bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Korean Rice Cake (Seolgitteok) on Plasma Glucose, Insulin, and Satiety Hormones
Authors: Pottgen Ellen , Yoon Suk Hoo , Pham Tung , Gu Xuan , Seo Han‐Seok , Lee Sun‐Ok
Published: June 2021

A human study was conducted to investigate the efficacy of Korean rice cake (Seolgitteok) on postprandial plasma glucose, insulin and satiety hormone levels. Using a randomized‐crossover design, subjects consumed one of three rice Seolgitteok (white rice, brown rice, and mixture of white and brown rice) with a one‐week washout period. Seolgitteok contained 50 g of total starch based on rice flour analysis, and additional ingredient were consistent. Blood samples were collected intravenously at 15 minutes prior and 0, 30, 60, 90, 120, and 180 minutes after consumption. Compared to the white rice cake, plasma glucose incremental area under the curve (iAUC) reduced ~32% and 30‐120 min intervals were lower with the brown rice cake. Incremental insulin concentrations for the brown rice cake treatment were lower at 60, 90, and 120 minutes and were also reduced about 34%. The degree of reduction of plasma ghrelin, a circulating appetite‐promoting hormone, was suppressed by consumption of brown rice cake. The results suggest that brown rice Seolgitteok has a potential for use as functional food to control blood glucose and insulin levels. Funding provided by Korean Food Research Institute is greatly appreciated.
Differences in pregnancy outcome and its predictors among various nationalities residing in Kuwait
Authors: Rahman Abdur , Al‐Rashidi Haila , Akhtar Saeed
Published: June 2021

We investigated differences in maternal and infant related birth variables among various nationalities residing in Kuwait and evaluated major predictors of pregnancy outcome. In a cross‐sectional study, pregnant women admitted for deliveries between 7:00 am and 7:00 pm over a three‐month period were recruited from Al‐Sabah Maternity Hospital, Kuwait. Data were collected from 369 mother‐infant dyads on mother height (MH), mother hemoglobin (mHb), child hemoglobin (cHb), mid‐upper arm circumference (MUAC), gestational age (GA), placental weight (PW), birth weight (BW), crown‐heal length (CHL), and head circumference (HC). PW/BW ratio was analyzed. Study sample comprised of 21.7% Kuwaitis, 22% Egyptians, 12.2% Indians, 11.4% Syrians, 7.6% Lebanese and 25.1% others. Significant differences among various nationalities were observed in mHb, HC, CHL, PW and MH. Kuwaiti and Indian women had lower PW and their babies had lower HC and CHL. Prevalence of anemia (Hb <11.0 g/dL) ranged from 29.6% (Egyptians) to 57.5% (Kuwaiti) with the overall prevalence of 42.7%. Prevalence of low birth weight in this sample was 3.5%. Multiple linear regression analysis revealed PW as a significant predictor of BW, CHL and HC. In conclusion, significant differences among different nationalities were observed in mHb, HC, CHL, PW and MH and these differences were significantly predicted by PW and MH. The significance of these differences and their contributing factors needs further research.
Becoming Food Aware in Hospital: A Narrative Review of Best Practices for a Multi‐level Approach to Improve the Culture of Nutrition in Hospital
Authors: Laur C , McCullough J , Keller H
Published: June 2021

Malnutrition can develop in the community, contributing to hospital admission and may persist post‐discharge thus increasing the chance of readmission. The Nutrition Care in Canadian Hospitals (2010‐13) study identified the prevalence of malnutrition on admission to medical and surgical wards as 45%, with older patients more likely to be malnourished. Nutrition practices in these hospitals including diagnosis, treatment, monitoring and primary care follow up were ad hoc. This lack of a systematic approach has demonstrated the need for knowledge translation of best practice. A multi‐level approach is proposed to address this complex issue. Care practices discussed are based on grey literature and evidence to date, which encompasses training and methods of action for hospital staff including hospital management. Patients and their families also need to be included in this approach as being aware of the importance of nutrition can make a patient more involved in their recovery. This approach encompasses but goes beyond the UK Seven Steps to End Malnutrition and the Australian Eight‐Step Interdisciplinary Framework on the Prevention of Undernutrition. Improvements to organizational practices include use of screening tools, protected mealtimes, additional nursing or nutritional assistants during mealtimes and discharge communications to support successful transition to the community. Everyone in the hospital should be aware of the importance of nutrition and their role in the optimization of nutrition care. Overall, this proposed multi‐level approach aims to improve the culture of nutrition in hospitals so issues are addressed before patients are discharged. Funder: Technology Evaluation in the Elderly Network
Synaptic Abnormalities of Mice Lacking Toll Like Receptor (TLR)‐9
Authors: Patel Vishwendra , Patel Axay , McArdle Joseph
Published: June 2021

