in a MDR human cancer cell line which overexpresses P‐gp (Follit CA, Brewer FK, Wise JG, Vogel PV (2015) Pharmacology Research & Perspectives 3(5): e00170). In this study, we expand our cell culture screening to evaluate 46 potential inhibitors identified by our in silico screening method and report that over 30% of these compounds restored chemosensitivity in MDR human cancer cell lines. Lead compounds will be further investigated to determine toxicity and specificity. With future chemical optimization, we are working towards developing powerful co‐therapeutics to treat and prevent MDR in human cancers.

Support or Funding Information

This work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzann Ruff of Dallas, Texas.
Fragment‐Based Drug Design with CNS Therapeutic Targets
Authors: Surratt Christopher K. , Pellegrene Kendy A. , Wasko Michael J. , Jean Bernandie K. , Madura Jeffry D.
Published: November 2023

Hundreds of millions of U.S. dollars are invested in the research and development of a single drug, a process fraught with failure. Lead compound development is an area ripe for new design strategies. Therapeutic lead candidates have been traditionally found using high‐throughput in vitro pharmacologic screening, a costly method for assaying thousands of compounds. This approach has more recently been augmented by structure‐based virtual screening, which employs computer models of the target protein to narrow the search for possible leads. A variant of virtual screening is fragment‐based drug design (FBDD), an emerging in silico lead discovery method that introduces low molecular weight fragments, rather than intact compounds, into the binding pocket of the receptor model. These fragments serve as starting points for “growing” the lead compound candidate. The objective of this study is to develop monoamine transporter and receptor computational models and employ their relevant ligand binding pockets as enclaves within which novel‐scaffold ligands can be constructed. Homology models of the human serotonin transporter (hSERT) and dopamine transporter (hDAT) proteins were constructed using as template a Drosophila dopamine transporter crystal structure; creation of a D3 dopamine receptor (D3R) model employed a human D3R crystal structure as template. The MedChem Transformations (MCT) module of Molecular Operating Environment 2015.10 software was used for FBDD. Applied to the generated D3R model, the MCT method successfully built using a fragment library the D2/D3 antagonist eticlopride and other potential D3R ligands. MCT places special emphasis on creating synthesizable, nontoxic molecules, suggesting that these hit compounds are viable ligand candidates (to be tested in subsequent in vitro pharmacologic assays). Fragment‐based methods should provide a viable, relatively low‐cost alternative for therapeutic lead discovery and optimization that can be applied to CNS targets to augment current design strategies.

Support or Funding Information

Supported by NIH grant DA027806.
Calmodulin regulates GPER/GPR30‐mediated signaling
Authors: Matnishian Vahe , Jasurda Jake , Tran Phuong , VerMeer Mark , TRAN QUANG‐KIM
Published: November 2023

The G protein‐coupled estrogen receptor 1 (GPER/GPR30) was recently identified as a novel receptor for estrogen, and has been demonstrated to be involved in numerous processes in various organ systems. However, its regulatory inputs are not entirely understood. Calmodulin (CaM) is the ubiquitous transducer of Ca2+ signals and is required for the functions of numerous cellular proteins. We recently developed FRET biosensors to identify and characterize four distinct CaM‐binding domains in GPER/GPR30. These domains are located separately in GPER/GPR30's submembrane domains (SMDs) 1–3 and the juxtamembranous (JM) segment of SMD4 (SMD4JM) (Tran et al., PLoS ONE 9(2): e89669). To investigate the impact of CaM binding at these domains on GPER/GPR30‐mediated signaling, we generated a series of mutations in SMD2, SMD3 and SMD4JM to alter hydrophobicity and charges of the original domains. Resultant biosensors developed on the mutant domains, BSGPER150–175mut, BSGPER242–259mut, and BSGPER330–351mut still display classic conformational changes upon CaM binding, yet with substantially reduced affinities. The Ca2+ sensitivities of the interactions between CaM and these domains also are decreased. Heterologous expression of wild‐type GPER/GPR30 increases phosphorylation of the extracellular related kinase (ERK1/2). CaM‐binding reducing mutations in SMD2, SMD3, or SMD4 do not significantly affect pre‐association between GPER/GPR30 and Gbg, yet substantially block GPER/GPR30‐mediated ERK1/2 phosphorylation. These data demonstrate that CaM binding to GPER/GPR30 plays an important role in GPER/GPR30‐mediated signaling.

