bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship of blood levels of homocysteine, folate and vitamin B12 with dietary nutrient density in Korean middle‐aged women with type 2 diabetes
Authors: Kim Eun‐kyung , Kim Hyesook , Oh Ji Soo , Choi Young Ju , Huh Kap Bum , Chang Namsoo
Published: November 2023

This study was conducted to examine the association between blood levels of homocysteine, folate, and vitamin B12 and nutrient density (nutrient content/1000kcal) in diet among middle‐aged women with type 2 diabetes (n=162, mean age=57.6±4.0 years). Dietary intake was assessed by semiquantitative food frequency questionnaire method. Multiple regression analysis showed that blood homocysteine concentration was positively correlated with dietary fat (p=0.0466) and negatively correlated with dietary fiber (p=0.0078) after adjusting for age, BMI, occupation, education, smoking and alcohol intake. Blood folate was positively correlated with dietary folate (p=0.0478). Blood homocysteine was negatively correlated with blood folate (p=0.0092) and vitamin B12 (p=0.0002), and a positive correlation (p=0.0228) was observed between blood folate and vitamin B12. Previous studies have reported on the association between levels of blood homocysteine and elastic properties of the arterial wall where high levels of blood homocysteine increases CVD risk. Through explaining the relationship between dietary fiber and folate and levels of blood homocysteine and folate, this study further proves an inverse relationship between consumption of foods rich in fiber and folate and CVD risk.

Support or Funding Information

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF‐2013R1A1A2011544) and Brain Korea 21 Plus.]
Association between beverage intake with urinary bisphenol A levels in Korean women of reproductive age
Authors: Jo Ara , Kim Hyesook , Kim Hyejin , Chung Hyewon , Chang Namsoo
Published: November 2023

This study was performed to investigate the health effects of exposure to environmental risk factors in Korean women of reproductive age. We analyzed the relationship between daily food intakes and endocrine disrupting chemical such as bisphenol A (BPA). A total of 302 female subjects (30–49 years) participated in this study. Dietary intake was assessed using a single 24 hour dietary recall method. The average daily intake of beverage was 114.2 ± 159.1 g/d and the mean urinary BPA level was 1.7 ± 1.5 μg/g creatinine. The partial correlation adjusted for BMI, total energy intake, cigarette smoking status and education showed that urinary BPA levels was positively correlated with beverage consumption. For this study, soda, canned and sachet coffee, plastic and can packed tea and alcohol were included as beverages. The transfer of BPA into food and beverages from BPA containing packaging materials such as polycarbonate and epoxy resins has been identified by several previous studies as the most likely source of urinary BPA. The study results indicate a leakage of BPA from the polycarbonate and epoxy lining in the plastic or canned containers of beverages, and suggest that increased beverage intake is associated with increased urinary BPA concentration in women of reproductive age.

Support or Funding Information

[This research was supported by Ministry of the Environment and the Brain Korea 21 Plus.]
The stem cell gene ABCB5 regulates DNA methylation in colorectal cancer
Authors: Guo Qin , Frank Alexander , Liu Robert , Berg Gretchen , Xu Shuyun , Zhan Qian , Murphy George , Lian Christine
Published: November 2023

ABCB5 marks cancer stem cell subpopulations in diverse human malignancies and is responsible for several features of tumor aggressiveness, including therapeutic drug resistance and metastasis. Exactly how these pleiotropic functions are exerted is currently unknown. We hypothesized that ABCB5 might regulate an epigenetic DNA hydroxymethylation pattern, i.e. loss of 5‐hydroxymethylcytosine (5‐hmC), which has previously been shown to correlate with cancer aggression in diverse malignancies, including colorectal cancer. To test this hypothesis, we examined the functional relationship between ABCB5 expression and 5‐hmC levels, utilizing four distinct mouse and human colorectal cancer models in which ABCB5 function was either genetically ablated or blocked by anti‐ABCB5 monoclonal antibody. In support of our hypothesis, we found that both genetic and antibody‐mediated ABCB5 blockade resulted in restoration of 5‐hmC levels and diminished tumor aggressiveness features. These results suggest a novel role of ABCB5 as a regulator of colorectal cancer aggressiveness through epigenetic regulation, with important implications for novel therapeutic strategies in this highly prevalent human malignancy.
Effect of Sex Steroids on expression of genes regulating adipose tissue
Authors: Alshammari Ghedeir M , Brameld John , Lomax Michael
Published: November 2023

Obesity represents an important health issue, since it is a risk factor for the development of a number of medical conditions, including type 2 diabetes, hypertension and cardiovascular disease. In men, visceral adipose tissue has been related to low plasma testosterone levels and low levels of oestrogens are associated with increased adipose tissue. In female, testosterone and estradiol plasma levels are related to fat cell morphology and expression of genes regulating adipocyte differentiation and adipocytokines secretion. In this present study we have analysed the effect of testosterone and estradiol on gene expression of adipose tissue regulating genes in adipocytes. The human preadipocyte cell line (SGBS) was differentiated into adipocytes and exposed to different concentrations (range 0.25–3nM) of testosterone and estradiol. The gene expression was analysed using quantitative real time PCR (qRT‐PCR). Our results indicate that physiological doses of testosterone and estradiol at do not alter expression of PPARγ, CEBPα, and adiponectin in differentiated adipocytes. Moreover, expression of cytokines (resistin, IL‐6, and TNFα) was very low in SGBS and not altered by sex steroids.