TLR9 is abundant in the central as well as the peripheral nervous systems. Although, motor and sensory abnormalities are reported for TLR9 knock‐out (KO) mice, a physiological role of TLR9 in the nervous system is largely unknown. Since altered synaptic transmission can contribute to sensory and motor abnormalities, we evaluated neuromuscular junction (NMJ) function and morphology of TLR9 KO mice. Triangularis sterni nerve‐muscle preparations were dissected from TLR9 KO and age‐matched control mice. Two electrode voltage clamp of the motor endplate revealed that the amplitude (nAmp) and frequency (sec‐1) of miniature end plate currents (mEPCs) for TLR9 KO NMJs (N=22) increased (p<0.001) to 4.7 ± 0.21 (mean ± SEM) and 52.7 ± 15.04 from the control (N=31) values of 3.5 ± 0.12 and 10.4 ± 1.03. In contrast, mean endplate current (EPC, 1Hz) amplitude was equivalent to control. The ratio of mean EPC to mean mEPC amplitude indicated a decline (p<0.006) of quantal content (m) from the control value (N=14) of 104 ± 5.9 to 80 ± 5.3 for TLR9 KO NMJs (N=13); furthermore, m declined quicker during 50 Hz stimulus trains. A rightward shift of the mEPC amplitude distribution (6240 mEPCs from 22 NMJs of both groups) suggested formation of vesicles containing larger amounts of acetylcholine (ACh). Staining with rhodamine α‐bungarotoxin revealed a decline of endplate volume from 2201 ± 116.0 µm3 (N=36) in control to 1751 ± 89.6 µm3 (N=44) in TLR9 KO mice. This decline of AChRs may result from ACh‐induced decline of AChR expression resulting from increased frequency and amplitude of mEPCs at TLR9 NMJs. At the same time, excessive ACh may induce retrograde suppression of the exocytosis process. Overall, these data suggest a novel role of TLR9 in the maintenance of motor nerve terminal and endplate properties. Supported by The Kirby Foundation
Vitamin D and Cognition: Findings from a Mendelian Randomisation Study
Authors: Maddock Jane , Cavadino Alana , Power Chris , Hyppönen Elina , Study Group Vitamin D and Cognition
Published: June 2021

Hypovitaminosis D has been associated with poor cognitive function, but questions surrounding confounding and reverse causality remain. We used Mendelian randomisation (MR) to estimate the relationship between 25(OH)D, a marker of vitamin D status, and cognition. Since genetic variants are randomly assigned and present from birth, confounding and reverse causality are minimised.

We used data from 9 European cohorts (N=43,954). DHCR7 (rs12785878), CYP2R1 (rs12794714) and their combined genetic score provided a proxy for 25(OH)D. Cognitive tests were standardised into global and memory scores. All analyses were stratified by study‐specific 25(OH)D tertiles to assess non‐linearity. Meta‐analyses assessed the observational and genetic associations between 25(OH)D concentrations and cognitive function. Random effects models were used where pheterogeneity<0.05, otherwise fixed effects meta‐analysis was applied.
Association of 25(OH)D with global cognition and memory was non‐linear, where both high and low levels were associated with poorer cognition. However neither DHCR7, CYP2R1 nor their combined score were associated with global cognition or memory.

While there was no evidence for a causal association between 25(OH)D and cognitive function in genetic analysis, the observational association was non‐linear. Analyses may be underpowered to detect small causal effects operating at the extremes of the distribution.
New Insights into Macaque Hyolingual Anatomy: A Preliminary microCT Study
Authors: Orsbon Courtney , Nabavizadeh Ali
Published: June 2021

The macaque (Macaca mulatta) is a promising model organism for the integrated study of the neurological and biomechanical basis of human feeding disorders. However, while feeding behavior has been extensively studied in the macaque, we have limited knowledge about its three‐dimensional hyolingual myoarchitecture, especially of the intrinsic musculature of the tongue. This preliminary study reports the results of a digital dissection utilizing radiopaque staining techniques and micro‐computed tomography. A 1:1 solution of 5% Lugols and 190 proof ethanol increased muscle contrast in the isolated hyolingual apparatus of a female Rhesus macaque, permitting segmentation of these tissues to resolve their three‐dimensional morphology. The scans showed that the macaque hyolingual apparatus shares many similarities with that of humans, excepting well‐known differences such as a more cranial hyoid resulting in a flatter tongue. However, the posterior third of the tongue has significantly higher levels of connective tissue relative to the anterior two‐thirds of the tongue. Given the importance of the posterior tongue in the “squeeze back” mechanism of swallowing seen in macaques and humans, such architecture may have important functional implications during feeding. Future directions include utilizing similar methods for comparative study among other mammals (including humans), marker‐based study of three‐dimensional soft tissue kinematics, and development of a computational model for feeding behaviors.
Establishing Self‐regulatory Guideline for the Functional Claims of Health Foods Including Dietary Supplements in Japan
Authors: Amagase Harunobu
Published: June 2021

Within the scopeof the Revitalization Strategypolicies and elongating healthy life span, government of Japan has been in the process to set a new labeling system of health claims for processed foods and fresh foods, in which industries can describe functionality based on scientific evidence under industry responsibility, without individual reviews by the government that is currently required for Foods for Specified Health Uses. This new labeling rule is referred to the labeling system of the dietary supplement in the US. Government of Japan explored the way of appropriate labeling. A consumer survey was conducted in 2014 to clarify basic knowledge regarding currently revealed issues such as the evidence level to determine functionality labeling and the readability of the labeling among general consumers including specific population assumed to easily misunderstand health claims. Based on the survey results, issues and the new system will be discussed by relevant experts. The new rule is planned to be finalized and implementedby March 2015. This will be a major shift in the food functionality labeling system. An overview of the perspective of the studies on the new functionality labeling system, with focuses on the meaning of the cabinet decision statement and the main issues that are anticipated in the future, will be presented.
Cerebellar Malformations in the Tmem67 Ciliopathy Mouse Model are Caused by Combined Wnt and Shh Signalling Systems Dysregulations
Authors: Abdelhamed Zakia , Toomes Carmel , Inglehearn Chris , Johnson Colin
Published: June 2021

Objectives

This study focuses on the characterization of the cerebellum in the newly‐described Tmem67tm1(Dgen) knockout ciliopathy mouse model.

Methods

various molecular, immunohistochemical,primary neuronal cell culture methods and RNA next generation sequencing were used in this study.