Support or Funding Information

National Institutes of Health Grant HL112184
Lesphedeza bicolor Ameliorates Methylglyoxal‐induced Dysfunction in Human Aortic Endothelial Cells via Regulation of Oxidative Stress and Apoptotic Signaling Cascades
Authors: Do Moon Ho , Lee Jae Hyuk , Subedi Lalita , Kang Min Cheol , Kim Sun Yeou
Published: November 2023

Reactive intermediates of glucose, especially methylglyoxal(MGO) can form advanced glycation products, which relate to the diabetic complications. Methylglyoxal is known to cause damage and induce apoptosis in endothelial cells. Endothelial cell apoptosis is implicated in the progression of diabetes complications and atherosclerosis. Lesphedeza bicolor (LB) has been used as a traditional folk medicine for the removal of various toxic materials, the replenishment of energy stores, and for the regulation of diabetes. The effects of LB on MGO‐induced apoptosis were confirmed by annexin V/FITC staining and western blot. Inhibition of LB on MGO‐induced ROS generation was measured using a JuLI live‐cell imaging system. The present study was conducted to investigate the antiglucotoxicity of the stalk of LB on MGO‐induced apoptosis in human aortic endothelial cells (HAoECs). Pretreatment with LB significantly inhibited MGO‐induced cellular apoptosis and generation of intracellular ROS. LB prevented MGO‐induced apoptosis in HAoECs by increasing anti‐apoptotic Bcl‐2 expression and decreasing pro‐apoptotic Bax expression. Pretreatment with LB strongly inhibited the activation of all MAPK signals. Our study suggests that LB prevents apoptosis induced by MGO in HAoECs via inhibition of the oxidative stress and MAPK signaling pathways. In conclusion, LB may prove beneficial for the treatment of diabetic complications.

Support or Funding Information

This research was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry of Agriculture, Food and Rural Affairs (No.115045‐3)
Essential Opposite Roles of ERK and Akt Signaling in Cardiac Steroid‐Induced Increase in Heart Contractility
Authors: Lichtstein David , Rosen Haim , Ami Hagit Cohen‐Ben , Inbal Adi , Buzaglo Nahum
Published: November 2023

Interaction of cardiac steroids (CS) with the Na+, K+‐ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as ERK and Akt. We hypothesized that the activities of these pathways are involved in CS‐induced increase in heart contractility. This hypothesis was tested using in‐vivo and ex‐vivo wt and SERCA1a deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in‐vivo, and in primary cardiomyocytes, in wt zebrafish larvae and acc mutant. Ca2+ transients were determined ex‐vivo, in and adult zebrafish hearts. CS dose‐dependently, augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca2+ transients, in wt, but not in acc mutant. CS in‐vivo, increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of wt zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS‐induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS‐induced increase in heart contractility both, in‐vivo and ex‐vivo without affecting CS‐induced Ca2+ transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS‐induced increase in heart contractility also without affecting Ca2+ transients. These results support the notion that the activity of MAPK pathway is obligatory for CS‐induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca2+ independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy.

Support or Funding Information

This work was supported by grants from the Ministry of Trade and Industry (NOFAR), Israel and the Szolts Foundation, The Hebrew University of Jerusalem, and the Walter and Greta Chair in Heart Studies (to D.L).
Non‐transporter mediated drug permeability across MDCK cell monolayers: Towards an explicit definition of the partition constant for passive drug interactions with cell membranes
Authors: Humphreys Sara
Published: November 2023

Drug transport models aim to simulate the passage of a drug through the human body. One transport parameter requiring further refinement is the partition constant for drug partitioning into membranes (Kp). In transport model terminology, Kp is defined as the relative rates of diffusion clearance into (CLi) and out (CLo) of an explicit membrane compartment. Here, we use a scanning laser confocal microscope to perform in vivo kinetics experiments where we monitor the diffusion clearance rates Coumarin 152 as it permeates confluent MDCK cell monolayers grown on Transwell® supports.
Suvorexant, a Clinically Used Dual Orexin Receptor Antagonist, Induces Human Pregnane X Receptor‐Mediated Expression of Cytochrome P450 3A4
Authors: Abbott Kodye , Pondugula Satyanarayana
Published: November 2023

Activation of human pregnane X receptor (hPXR)‐regulated expression of cytochrome p450 3A4 (CYP3A4) plays an important role in mediating adverse drug interactions. Here, we studied whether suvorexant, a dual orexin receptor antagonist used to treat insomnia, could induce hPXR‐mediated regulation of CYP3A4. hPXR transactivation assays were performed to determine CYP3A4 promoter activity and in HepG2 human liver carcinoma cells as well as in LS174T and LS180 human colon cancer cells. Quantitative RT‐PCR assays were conducted to study gene expression of CYP3A4 in human primary hepatocytes and hepatocells. Suvorexant, at its therapeutically‐relevant concentrations, induced hPXR transactivation of CYP3A4 promoter activity. Similar to a known PXR agonist rifampicin, suvorexant also induced CYP3A4 gene expression in human primary hepatocytes and hepatocells. These results suggest that suvorexant induces hPXR‐regulated CYP3A4 gene expression. As insomnia often coexists with other medical and psychiatric conditions, it is treated concurrently with the coexisting conditions. Therefore, the inductive effects of suvorexant on CYP3A4 expression caution the use of suvorexant in conjunction with other medications metabolized via CYP3A4. Future studies will be directed to investigate the mechanisms of suvorexant induction of hPXR‐mediated CYP3A4 expression.

Support or Funding Information

This work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant, and Auburn University Startup Funds to Pondugula SR. We thank Drs. Tao, Coleman, and Schwartz for sharing their facilities.