Support or Funding Information

King Saud University
The Development of a Genomic Health Disparity Database and Analytic Tool
Authors: Hughley Raymond Welsey , Tripathi Shweta , Davis Mellissa , Yates Clayton
Published: November 2023

Publically available datasets such as The Cancer Genome Atlas (TCGA) provides scientific investigators and clinicians access to multiple NGS applications of patients including exome sequencing, comparative genomic hybridization ‐arrays, DNA methylation arrays, RNA sequencing, and reverse protein phase arrays. Many tools are being developed to allow basic researchers and clinicians without bioinformatics training to envisage, ‐analyze and understand the data, however, at the moment there is no centralized location to obtain sequenced genomic data that permits analysis of cancer patients based on health disparities. Interestingly, genomic analysis tools such as Oncomine and KM plot do not provide ethnicity as search criteria. Whether, this is a lack of ethnicity obtained at during sample acquisition or lack of including in the study, the result is that very few dataset have racial information, limiting the large scale analyses due to insignificant sample populations.

Therefore, we proposed to create a data base that combines genome sequenced information from multiple locations that include racial information. Additionally, we have developed analytical tools to access files from databases like GSE files and the TCGA data, which contain information on race. Genes from each study where identified using David or the direct probe sequence information for each sequencing machine. R statistical language and multiple packages where used to normalize data sets and merge them into one centralized form. Ancestry markers (AIMS) and SNPs where used to verify ethnicity. We further developed analytic tools analyze Gene Expression and patient survival. The development of such a resource can greatly contribute in solving many of the health disparities questions being asked today and in the future

Support or Funding Information

Supported by Department of Defense Prostate Cancer Research Program (PC120913) [CY], G12 RR03059–21A1 (NIH/RCMI) [CY], (NIH/NCI) 1 R21 CA188799–01, and a pilot project on U54 CA118623 (NIH/NCI) [CY]
Effects of Artemisia Annua leaf extract on hepatic steatosis and inflammation in high‐fat diet‐fed mice
Authors: Jeong Eun Ae , Kim Kyung Eun , Yi Chin‐ok , Lee Jong Youl , Roh Gu Seob
Published: November 2023

Artemisia Annua L. (AA) is a well‐known source of the antimalarial drug artemisinin. AA also has an antibacterial and antioxidant activity. However, the effect of AA extract on hepatic steatosis induced by obesity is unclear. We investigated whether AA extract prevents obesity‐induced insulin resistance and hepatic steatosis in high‐fat diet (HFD)‐fed mice. Mice were randomly divided into groups that received a normal chow diet (ND) and HFD with or without AA for 12 weeks. We found that AA extract reduced insulin resistance and hepatic steatosis in HFD‐fed mice. Western blot analysis showed that HFD‐induced expression of nuclear sterol regulatory element‐binding protein 1 (SREBP1) and carbohydrate response element‐binding protein (ChREBP) in the livers was decreased by AA extract. In particular, dietary administration of AA extract decreased hepatic high‐mobility group box 1 (HMGB1) and cyclooxygenase‐2 (COX‐2) expression in HFD‐fed mice. AA extract also attenuated HFD‐induced collagen deposition and fibrosis‐related transforming growth factor (TGF)‐β1 and connective tissue growth factor (CTGF). These data indicate that dietary AA extract has beneficial effects on hepatic steatosis and inflammation in HFD‐fed mice.

Support or Funding Information

National Research Foundation of Korea (No.2014R1A2A1A11049588)
Nef mediated learning impairment is associated with a TGF‐β inflammatory environment in astrocyte cultures in vitro
Authors: Manoharan‐Valerio Michael Anand , Martinez‐Orengo Neysha , Noel Richard
Published: November 2023

The worldwide epidemic of HIV has been ameliorated with the development of antiretroviral therapies. Modern treatment has been efficient in impairing viral replication, resulting in an undetectable viral load in patients. Unfortunately, the process of viral protein production is still functional contributing to detrimental conditions such as HIV associated neurocognitive disorders (HAND). Recent studies have associated HAND with astrocyte mediated toxicity of neurons, stimulating further investigations regarding the mechanisms of these effects. Studies conducted in our lab have shown loss of neurons and deficiency in the spatial learning of rats infused with astrocytes expressing the viral protein Nef. The release of inflammatory cytokines and modulation of TGF‐b pathway could be a plausible explanation for the pathology herein observed. We hypothesize that astrocytes transfected with Nef increase the expression of TGF‐b and promote the secretion of inflammatory cytokines. Therefore, we aim to investigate the possible molecular explanation to the relation between learning impairment, inflammation and modulation of TGF‐b pathway in the presence of HIV‐Nef. In order to verify the production of these factors, astrocytes were transfected by electroporation with a Nef or GFP plasmid. During the following 48 and 72 hours samples were collected and assessed by western blot and immunofluorescence. Resulting from these studies we found an increased expression of iNOS in supernantants and increased expression of TGF‐b in lysates of astrocytes expressing Nef compared to GFP controls. These results were more noticeable at 48 hours. We also observed that cellular localization of TGF‐b varied depending on treatment. In astrocytes transfected with GFP, TGF‐b labeling was primarily nuclear whereas in astrocytes transfected with Nef there was a distribution of the labeling more towards the cytoplasm. These results suggest a stimulation of a pro‐inflammatory environment by HIV‐Nef that could lead to harmful effects to neurons. We are also conducting in vitro co‐cultures of neurons exposed to astrocytes expressing Nef to study the extent of the inflammation and toxicity in this environment. Because TGF‐b can be either beneficial or negative to cell processes and cell response can vary depending on the type of TGF‐b receptor, further studies include understanding more in depth activation of the pathway. Studies have shown that the production of TGF‐b ligand can correlate directly or inversely to the receptors. Thus we are interested in measuring SMADs proteins, expression of TGF‐b receptors, and include a TGF‐b receptor blocker to better understand the role of this pathway.