Results

:The Tmem67 mutant cerebellar vermis was hypoplastic with foliation defects. Mutant cerebellar hemispheres had severe foliation defects and malformation of the inferior lobe, concomitant with aberrantly low canonical Wnt/β‐catenin signalling. Shh transcript levels were unaffected, but the levels of Shh signalling pathway modulators such as Ptch1 and Gli1 were significantly reduced. This resulted in reduced proliferation of granule cell precursors (GCP), with exogenous Shh stimulation failing to rescue the GCP defect. RNA sequencing and hierarchical clustering analysis identified a total 28 differentially expressed genes between Tmem67 mutant and wild type control cerebellum. Unexpectedly, a single clustered group of homeobox‐type transcription factors (Hoxa5, Hoxa4, Hoxb5 and Hoxd3) were all significantly up‐regulated in the mutant cerebellum. Immunohistochemical analysis confirmed this finding.

Conclusion

Results suggest that cerebellar hypoplasia phenotype in the Tmem67 model is a combination of Wnt/β‐catenin and Shh signalling defects, causing significant changes to downstream gene expression patterns and cell‐fate specification.
LR‐1 Antibody Staining in Kisspeptin Receptor Knockout Mice Brains Reveals Axonal Diameter Differences in GnRH Neurons Due to Reduced GnRH Protein Levels
Authors: Murphy Kelley , Wu Sheng , Novaira Horacio , Wolfe Andrew , Radovick Sally , Hoffman Gloria
Published: June 2021

Kisspeptin, through its receptor, GPR54, regulates gonadotropin releasing hormone (GnRH) mRNA. However, the relationship between mRNA and protein is controversial and confounded by the lack of a method for analyzing protein levels from tissue sections. GnRH mRNA is markedly suppressed in GPR54GnRH knockout (KO) mice. We determined whether reduced mRNA dictated less protein expression in the neurons. We examined two features: axon diameter and somal size. We hypothesized that the absence of GPR54 would produce decreased GnRH levels reflected by the thickness of the immunostained axons and the size of the cell bodies. Sections from global and conditional GPR54 KO mice were stained using the ABC immunoperoxidase method and compared to sections from wild type controls. We first learned that the optimum staining conditions for the KO mice grossly overstained the WT. When conditions were adjusted to be in the linear part of the titration curve, obvious differences in protein levels emerged. Axons containing GnRH were imaged at a magnification of 200x, then skeletonized to 1 µm width, enabling calculation of total axon length in the field. The axon area divided by the skeletonized length would then be equivalent to the axon diameter. Axon volume was calculated. For cells, somal width was determined. Our results demonstrate that the GnRH+ axons in GPR54 KO mice are approximately three times thinner than WT axons and somal diameter was proportionately reduced. The results stress the importance of primary antibody titrations.

Funded by NIH grants R01HD370246, U01HD66435 and U01HD66432 to SR and GEH
Inhibition of Polyamine Oxidase Reduces Excitotoxicity‐mediated Retinal Neuro‐inflammation
Authors: Narayanan S.Priya , Patel Chintan , Xu Zhimin , Xing Ji , Rojas Modesto , Caldwell Ruth
Published: June 2021

Cellular damage resulting from polyamine catabolism has been demonstrated to be a major player in many neurodegenerative conditions of the central nervous system. We have previously shown that inhibition of polyamine oxidase (PAO) using MDL 72527 significantly reduced neurodegeneration and cell death signaling pathways in hyperoxia‐mediated retinal neurodegeneration (Narayanan et al 2014). In the present study we investigated the impact of PAO inhibition in NMDA (N‐Methyl‐D‐aspartate)induced excitotoxic retinal neurodegeneration. A marked increase in retinal ganglion cell (RGC) death was observed in NMDA treated retinas compared to their sham and NMLA (N‐Methyl‐L‐aspartate) treated controls, as studied by quantitation of NeuN‐positive RGCs. Treatment using MDL 72527 reduced this RGC loss in NMDA treated retinas. This was confirmed by TUNEL labeling studies and morphometry analysis on retinal sections. Further studies demonstrated increased activation of microglia (studied by Iba1 immunostaining) and elevated ROS (reactive oxygen species) formation in NMDA treated retina, suggesting the involvement of inflammatory signaling in NMDA mediated neuronal injury. These changes were abrogated in response to PAO inhibition. Analysis of signaling pathways activated in response to excitotoxicity, showed increased level in BID expression, which was reduced by MDL 72527 treatment. Taken together, our data demonstrate that PAO inhibition offers a new therapeutic target for limiting central nervous system neuroinflammatory diseases.
Concurrent Lung Pathology in Dogs with Chronic Liver Disease
Authors: Montgomery Julia , Montgomery James , Simko Elemir , Hendrick Steve
Published: June 2021

Remote lung injury is a frequent sequela of chronic liver disease, especially liver cirrhosis, in people. Anecdotal evidence of concurrent lung pathology in dogs with chronic liver disease warrants further investigation in this species. The objective was to perform a retrospective analysis of dogs diagnosed with cirrhotic and non‐cirrhotic chronic liver disease and determine if there is radiographic and/or histopathologic evidence of concurrent pulmonary pathology. Dogs were diagnosed with chronic liver disease based on history, clinical signs, clinicopathological abnormalities, diagnostic imaging, biopsy, and/or necropsy. Only dogs where liver disease was confirmed with diagnostic imaging, liver biopsy, and/or necropsy were included in the study (n=28). Evidence of concurrent lung pathology was based on radiographic and/or histopathologic findings. 23/28 dogs (82%) (95% CI: 63‐94%) had evidence of lung pathology. 18/28 dogs (64%) (95% CI: 44%‐81%) had an interstitial pattern on thoracic radiographs. A specific diagnosis of liver cirrhosis was not related to concurrent lung pathology (P=0.53) or an interstitial lung pattern (P=0.42). In conclusion, lung pathology was quite common (>80%) in dogs with liver disease. However, there was no clear relationship between liver cirrhosis and concurrent lung pathology or interstitial lung pattern in our group of dogs. Prospective research is needed to further evaluate dogs as an animal model for study of remote lung injury in chronic liver disease.
The Effects of Reactive Oxygen Species (ROS) on Human Malignant Mesotheliomal Cells
Authors: Kim Na‐yon , Kim Hang‐ah , Kim Myung‐chul , Seo Yu‐ri , Kim Yongbaek
Published: June 2021