Support or Funding Information

R25GM082406, GM106970, MD007579
A physiological model of a subset of compounds that regulate circadian rhythms in humans
Authors: Garan Steven Andrew , Peng Justin , Barakat Rita , Efe Gizem , White Randall , Preecha Andrew , Mehdizade Morvarid , Sun Shuzhang , Mehdizadeh Sadaf , Gouw Arvin , Brooks George
Published: November 2023

Circadian rhythms allow organisms to regulate daily processes, such as sleep, feeding and body temperature. A subset of the compounds that regulate circadian rhythms in humans has been simulated in a quantitative temporal physiological model using the Center for Research and Education in Aging Language (CREAL). CREAL is a language that can be used to describe biological systems from a macro to a molecular scale, and simulations can take place over time scales such as an hour or a day or a longer period of time. The simulation of the circadian rhythms shows the concentration of compounds throughout the model, during a 24 hour period. The release of melatonin, which modulates the sleep cycle is simulated with respect to events that take place in the suprachiasmatic nucleus (SCN). The model also shows changes in cortisol levels as well as many other compounds that are involved in regulating daily processes.
Small molecule GCPII inhibitor ameliorates disease activity in murine models of IBD
Authors: Rais Rana , Jiang Weiwei , Wozniak Krystyna , Stathis Marigo , Rojas Camilo , Li Xuhang , Slusher Barbara
Published: November 2023

Background

Folate Hydrolase 1 (FOLH1) gene encodes a type II transmembrane glycoprotein termed glutamate carboxypeptidase II (GCPII) that cleaves terminal glutamate moieties from the abundant neuropeptide N‐acetylated aspartate‐glutamate (NAAG) in nervous tissues and sequentially from folate poly‐γ‐glutamate in the gut. Recent gene‐profiling and immunohistological analyses have shown a selective and robust upregulation of FOLH1/GCPII mRNA and protein in the intestinal mucosa of patients with Crohn's disease.

Methods

Using a functional FOLH1/GCPII radioenzymatic assay, we first quantified the glutamate carboxypeptidase activity in normal and diseased mucosa of 31 surgical intestinal specimens from 20 subjects including healthy controls and patients with inflammatory bowel disease (IBD). Using two validated murine models of IBD ‐dextran sodium sulfate (DSS)‐induced and IL10−/− spontaneous colitis models ‐we next explored the therapeutic potential of chronic GCPII inhibition using the selective GCPII inhibitor 2‐PMPA (IC50=300 pM).

Results

We report a robust 300–3,000% increase in FOLH1/GCPII enzymatic activity in the intestinal mucosa of patients with active IBD when compared to that in an uninvolved area of the same patients, or the intestine from healthy controls. In the DSS‐colitis model, daily 2‐PMPA inhibited the glutamate carboxypeptidase activity in the colonic mucosa by over 90% and significantly reduced the disease activity index. These findings were corroborated in FOLH1−/− mice which showed significant protection to DSS‐induced colitis compared to wild type mice. In the murine IL‐10−/− model of colitis, daily 2‐PMPA treatment also significantly reduced both macroscopic and microscopic disease severity.

Conclusions

We have demonstrated, for the first time, a robust upregulation of FOLH1/GCPII enzymatic activity in diseased mucosa from IBD patients and significant attenuation of IBD symptoms in murine models using a selective FOLH1/GCPII inhibitor and in FOLH1−/− mice. These data strongly implicate FOLH1/GCPII in the etiology of IBD, providing a new therapeutic target.

Support or Funding Information

This research was supported by NIH Grant RO1CA161056
Conjugation Route Regulates the Abundance of Protein Antigen Epitopes Presented by Dendritic Cells Post KLH‐Protein Antigen Conjugates Pulsing
Authors: Bi Shuguang , Bailey William , Brisson Catherine
Published: November 2023

Owing mainly to its large size, and abundant and complex carbohydrate structures on its peptide chains, Keyhole Limpet Hemocyanin (KLH) has been widely used for decades as an antigen carrier to generate antibodies or to build vaccines for immunotherapy. In both circumstances, an antigen is conjugated to KLH. Depending on the nature of the antigens, there are many conjugation methods utilized in both academia and industry. At Stellar, we are also exploring the conjugation methods used to conjugate various types of antigens to KLH. We chose ovalbumin (OVA) as a model antigen to build KLH‐protein antigen conjugates. We conjugated OVA to KLH, both the 400 KDa monomer (subunit KLH) and the 8 MDa didecamer (high molecular weight KLH), via disulfide bonds or primary amine groups on OVA polypeptide chains. We also conjugated OVA to KLH via the carbohydrate structures on OVA or the carbohydrate structures on KLH. For each of these methods, we utilized different ratios of KLH to the linker, KLH to OVA. The KLH‐OVA conjugates were used to pulse mouse bone marrow‐derived primary dendritic cells (DCs) and immature mouse DC line JAWSII cells. After being pulsed with conjugates, DCs were stained with fluorescence tag conjugated antibodies that recognized and bound to the OVA epitopes presented by MHC II molecules on the surface of the DCs. The abundance of OVA epitopes on DC surface was analyzed with flow cytometry. We found that the conjugation route affected the capability of KLH to serve as a carrier for protein antigens. We also found that, for certain KLH samples, conjugating a protein antigen via carbohydrate structures (instead of disulfide bonds or primary amines) did not interfere with protein antigen processing and even increased the amount of protein antigens to be successfully presented by DCs.
The Virginia Tech Center for Drug Discovery and the Virginia Drug Discovery Consortium
Authors: Kingston David George Ian
Published: November 2023

This poster presents the work of two Virginia‐based drug discovery centers.

The Virginia Tech Center for Drug Discovery, or VTCDD, was established in 2011 to bring together the expertise of scientists from several different departments at Virginia Tech with the mission to continue the growth and advance the stature of the existing drug discovery and development programs of the university. It operates as a “virtual center” with a small screening laboratory and a modest budget, but it has been successful in catalyzing several research collaborations and increasing grant funding. Two VTCDD members have drugs in clinical trials based on earlier work, and other members have developed new drug formulations and new drug candidates. The presentation will summarize the work of several VTCDD members.

The Virginia Drug Discovery Consortium (VDDC) is a new Consortium between the major research universities in Virginia, including the University of Virginia, Virginia Commonwealth University, Virginia Tech, and the Virginia Tech Carilion Research Institute. Its initial focus is on diseases of the brain, and it has planned a BrainRx Symposium in Richmond, Virginia, for May 23–24 to promote this collaborative research focus. Details of this Symposium will be presented.

Support or Funding Information

Support of VTCDD by the Fralin Institute for Life Sciences at Virginia Tech is gratefully acknowledged
Development of a multiplexed plasmonic nanoparticle‐based assay to identify inhibiters of HuR protein‐RNA binding
Authors: Yoshida George , He Jie , Gabanic Andrew , Sagle Laura , Tranter Michael
Published: November 2023

Introduction

RNA binding proteins such as HuR are becoming recognized as potential mediators of cardiac physiology and pathology, but this class of proteins has traditionally been considered to be “un‐druggable” targets. Recent work from our laboratory using a cardiomyocyte‐specific HuR‐deficient mouse model has identified HuR as a novel therapeutic target for heart failure. Specifically, deletion of HuR preserves cardiac function and decreases pathological ventricular remodeling in a mouse model of heart failure. In order to identify novel HuR inhibitors, we are collaborating with the Sagle laboratory, which has significant expertise in localized surface plasmon resonance (LSPR) biosensing and recently developed a nanoparticle fabrication technique applicable to high‐throughput drug screening. Thus, the objective of this study is to adapt a multiplexed plasmonic nanoparticle platform developed by the Sagle laboratory for the development of a novel plasmonic nanoparticle‐based drug screening assay to identify small molecule inhibitors of HuR.

Methods and Results

Gold nanoparticle arrays, 100 nm in diameter and 60 nm in height, were fabricated and thiolated DNA was covalently bound to the surface of the gold. Next, a single‐stranded RNA containing a HuR target binding site was hybridized to the nanoparticle‐bound DNA. We are then able to detect HuR protein binding to its target site within the RNA by observing a LSPR shift induced by the increased mass bound to the gold nanoparticle surface. For each step of the assembly, a significant LSPR absorbance shift was observed with the addition of DNA, RNA, and HuR protein to the nanoparticle surface. Importantly, we are able to quantitatively demonstrate a reversal of HuR‐RNA binding in the presence an experimental new inhibitor.

Conclusion

Our results describe a novel multiplexed plasmonic nanoparticle‐based assay suitable for high‐throughput screening of drug candidates to inhibit RNA binding proteins through a simple colorimetric (LSPR) detection method. Here we demonstrate the initial application of this approach to specifically screen for inhibitors of the RNA binding protein HuR, but this assay provides a flexible platform applicable to many RNA binding proteins by simply changing the RNA target sequence.
Functional and Transcriptional Interactions Between Regulator of G‐protein Signaling Protein 10 (RGS10) and Tumor Suppressor Proteins in Cancer Chemosensitization
Authors: Hooks Shelley , Alqinyah Mohammed , Webster Andrew , Sakamuro Daitoku
Published: November 2023

Ovarian cancer is the deadliest gynecologic cancer, accounting for approximately 15,000 deaths per year. Chemotherapeutic drugs are initially very effective, but more than 80% of patients whose tumors initially respond to chemotherapeutics will relapse within two years with drug‐resistant, terminal disease. Understanding the molecular changes that ovarian cancer cells undergo to gain the ability to survive in the presence of DNA‐damaging chemotherapeutics is critical to develop therapies for chemoresistant cancers. Our studies are focused on the protein Regulator of G‐protein Signaling protein 10 (RGS10), which we have shown is critical in maintaining sensitivity to cisplatin‐induced cell death. Analysis of gene and protein expression in multiple ovarian cancer cell lines demonstrates that expression of RGS10 is suppressed during progression to chemoresistance. Further, we show that acute loss of RGS10 expression directly induces chemoresistance. RGS10 is a nucleo‐cytoplasmic protein with unknown nuclear functions. In order to define the mechanism of RGS10's role in chemoresistance, we probed the functional interaction between RGS10 and the nucleo‐cytoplamic tumor suppressor protein Bridging Integrator 1 protein (BIN1). Our results reveal a functional interaction between RGS10 and BIN1. The ability of BIN1 to regulate cell survival in the presence of cisplatin is partially reversed by normalizing RGS10 expression levels. The mechanism of suppression of RGS10 during chemoresistance is not fully understood, but includes recruitment of histone deacetylase enzymes. To explore upstream signaling that may trigger loss of RGS10 expression, we further determined if there exists an effect of BIN1 expression on RGS10 expression. Our results show that manipulation of BIN1 expression in cancer cells acutely regulates RGS10 expression. Together, our results suggest that the expression and function of RGS10, a novel regulator of ovarian cancer cell survival, is modulated by the established tumor suppressor protein BIN1. Ongoing work will further define the molecular mechanisms linking these two regulator proteins.