It is widely accepted that reactive oxygen species (ROS) are associated with the acquisition of malignancy in cancer cells. The ROS are also known to contribute to the mesothelial carcinogenesis linked to asbestos exposure and accompanying inflammation. The present study was performed to determine the effects of ROS on the biological phenotypes of human malignant mesotheliomal (HMM) cells.

HMM cell lines were cultured and treated with H2O2 at different concentrations, and subjected to the analysis of the cancer‐related characteristics. Treatment of HMM cells with H2O2 decreased cell survival. In wound healing assay, increased number of migrated cells was observed as H2O2 concentration increased. Clonogenicity assay revealed that the number of small sized colony was increased in a dose‐dependent manner. Cell cycle analysis by PI staining showed that H2O2 induce S phase arrest in HMM cells. The study results suggest that increase in ROS concentration to some extent influences the overall biological characteristics of HMM. Though treated H2O2 did not highly affect the viability of cells, it induced change in mobility, clone forming ability, and cell cycle distribution, indicating that ROS is not a passive bystander but a driving force of acquirement of aggressive phenotypes.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant #: NRF‐2013R1A2A2A01068237)
Keratin 17: Cervical Cancer Prognostic Marker Promotes p27‐Nuclear Export and Tumor Growth
Authors: Escobar‐Hoyos Luisa , Shah Ruchi , Roa‐Peña Lucia , Vanner Elizabeth , Akalin Ali , Shroyer Kenneth
Published: June 2021

Recently, we identified keratin 17 (K17) as a negative prognostic marker for cervical cancer and based on preliminary data, we set out to determine if K17 is related to tumor stage and sustained proliferation. K17 and tumor suppressor p27 were detected by immunohistochemistry in cervical cancer specimens and multivariate survival analysis was conducted. Loss and gain of function approaches were performed by in vitro and in vivo by RNAi and transgenic expression of wild type and mutant K17 in cervical cancer cell lines. The five‐year survival for low K17 cervical cancer cases was 90% versus 52% for high K17 cases; the differences were consistent across low and high stage groups. K17 expression promoted cell proliferation and tumor growth and silencing of K17 induced G1 arrest and decreased tumor mass by nuclear stabilization of p27, a key regulator of G1/S transition. During G1, K17 was found in the nucleus bound to nuclear p27. Nuclear p27 was lost in cells expressing wild‐type K17 but nuclear p27 levels were 3‐fold higher in cells expressing K17 with either mutated nuclear localization signal (NLS) or mutated nuclear export signal (NES). To our knowledge, this is the first report of the presence of NLS and NES in any keratin. Finally, we determined that K17 and p27 are inversely expressed in human cervical cancer specimens, intratumorally and intertumorally. These data suggest a model in which nuclear K17 acts as an oncoprotein regulating sustained proliferation in cancer cells by acting as a bridge between p27 and Exportin 1 binding, controlling nuclear export and degradation of p27, contributing to poor patient outcome of cervical cancer patients.
Effect of vascular endothelial growth factor receptor inhibition on the regenerating axolotl tail
Authors: Dickie Renee , Ritenour Angela
Published: June 2021

Vascular endothelial growth factor (VEGF) signaling plays a crucial role in regulating angiogenesis, but its effect in regenerating salamander appendages has not been examined. The objective of this study was to evaluate the effect of vatalanib, a small molecule inhibitor of vascular endothelial growth factor receptor, on tail regeneration in Ambystoma mexicanum. Following partial tail amputation, juvenile axolotls were exposed to either 100nM vatalanib, 500nM vatalanib, or the equivalent volume of dimethyl sulfoxide as a vehicle control. Treatment was continued and animals imaged weekly until the tail had fully regenerated in the control animals. Both dosages of vatalanib inhibited regenerative angiogenesis but had little effect on regenerative outgrowth of the tail. We conclude that in this system, significant tissue regrowth is possible in the absence of vasculature.
SciCats: An Integrative Outreach Initiative to Enhance STEM Education in Our Community
Authors: Krentz Madeline , Garneau‐Tsodikova Sylvie
Published: June 2021

Objective

To bolster the Science, Technology, Engineering, and Math (STEM) education of K‐12 students by providing dynamic, hands‐on experiences aligned with national education standards while providing professional‐development for university students.

Methods

University of Kentucky (UK) students in the allied‐sciences programs volunteer to work with an elementary school class for a calendar year. Graduate students collaborate with UK professors and their assigned teachers to design lesson plans in line with the core standards tailored to be palatable to specific grade levels. Lessons are led by UK students in order to offer advanced science students an experience in teaching and conveying difficult concepts at a lay level. Gifted high‐school students also work on developing exciting experiments for primary school students.

Results

Elementary student science interest increased drastically in the first four trial months of this initiative. Interest in science for students that originally reported that they did not like science has also increased. UK graduate students involved in the program have noticeably enhanced communication skills over students who have not yet chosen to participate in the program. Post‐secondary science students are not commonly taught how to teach, and participation in this program allows them to learn, practice, and perfect the art of teaching and communicating with the mentorship of a professional educator.