Support or Funding Information

Marsha Rivkin Center for Ovarian Cancer Research (SBH)

NCI R01CA140379 (DS)
Liver lipid profiling of chemical‐induced phospholipidosis
Authors: Saito Kosuke , Ishikawa Masaki , Yamada Hiroshi , Nakatsu Noriyuki , Maekawa Keiko , Saito Yoshiro
Published: November 2023

Lipids play important roles in multiple biological processes and its disorder is well associated with diseases and injuries. Thus, lipid profiling would be a useful to understand pathophysiology of but also to identify biomarker for chemical‐induced phospholipidosis. In the present study, we used rat model to overview liver lipid profiles of two types of phospholipidosis. Hepatic phospholipidosis were induced by treatment of clomipramine (CPM), imipramine (IMI), amitriptyline (AMT) and ketoconazole (KC) for 28 days. Phospholipidosis were determined by histopathological evaluation. CPM, IMI and AMT induced phospholipidosis in hepatocyte, while KC induced phospholipidosis in bile duct. To overview liver lipid profiles, we employed recently developed lipidomics platform with liquid chromatography and mass spectrometry (LC/MS), whic hsimultaneously determine the levels of broad spectrum of lipids. In total, we determined and examined 138 lipids (62 phospholipids, 10 sphingolipids and 65 neutral lipids). Each chemical treatment have their corresponding control treated with vehicle. To identify specific lipid alterations in the liver, we employed multivariate orthogonal partial least squares discriminant analysis. A bis‐monophosphatidic acid (BMP) (BMP[44:12]) and several triacylglycerols (TGs) were identified as specific lipids separating control and chemical‐treated rats in all models of phospholipidosis in the liver. The levels of BMP(44:12) were increased in the liver by chemical‐induced phospholipidosis. On the other hand, several TGs were increased in the liver by CPM, IMI and AMT but decreased by KC. Therefore, increased levels of BMP is common features of hepatic phospholipidosis and alteration of TGs might be associated with zone‐specific events in the liver. Our study provides the new insight into the alteration of liver lipidprofiles which probably reflecting chemical‐induced hepatic phospholipidosis and may help to understand its pathophysiology.

Support or Funding Information

This work was supported by the intramural research grant of National Institute of Health Sciences and the Health and Labour Sciences Research Grants (Grantnumber H028) from the Ministry of Health, Labour and Welfate of Japan.
The Use Of A Novel Stereological Sampling Approach To Estimate Islet, Duct And Acinar Total Cell Number And Compartment Volume In Nonhuman Primate Pancreas
Authors: Mirabile Rosanna , Kambara Takahito , Maier Curtis , Gundersen Hans Jorgen
Published: November 2023

Design‐based stereology is used to obtain quantitative estimates of biological structures from sections with known precision and unlike model‐based stereology, is free of any assumptions concerning tissue structure. Practical solutions for incorporation of stereological estimates of total number in regulatory toxicology studies should provide appropriate sections for both the estimation of cell or particle number and routine histopathology evaluation in standard paraffin format from the same animal. Development of novel sampling principles, stereological software and advancements in computer hardware technology make it possible to fulfill both these criteria. A novel form of sampling based on non‐uniform statistical sampling principles known as the Proportionator has been recently developed that provides precise estimates of non‐uniformly distributed structures efficiently. In this test system, the pancreas was used because of the inhomogeneous distribution of cell types with in the organ. Coupled with the Fractionator sampling principles, estimates of total acinar, duct and islet cell number and compartment volume can be efficiently quantified. Using a systematic uniform sampling strategy, entire pancreas from 6 nonhuman primates were sampled and processed to paraffin blocks. Disectors were generated and immunostained to delineate the islet and duct cells. Virtual images of disectors were captured and imported into the Visiopharm Integrator System (VIS) and sampled using the Autodisector/Proportionator software. Matched fields of view (FOV) were superimposed with either an unbiased counting frame (test probe for cell number) or a set of test points (test probe for compartment volume), and counts were performed. Mean total cell estimates were 656.78 × 106 (acinar), 267.7 × 106 (duct) and 49.47 × 106 (islet). Mean total compartment volumes were 1575.46 mm3 (acinar), 477.04 mm3 (duct), 35.91 mm3 (islet). Application of the Proportionator Fractionator sampling principles allowed for a more efficient, less labor intensive approach to quantitate organ sub‐structures and heterogeneously distributed cell populations. This study was conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and was reviewed by the ethical review process at the institution where the work was performed.
The New Algorithm For Calculation Of Median Lethal Dose (Ld50) And Effective Dose Fifty (Ed50) Of Micrarus Fulvius Venom And Anti‐Venom Using Mice
Authors: Saganuwan Saganuwan Alhaji
Published: November 2023

Background

One million people throughout the world are bitten each year by poisonous snakes. Of this, one‐tenth died and three‐tenth suffer some forms of disabilities. In view of this, anti‐snake venoms are currently being developed against viper and colubrid snake venoms using mice. Therefore, a new algorithm for calculation of median lethal dose (LD50) and effective dose fifty (ED50) was developed for Micrarus fulvius venom and antivenom respectively.

Methods

Therefore, this paper compared the formula of Effective Dose Fifty (ED‐50) developed by Spearman and Karber with ideal median lethal dose (IMLD‐50) formula developed by Saganuwan with a view to bringing out their difference and similarity in calculation of ED‐50 that could be used to develop a new median lethal dose formula for calculation of Micrarus fulvius venom in mice. To achieve this, various reported antivenom concentrations (17.2, 8.6, 4.3, 21.5 and 5.3 milligram per 20‐gram body of mice) were used for calculation of LD50 and ED50 of the snake venom and antivenom respectively.

Result

The LD50 value of coral snake venom using the new formula was 0.29 mg/kg body weight of mice whereas the ED50 was 428.75 mg/kg body weight respectively. The findings revealed that ED50 value (477 mg/kg) from Spearman and Karber's formula (ED50 = (logf()ED)_50 = (logf()X)_100 ‐ logf()FD/n (·t ‐ n/2)) is comparatively similar with ideal median lethal dose value (428.75 mg/kg) from Saganuwan's formula (MLD50 + MSD50/2). The new LD50 formula (LD50 = ((ED)_50/3) × Wm × 10‐4) yielded value (0.29 mg/kg) of comparative significance with reported value (0.32 mg/kg). When ED50 is equal to 2LD50, the denominator of (ED)_50/3 becomes 2.

Conclusion

Ideal median lethal dose formula of Saganuwan can be used to calculate an effective dose of Micrarus fulvius anti‐snake venom whereas the new median lethal dose formula can be used to calculate a dose of the snake venom that can kill 50% of test mice.

Support or Funding Information

This study was funded using my monthly salaries
The effect of antibiotics on the mitochondrial function in rat skeletal muscle
Authors: Morina Edon
Published: November 2023

Background

Mitochondria are the main energy producing organelle of the cell through oxidative phosphorylation (OXPHOS) and the electron transport system, and major sources of reactive oxygen species (ROS) result from superoxide (O2−) and hydrogen peroxide (H2O2). Accumulation of ROS has the potential to harm macromolecules such as DNA and RNA. How antibiotics contribute to oxidative damage in cells have not been sufficiently investigated, although Kohanski et al. (1) have proposed a mechanism of how bactericidal antibiotics increases O2− production causing an excessive production of hydroxyl radical (OH−) via the Fenton reaction. This study has therefore investigated the effect of clarithromycin, trimethoprim and penicillin on the mitochondrial respiration and H202 production in rat skeletal muscle to see if our findings correlate with Kohanski's proposal.

Method

An approach combining high‐resolution respirometry and fluorometric measurement of H2O2 production by the Oroboros Oxygraph 2K instrument was used. Skeletal muscle tissue from mice (C57BL/6JRJ) was prepared and permeabilized using saponin stock solution. A stepwise multiple substrate‐uncoupler‐inhibitor titration (SUIT) protocol was used to analyze the oxidative phosphorylation, the mitochondrial respiratory control, and H2O2 production. The drug concentrations of antibiotics were equivalent to those from clinical use. 6 muscle samples were exposed to each antibiotic and 6 were used as control. The software Datlab was used to calculate mitochondrial oxygen flux per mass [pmol/(s*mg)] and the H2O2 production using a fluorescent probe.

Results

Our findings showed that clinical doses of clarithromycin increased the mitochondrial respiration by 3.62 pmol, SD= ±4.71 compared to control which showed a decrease in respiration by −0.84 pmol, SD= ±1.16 (p‐value = 0.063). There were not observed any significant alterations in H202 production with the addition of clarithromycin as the H2O2 production increased by 0.52 pmol, SD= ±0.75 compared to control 0.011 pmol, SD= ±0.037 (p‐value = 0.164). Trimethoprim and penicillin on the other hand caused a decrease in mitochondrial respiration by −1.11 pmol SD= ±3.01 and −4.65 pmol SD= ±25.09 compared to their respective control −0.46 pmol SD= ±1.27 and 3.46 pmol SD= ±5.38, (p‐value = 0.706). No significant findings were observed with the H2O2 production.

Conclusion

Our findings suggest that clarithromycin possibly increases the mitochondrial respiration and leads to an increased production of O2−. Clarithromycin did increase the H202 production slightly. This study does not finally elucidate that antibiotics contribute to the formation of ROS, but can on the other hand assist the further understanding of drugs and mitochondrial dysfunction.

Support or Funding Information

Edon Morina1, Vanja Cejvanovic1, Laura Kofoed Kjær1, Henrik Enghusen Poulsen1, Søren Nielsen21Q7642, Department of Clinical Pharmacology, Bispebjerg Hospital; 2Department of Health Science and Technology Aalborg University
Quercetin protects against the Aβ‐induced amnesic injury through inhibiting RAGE‐mediated pathway and preserving the neurovascular unit
Authors: Liu Rui , Zhou Dan , Bai Xiaoyu , Du Guanhua
Published: November 2023