Conclusion

Participation in the program increases scientific education of K‐12 students. UK students who participate in the program gain the teaching and communication skill, organization and preparedness, and the community‐outreach commitment qualities valued in academic and industrial researchers alike.
Maternal Delay and Social Discounting Behavior Predicts Impaired Mother‐Infant Bonding, Maternal Stress, and Paternal Involvement
Authors: Brents Lisa , Young Jonathan , Knight Bettina , Coker Jessica , Ray‐Griffith Shona , James Andrew , Stowe Zachary , Kilts Clint
Published: June 2021

Maternal care is a major determinant of offspring well‐being, but psychiatric conditions, such as depression and addiction, can interfere with maternal behavior and lead to poor offspring outcomes. We report an ongoing pilot study performed to develop tasks that objectively quantify maternal behavior in three groups of early postpartum women: healthy, depressed and opioid dependent. These innovative tasks are modified classical behavioral paradigms that exploit the Stroop effect (infant Stroop task, iStroop), and delay and social discounting (Maternal Baby Discounting Task, MBDT). Preliminary pooled data (n=4) show strong correlations between multiple MBDT discounting behaviors and mother‐infant bonding (Postpartum Bonding Questionnaire), stress (Parenting Stress Index‐Short Form), and paternal involvement (Parental Responsibility Scale). Lower delay and infant discounting was associated with impaired bonding, pathological anger and rejection, total stress, and paternal involvement. As expected in the iStroop, infant emotion (distressed versus neutral) had no effect as a distractor in this small, primarily depressed sample.

Early results suggest that the iStroop and MBDT predict bonding, stress, paternal support and sensitivity to infant cues. The format of these tasks makes them ideal for use in neuroimaging studies to provide insight into the neural mechanisms underlying normal and disrupted maternal brain‐behavior development. This study was supported by NIH 2T32DA022981‐06 and a BIRC Endowment Fund grant.
Mediation of Opioid Analgesia by a Truncated Six Transmembrane G‐Protein‐Coupled Receptor
Authors: Lu Zhigang , Xu Jin , Rossi Grace , Majumdar Susruta , Pasternak Gavril , Pan Ying‐Xian
Published: June 2021

Potent opioid analgesics lacking side‐effects may be possible by targeting truncated mu receptor splice variants. Mu opioids act through G‐protein coupled receptors generated from the mu opioid receptor (Oprm1) gene which undergoes extensive alternative splicing. The most abundant set of variants encode classical full‐length 7 transmembrane domain (TM) mu opioid receptors that mediate the actions of the traditional mu drugs morphine and methadone. In contrast, IBNtxA (3‐iodobenzoyl‐6β‐naltrexamide) is a potent analgesic against thermal, inflammatory and neuropathic pain whose actions are independent of the 7TM mu opioid receptors and lost in a knockout mouse lacking a set of 6TM truncated mu receptor splice variants. Unlike traditional opioids, IBNtxA has no respiratory depression, physical dependence or reward behavior, suggesting it acts through a novel mu opioid receptor target. Here we demonstrate that a truncated 6TM splice variant, mMOR‐1G, can rescue IBNtxA analgesia in a mu opioid receptor knockout mouse lacking all Oprm1 splice variants due to disruptions of both exons 1 and 11. No mu opioids were active in these mice. A lentivirus containing the 6TM variant mMOR‐1G administered intrathecally restored IBNtxA, but not morphine, analgesia, confirming that a truncated 6TM GPCR is both necessary and sufficient for IBNtxA analgesia.

This work was supported by the NIH with grants from NIDA (DA013997 and DA02944) to Y‐X.P. and (DA06241 and DA07242) to G.W.P. and a core grant from the NCI to MSKCC (CA08748).
Investigation of the Properties of the CART Receptor in PC‐12 Cells
Authors: Prinster Steven
Published: June 2021

The cocaine‐ and amphetamine‐regulated transcript (CART) was first isolated from rat striatum following the acute administration of cocaine or amphetamine. It was quickly determined that CART or CART peptide was expressed in a variety of tissues affecting a wide range of important processes. The function of these areas have grown to include feeding behavior, pleasure and reward, diabetes, ovarian follicle development, bone remodeling and anxiety. In spite of the relevance of these conditions as therapeutic targets, development of small molecules to modulate these systems has been impeded by the lack of an identified receptor for CART peptide. Localization studies suggest that CART and CART peptide are located in similar areas: hypothalamus, pituitary and adrenal gland. Cells isolated from these tissues have provided information delineating much of the current state of knowledge regarding the CART peptide receptor. We have characterized the signaling pathways activated by CART peptide in rat PC‐12 cells. We observed a pertussis‐toxin sensitive alteration of cAMP production, and phosphorylation of extracellular signal regulated kinase 1 and 2. Effects were also observed on the phosphorylation of protein kinase B. These results provide addition evidence that the receptor responsible for mediating the effect of the CART peptide is a Gi/o‐coupled receptor. These CART peptide studies provide important information that will aid in the identification of CART peptide receptor.
Use of Pharmacological Modeling to Understand Key Questions on the GOAT Ghrelin Processing In Vivo
Authors: Yang HsiuChiung , Kuo Ming‐Shang , Tong Jenny , Reynolds Nichole , Shockley Travis , Chen Yanyun , Mosior Marian
Published: June 2021