Quercetin is one of the most common flavonoids existing widely in many edible plants. It has demonstrated protective effects against Aβ‐induced toxicity on both neurons and endothelial cells. However, whether or not quercetin has an effect on the neurovascular coupling is unclear. In the present study, we aim to investigate the anti‐amnesic effects of quercetin and to explore the underlying mechanisms. The aggregated Aβ25–35 was injected into the right lateral ventricle in mice. Quercetin was administrated orally for 8 days after injection. Learning and memory behaviors were evaluated by measuring spontaneous alternation in Morris Water Maze test and the step‐through positive avoidance test. The regional cerebral blood flow was monitored before the Aβ25–35 injection and on seven consecutive days after injection. Mice were sacrificed and cerebral cortices were isolated on the last day. The effects of quercetin on the neurovascular unit (NVU) integrity, microvascular function and cholinergic neuronal changes, and the modification of signaling pathways were tested. Our results demonstrate that quercetin treatment for Aβ25–35‐induced amnesic mice improved the learning and memory capabilities and conferred robust neurovascular coupling protection. In these effects, quercetin improved the spatial learning and memory effectively across the acquisition training period and the memory capability trials in behavioral tests. Furthermore, quercetin maintained the integrity of NVU major components, reduced the level of neurovascular oxidation, modulated the rCBF value of cerebral microvessels, improved the function of cholinergic system, and regulated the neurovascular RAGE signaling and ERK/CREB/BDNF pathways. In conclusion, in Aβ25–35‐induced amnesic mice, optimal doses of quercetin administration were beneficial. Quercetin protected the NVU through reduction of oxidative damage, inactivation of RAGE‐mediated pathway and preservation of cholinergic neurons, offering an alternative medication for Alzheimer's disease.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (No. 81102830, 81073120), Beijing New‐star Plan of Science and Technology (xx2013065), and Beijing Organization Department Excellent Talents Project (2013D009008000007).
Identification of a new sesquiterpine component from Schisandra chinensis and its anti‐inflammatory effect
Authors: Park Soojin , Im Dongsoon
Published: November 2023

Background

In Oriental countries, extracts of Schisandra chinensis have been used as an anti‐fatigue and tonic agent. Because inflammatory and oxidative stress are related to chronic fatigue syndrome, it was hypothesized that Schisandra chinensis has anti‐inflammatory constituents. The effects of new compounds isolated from dry fruits of Schisandra chinensis were studied, to elucidate the anti‐fatigue effect of Schisandra chinesis.

METHODS

From an extract of Schisandra chinensis fruits, SC‐3162 was isolated. Inductions of inducible nitric oxide synthase and cyclooxygenase 2 by lipopolysaccharide were quantified by Western blotting in peritoneal macrophages isolated from thioglycol‐treated mice. A peritonitis animal model was used to test the in vivo efficacy of SC‐3162.

RESULTS

SC‐3162 inhibited the inductions of inducible nitric oxide synthase and cyclooxygenase 2 in peritoneal macrophages isolated from thioglycol‐treated mice at the mRNA and protein levels. lipopolysaccharide ‐induced productions of nitric oxide and prostaglandin E2 were inhibited by SC‐3162. Furthermore, SC‐3162 inhibited the lipopolysaccharide‐induced activations of p38 MAPK, but not that of ERK and JNK in mouse peritoneal macrophages. In addition, SC‐3162 strongly inhibited the accumulation of neutrophils, eosinophils, and basophils in the peritoneal cavity in the lipopolysaccharide‐induced peritonitis model,.

CONCLUSION

SC‐3162 suppresses inflammatory responses in peritoneal macrophages and could be a potential therapeutic agent for treatment of inflammatory diseases.
Mechanistic Investigation into Signaling Specificity of Akt/mTOR Signaling
Authors: Zhou Xin , Zhang Jin
Published: November 2023

The Akt/mTOR signaling pathway regulates critical cellular processes including cell growth, metabolism, and survival. To achieve the signaling specificity for diverse cellular functions, spatial compartmentalization has been suggested to be an important mechanism. However, the spatial regulation of Akt/mTOR is not well defined and the underlying mechanisms remain poorly understood. We characterize the subcellular Akt/mTOR activity using the newly developed genetically encoded biosensors and further examine the underlying molecular mechanisms of the subcellular Akt/mTOR regulation. We also examine the isoform‐specific regulation of Akt using Akt1 and Akt2 activation reporters. We found conformational changes occur during the activation of Akt isoforms, which are dependent on the presence of the phosphorylatable threonine 308/309 in the activation loop. Mutants of Akt1 and Akt2 carrying phosphomimetic mutations at this residue behave differently in terms of membrane translocation. Take together, our data unveil the location‐specific Akt/mTOR signaling and suggest a model for isoform‐specific activation of Akt.

Support or Funding Information

NIH grants R01 DK073368 and DP1 CA174423
The Role of VE‐PTP in Stabilizing VE‐cadherin Adhesion
Authors: Juettner Vanessa V , Dan Arkaprava , Leckband Deborah , Komarova Yulia , Malik Asrar
Published: November 2023

Vascular Endothelial (VE)‐cadherin is the main adhesion molecule of adherens junctions (AJs) that regulate permeability of the endothelial barrier. It is crucial for maintaining tissue‐fluid homeostasis by restricting the transport of plasma proteins across the endothelium. VE‐cadherin bridges endothelial cells by forming trans interactions with opposing VE‐cadherin molecules at the AJs. VE‐cadherin steady‐state kinetics is described by the rate of VE‐cadherin exchange between junctional and cytosolic pools. In our studies, we determine the role of vascular endothelial protein tyrosine phosphatase (VE‐PTP) in regulating steady‐state kinetics of VE‐cadherin at AJs. Interestingly, overexpression of wild‐type and phosphatase dead VE‐PTP mutant decreased the rate of VE‐cadherin dissociation from AJs suggesting that VE‐PTP controls stability of VE‐cadherin adhesion in a phosphatase‐independent manner. VE‐PTP overexpression also led to activation of Rac1 at AJs thus relieving tension across VE‐cadherin adhesion. To complement these experiments, we measured distribution of intracellular forces at AJs of murine endothelial cells isolated form VE‐PTP knockout model using a biophysical approach. Consistent with the proposed role of VE‐PTP in regulating retraction forces, loss of VE‐PTP led to increased stress at AJs. These findings demonstrate that VE‐PTP stabilizes VE‐cadherin adhesion by regulating the intracellular tension at AJs independent of its phosphatase activity. Future studies will investigate the molecular and signaling mechanisms of VE‐PTP‐dependent regulation of VE‐cadherin dynamics at AJs.