Ghrelin (AG) is the only known orexigenic peptide in human and rodents. It is converted from its precursor, unacylated ghrelin (UAG), to the biologically active peptide, acylghrelin. Two independent groups (Yang et al., 2008; Gutierrez 2009) using different approaches identified the same protein, named GOAT (Ghrelin O‐Acyl Transferase), as the long sought enzyme which is responsible for the bioactivation of ghrelin peptide. Results from GOAT gene knockout mice confirmed that GOAT is the principal enzyme which is responsible for the acylation of ghrelin. We have developed a GOAT enzymatic assay. Using this assay, we have demonstrated the detailed molecular mechanism of the GOAT enzymatic reaction. In addition, we used genetic and pharmacologic data to build a model for GOAT/ghrelin processing in mouse. We also used plasma acylghrelin and desacylghrelin profiles from human volunteers, following three doses of acylghelin infusion, to enrich this model and improve understanding of the dynamics of ghrelin processing in humans. We will present the ghrelin processing models for human and mouse, as well as how we use these models to answer key questions regarding ghrelin metabolism and regulation. Our data suggest that plasma unacylated ghrelin is secreted from the stomach, and not generated through deacylation in the plasma.
Utilizing a Novel Human P450 Biocatalytic System for the Multi‐Cycle Production of Metabolites
Authors: Martinez Enrique , Das Shuvendu , Callewaert Denis , Subramanian Mani
Published: June 2021

The large‐scale preparation of drug metabolites is typically a lengthy and problematic step in pharmaceutical development. We have characterized a robust P450 Catalytic System (PCS) which contains unmodified recombinant human cytochrome P450 (CYP) and oxidoreductase enzymes. The engineered inclusion of cofactors and antioxidants within its semipermeable structure greatly stabilizes the PCS and increases its usable lifetime. The present study focused on evaluating the ability of PCS expressing different isoforms to absorb and sequester substrate and generate additional product in multiple reaction cycles. After incubation at 30°C with 500 µM substrate for an initial 4 hr. reaction cycle, the PCS was pelleted via low speed centrifugation and resuspended in fresh buffer containing glucose‐6‐phosphate but no additional substrate, and incubated for an additional 4 hr. (cycle 2). The substrate and product concentrations in the reaction supernatant and that sequestered within the PCS system after each reaction cycle were determined by reversed phase HPLC. The second cycle produced significant amounts of additional product.

Isoform
Substrate
Cycle 1 Yield, µM
Cycle 2 Yield, µM

1A2
Phenacetin
43
28

2C9
Diclofenac
68
308

2C19
Mephenytoin
43
95

2D6
Dextromethorphan
68
89

3A4
Testosterone
47
46

With vigorous agitation to provide sufficient oxygen, the PCS retained P450 activity for multiple reaction cycles performed at 30° ‐ 37°C and for substrate concentrations ranging from 200 – 1,000 µM. Similar results were obtained for reaction volumes ranging from 200 µL to 400 mL, with the larger scale reactions typically yielding several milligrams of product. After the first reaction cycle, the PCS could also be stored at 4°C overnight without significant loss of activity.
Ischemia‐Reperfusion‐Inducible Protein Modulates the Activity of Metabolizing Enzymes
Authors: Obianom Obinna , Shu Yan
Published: June 2021

Ischemia‐Reperfusion‐Inducible Protein (IRIP) belongs to the SUA5/YrdC/YciO protein family. Alterations in this protein have been shown previously to affect the gene expression and activity of various drug transporters via posttranscriptional mechanisms yet to be fully unraveled. Herein, we further evaluate the effect of IRIP on metabolizing enzymes. Microarray study was performed using cDNA obtained from liver tissues of liver specific IRIP knockout and wild‐type mice. We identified several metabolic genes, which were significantly changed in knockout mice compared to wild‐type mice.

Locus ID
Gene Symbol
Gene Description
P‐value
Fold Change

13088
CYP2B10
cytochrome P450
4.74E‐03
2.17

13087
CYP2A5
cytochrome P450
6.37E‐03
0.42

11761
AOX1
aldehyde oxidase 1
5.27E‐03
0.42

14859
GSTA3
glutathione s‐ transferase
5.59E‐03
0.40

13096
CYP2C37
cytochrome P450
7.53E‐03
0.37

14858
GSTA2
glutathione s‐transferase
4.52E‐03
0.37

56615
MGST1
microsomal glutathione s‐transferase 1
9.80E‐04
0.32

71773
UGT2B1
RIKEN cDNA 1300012D20 gene
1.74E‐03
0.30

14857
GSTA1
glutathione s‐transferase
7.10E‐04
0.29

14859
GSTA3
glutathione s‐transferase
1.00E‐04
0.26

14860
GSTA4
glutathione s‐transferase
8.30E‐04
0.25

66447
MGST3
microsomal glutathione s‐transferase 3
1.10E‐04
0.23

11522
ADH1
alcohol dehydrogenase
2.71E‐03
0.17

Subsequent studies using western blots, RT‐qPCR

image

and activity assays especially focusing on microsomal and cytosolic glutathione‐s‐transferases and alcohol dehydrogenases were performed. At the mRNA expression level, several genes including glutathione‐s‐transferases (GST) were upregulated, while their protein levels were significantly down‐regulated. GST activity assay measured by 1‐chloro‐2, 4‐dinitrobenzene metabolism showed a decreased GST activity in knockout mice compared to wild‐type littermates.

image

These findings suggest that IRIP can modulate the expression of some key metabolizing enzymes in the liver in addition to already identified transporters through a mechanism consistent with posttranscriptional modifications, with potential implications in the disposition and metabolism of endobiotics and xenobiotics.
Enhanced Expression and Purification of a SUMO‐Metallothionein Chimeric Protein Using an Improved Fermentation Method
Authors: Nguyen Phuc , Jordaan Gwen , Nguyen Hieu , Acey Roger
Published: June 2021

We have been developing a method for improving the expression and yield of Artemia metallothionein (MT). The protein has the unique ability to selectively bind heavy metals, e.g., lead, mercury, gold, and uranium. Therefore, MT could be used as a “heavy metal sponge” for the removal and/or recovery of toxic and precious metal from aqueous solutions. Preliminary evidence indicates the protein could also be used to remove metals from organic solvents. Our goal was to develop a large‐scale fermentation process to enhance the yield and simplify the purification of MT. This involved the use of an MT chimeric protein. An N‐his‐tagged SUMO (small ubiquitin‐like modifier) MT fusion construct was cloned and the recombinant protein expressed in an inducible T7 expression system in E. coli. The SUMO tag was incorporated to enhance the expression and solubility of MT. The media used for fermentation was supplemented with a trace metal solution and ammonium chloride, and fermentation was maintained at pH 7.0. There was an 8‐fold increase in collected cell mass. Purification was by affinity chromatography using Ni2+‐NTA resin. The chimeric protein retained its metal binding activity. The concentration of eluted protein was four times more than previously purified by other methods. Analysis of the eluted protein by SDS‐PAGE showed a 22 kDa protein consistent with the size of the MT fusion protein. Supported in part by a grant from CSUPERB and the CSULB Mini‐Grant Program.
Modulation of Intrinsic and Extrinsic Apoptotic Pathways by Macrolide Antibiotics in Chemically‐Induced Hepatocellular Carcinoma
Authors: El‐Azab Mona , Abdel Hamid Nagwa , Moustafa Yasser
Published: June 2021

Hepatocellular carcinoma(HCC) is one of the most common solid malignancies and a leading cause of cancer‐related deaths. Macrolide antibiotics demonstrated beneficial effects in several pathological conditions however; their therapeutic potential in cancer is not fully elucidated. The current study aimed to investigate the individual anti‐tumor effect of some macrolide antibiotics in chemically‐induced HCC. HCC was induced in rats by initiation‐selection‐promotion protocol using diethylnitrosamine (200 mg/kg, single i.p. injection)/2‐acetylaminofluorene (2‐AAF) (0.03%w/w supplemented diet for 2 weeks/carbon tetrachloride (2ml/kg diluted in corn oil 1:1, single intra‐gastric dose)/phenobarbitone sodium (0.05%w/w supplemented diet for 28 weeks). Macrolides were administered once daily from the beginning of the 3rd week until week 17 at a dose of 100mg/kg and study was terminated on week 32. Current data proved that clarithromycin suppresses HCC via induction of apoptosis through up‐regulation of both extrinsic and intrinsic apoptotic pathways proteins (TNF‐α, TNFR1, cytochrome‐c, caspase‐3, and Bax with increased Bax/Bcl2 ratio). Similarly azithromycin reduced the tumorgenic effect of the former carcinogenic agents, however to a lesser extent compared to clarithromycin. Surprisingly, erythromycin worsened the tumorgenic effect of the carcinogenic agents used in the current model with insignificant apoptotic outcome. It is concluded that the anticancer potential of some macrolide antibiotics is an individual effect rather than a group effect and the current detected anti‐tumor mechanism involved the activation of both intrinsic and extrinsic apoptotic pathways.
Antiproliferative and Cytotoxic Evaluation of Herbal Supplement SAABFAT6 on HT29 Colorectal Adenocarcinoma Cells
Authors: Kwende Syntia , Yakubu Momoh
Published: June 2021

The consumption of herbal supplements has been one of the remedy for several aliments including cancer for a long time. Cancers, especially colorectal adenocarcinoma are diseases with high morbidity and mortality and are often associated with suffering and poor quality of life. Herbal supplements are an attractive cancer therapy, we have investigated the antiproliferative and cytotoxic properties of the herbal supplement SAABFAT6 on colorectal adenocarcinoma cell (HT29). Ethanol extracts of SAABFAT6 (0.5‐2 mg/mL) was incubated with HT29 and in vitro antiproliferative/cytotoxicity activities were evaluated using MTT assay. Treatment of HT29 with SAABFAT6 significantly reduced cellular proliferation at 48 and 72 but not at 24 hrs except for 2 mg/mL concentration. Cytotoxic evaluation showed significant reduction in cell viability at 24 and 48 hrs (2 mg/mL) and at 48 hrs (0.5 and 1 mg/mL). This preliminary result indicates that SAABFAT6 supplement may have a potential for the regulation of HT29 growth and survival. Further study is required to identify the mechanism(s) by which the antiproliferative and cytotoxic effects of SAABFAT6 is mediated.
Microvascular Responses to Ischemic Stroke following Splenectomy
Authors: Seifert Hilary , Russell Janice , Granger Daniel
Published: June 2021

Splenectomy is known to protect the brain against the tissue injury that results from ischemia and reperfusion (I/R). The mechanisms that underlie this neuroprotective effect of splenectomy remain poorly understood. An early response to brain I/R is the recruitment of inflammatory cells and platelets into the cerebral microvasculature. The objective of this study was to determine if splenectomy alters the recruitment of adherent leukocytes and platelets in cerebral microvessels in the early phase (24 hr) of reperfusion following ischemic stroke. Wild type C57Bl/6 mice underwent splenectomy or sham splenectomy two weeks prior to 45 min middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. After 24 h of reperfusion, intravital fluorescence video microscopy was used to monitor and quantify the adhesion of leukocytes (rhodamine‐G6 labeled) and platelets (CFSE‐labeled) in cerebral venules. Infarct volume and peripheral blood counts were also measured. Splenectomy attenuates the recruitment of adherent platelets and leukocytes in the cerebral microvasculature and reduces infarct volume. The increased blood platelet count observed following MCAO was reduced to control levels in splenectomized animals. Peripheral leukocyte count did not differ between the splenectomized and sham splenectomized mice. These results implicate the spleen in I/R‐induced brain inflammation, thrombogenesis, and tissue necrosis as early as 24 h after reperfusion (Supported by a Malcolm Feist Postdoctoral Fellowship)
Disturbed Shear Stress Promotes Caveolae‐Mediated oxLDL Uptake Leading to Increased Endothelial Stiffness
Authors: LeMaster Elizabeth , Eddington David , Cho Michael , Minshall Richard , Levitan Irena
Published: June 2021

Introduction

Oxidized low density lipoprotein (oxLDL) is a major risk factor in the development of atherosclerotic plaques, which preferentially form at vascular bifurcations subject to bidirectional recirculating disturbed shear stress (DSS).