Support or Funding Information

Funding: National Institutes of Health grant R01 HL103922 to Y.A. Komarova, and T32 and RO1 HL45638 to A.B. Malik.
A Novel Parenteral Formulation For the Antiplatelet Agent Clopidogrel
Authors: Karim Zubair A , Venkatesan Natarajan , Ting Harold J , Betageri Guru , Khasawneh Fadi T
Published: November 2023

While platelets play a vital role in preventing excessive bleeding after injury (hemostasis), they also contribute to the genesis of thrombotic disorders. On this basis, research efforts have focused on identifying new targets and/or designing agents against these targets for therapeutic purposes. The P2Y12 receptor (P2Y12R) remains one of the most attractive antiplatelet targets given that it plays a central role in platelet function. Furthermore, clopidogrel (Plavix®), which is a P2Y12R antagonist, is one of the most commonly prescribed drugs. However, one of the limitations facing antiplatelet therapeutic strategies is the lack of safe and/or suitable parenteral antiplatelet agents or formulations in order to complement oral agents. Based on these considerations, we designed and characterized the biological activity of a liposomal formulation (i.e., soya phosphatidylcholine, 50 mg; and cholesterol, 5 mg) of clopidogrel bisulfate under ex vivo and in vivo experimental settings. The liposomal clopidogrel was then administered by intravenous route (IV) to C57BL/6 mice through their tail vein, and our results revealed that one (1) hour post injection this formulation relative to vehicle control: 1. has the capacity to dose dependently (2 or 6 mg/kg) inhibit platelet aggregation induced by the agonist ADP; 2. this inhibitory effect was found to be selective given that it produced no detectable effects on aggregation mediated by a non‐ADP dependent pathway (i.e., the thromboxane receptor pathway stimulated by the agonist U46619); 3. prolong the tail bleeding time (modulate hemostasis), again in a dose‐dependent manner; and 4. confer protection (at 6 mg/kg) against the development of occlusive events using a carotid artery FeCl3 injury‐induced thrombosis model. Collectively, we have successfully prepared and characterized a novel IV formulation for clopidogrel; which exhibits pharmacological activity on mouse platelets, at least 1 hour post administration, and under in vivo conditions. This formulation is expected to have a wide‐therapeutic application, given the limitations of the only currently available IV antiplatelet therapeutic agents (i.e., the platelet glycoprotein IIb IIIa antagonists).

Support or Funding Information

College of Pharmacy (to F.T.K), Western University of Health Sciences, Pomona, CA 91766
Studies on Pharmacokinetics/Pharmacodanymics of Enrofloxacin in Broiler chicken
Authors: Park Haechul , Kang JeongWoo , Jang Yangho , Song Jae‐Young , Lee Kwang‐Jick
Published: November 2023

Enrofloxacin(ENR) is a synthetic and second generated fluoroquinolone antibiotics, and has been showed broad spectrum antimicrobial activity. The mechanism of action of ENR is known that inactivation of DNA gyrase by interfering DNA synthesis. It is widely used in veterinary medicine in cattle, dogs, poultry, fish, and cats to treat diseases caused by various bacteria. It is reported that ENR sales amount is the highest among all of antibiotics used in chicken in Korea several years.

In this study, pharmacokinetic(PK) and pharmadynamic (PD) of ENR administered with PO and IV were carried out to understand pharmacology of ENR in chicken. To analyze PK parameters, sensitive detection method for ENR was developed and validated by liquid chromatography tandem mass spectrometry, and its metabolite ciprofloxacin in broiler chicken plasma, which was further analyzed to study the pharmacokinetics in broiler chicken. Animal study was carried out in chicken and ENR was administered with PO and IV routes with 10 mg/kg. Serum samples were taken for 20 times. The serum samples were treated for chromatography analysis. The LC‐MS/MS analysis was carried out mobile phase gradient consisting 0.1 % formic acid in D.W. (A) and 0.1 % formic acid in ACN (B) with C18 reverse phase column. Mass spectrometry was performed using the positive ion mode and the selected ion monitoring (MRM). The methods validation results showed that good linearity (R2>0.999) and the quantified average recovery of ENR was 99% at level of 10 ng g−1 ~100 ng g−1. The percent of coefficient of variation (CV) for the described method was less than 5.1 % over the range of concentrations. The limits of detection (LOD) and quantification (LOQ) were 5 and 15 ng g−1, respectively. Several PK parameters of ENR those are area under the curve (AUC), half‐life (T1/2), peak plasma concentration (C max) and so on were obtained from LC‐MS/MS analysis.

PD analysis was carried using in vitro (MIC) and ex vivo (time‐kill assay). On MIC studies, ENR(0.004~32 ug/ml) was incubated sensitive bacteria and the MIC concentration was determined by visible turbidity in incubation tubes. The ex vivo study was carried using serum samples which containing ENR from chicken. Integrated PK/PD analysis was carried out based on each PK and PD data. Based on PK/PD analysis, the resistance of ENR on chicken is very high on target bacteria such Campylobacter jejuni, we suggest that newly establishment of administration of ENR in broiler chicken.

Support or Funding Information

This research was supported by a fund(B‐1543073‐2015‐17‐01) by Research of Animal and Plant Quarantine Agency, South Korea