Methods

A microfluidic channel with a step barrier was designed, tested and fabricated to generate atheroprotective unidirectional laminar shear stress (LSS) and atheroprone DSS onto an EC monolayer for 48 hours. EC stiffness was assessed using atomic force microscopy.

Results

Our results show a statistically significant increase in oxLDL uptake in DSS vs. LSS regions in three EC types: human aortic (55% increase), human microvascular (40% increase), and mouse microvascular (50% increase). OxLDL uptake was reduced in cells lacking Cav1, the main protein in caveolae (n=4, p<0.05). The mechanism of differential oxLDL uptake was further investigated using siRNA treated ECs for CD36 and Lox1, two oxLDL receptors. Our experiments revealed that CD36 (n=4, p<0.05), but not Lox1 (n=4, p<0.05), is required for increased uptake under DSS. Furthermore, the increased uptake under DSS results in a 60% increase in EC stiffness (n=3, p<0.05). This data is further supported by ex vivo stiffness measurements of vessels from WT mice which show a 50% increase in EC stiffness in the aortic arch (DSS) than the LSS region of the descending aorta (DA) (n=5, p<0.05). In Cav1 KO mice, EC stiffness was markedly reduced (n=5, p<0.05) and no differences observed between the DA and arch.

Conclusion

Taken together, this study suggests that DSS‐induced increase in oxLDL uptake is mediated by caveolae and plays a significant role in increasing localized EC stiffness.
About system evaluation of health and diagnostics level of adaptable possibilities of an organism of teenagers and youth
Authors: Slivkina Nataliya
Published: June 2021

The problem of health condition studying of growing generation has an important state value, being the basic criterion of planning of social transformations in the society.

Research objective was to develop the system of prenosological diagnostics on the basis of evaluation of adaptable possibilities of the organism of teenagers and youth.

Features of functional condition of the organism of youth were: the increase in quantity of people with mixed tone and sympaticotonia, increase of irritability indicators, negativism, sensitivity and neirotism and aggression indicator reduction, authentically higher indicators of humoral immunity, relative vital capacity of lungs, lower values of physical working capacity, the maximum consumption of oxygen, authentic higher occurrence of boundary and high figures of arterial pressure whereas for teenagers there was the prevalence of persons with vagotonia, higher indicators of cellular immunity were characteristic.

It was established during the estimation of adaptation degree of teenagers that the quantity of youth with satisfactory adaptation was authentically higher than teenagers, among teenagers the condition of adaptation tension prevailed. At the same time, irrespective of age, the quantity of surveyed in a condition of unsatisfactory adaptation and its failure authentically didn't differ.

Diagnostics system of prenosological conditions among teenagers and youth which on the basis of functional indicators allowed to allocate criteria of the satisfactory state of health, pressure with preservation of functioning level and prenosological conditions was developed.
High Trans‐Fat but not Saturated Fat Beverage Causes an Acute Reduction in Vascular Endothelial Function and Insulin Sensitivity in Humans
Authors: Lane‐Cordova Abbi , Witmer Jordan , Dubishar Kaitlyn , Dubose Lyndsey , Chenard Catherine , Wehrle Andrea , Pierce Gary
Published: June 2021

A diet high in trans‐fatty acids (TFA) is associated with increased cardiovascular disease risk, mediated in part from dyslipidemia, endothelial dysfunction and insulin resistance. A high‐fat meal causes a transient (3‐4 hrs postprandial) reduction in endothelial function and insulin sensitivity, but studies are often confounded by meals high in carbohydrate, saturated fatty acids (SFAs) and/or TFAs. We hypothesized that a beverage high in TFA (but low in carbohydrate, SFA) would cause a larger acute reduction in endothelial function (brachial artery flow‐mediated dilation, FMD) and insulin sensitivity compared with SFA (but low in carbohydrate, TFA). Healthy non‐obese adults (n=11; 9M/2F; age=47±3 yrs) ingested a warm beverage (520kcal, 56g total fat, 5g carbohydrate), high in either TFA (42%TFA, 23%SFA; partially hydrogenated soybean oil), SFA (88%SFA, 0.2%TFA; coconut oil) or placebo (2g total fat, 5g carbohydrate) in a randomized, cross‐over study. TFA (0.19±0.02 to 0.06±0.03mm, P<0.01), but not SFA (0.14±0.03 vs. 0.11±0.04mm, P=0.49) or placebo (0.08±0.04 to 0.062±0.03mm, P=0.52), caused a reduction in FMD at 3‐4 hours (P<0.01 for time; p=0.02 for interaction effects). Postprandial glucose was reduced across groups (P<0.001), whereas the homeostasis model of insulin resistance (HOMA‐IR) decreased in SFA and placebo (P<0.001) but not TFA (1.4±0.2 vs 1.2±0.2, P=0.08, interaction P=0.02). These data suggest that ingestion of a beverage high in TFA, but not SFA, results in an acute reduction in endothelial function and insulin sensitivity in healthy adults. Supported by NIH 5T32HL007638‐28, 5T32HL007121‐3, U54TR001013.