bluuebunny/crossref_metadata_2025 huggingface dataset:
The Dual Specific Hemolysin‐X is a Bioactive Natural Product that Forms Pores on the Surface of Erythrocytes
Authors: Dam Tarun , Welch Christina , Edwards Jared , Kadav Priyanka , Hromada Michael , Bandyopadhyay Purnima
Published: June 2021

Hemolysins are natural cytotoxic agents that are capable of destabilizing plasma membrane structures of erythrocytes. We recently identified a plant hemolysin named Hemolysin‐X (HelyX) that showed dual specificity for cholesterol and certain serum glycoproteins, such as asialofetuin and fibrinogen. HelyX was isolated through two different approaches: protein purification protocols as well as organic solvent extraction. Ligand binding properties of HelyX were elucidated by inhibition assays using microtiter plates as well as spectrophotometer. HelyX demonstrated significantly fast kinetics (30–180 seconds) of lysis and ligand binding. Scanning electron microscopic imaging captured distinct pores on the surface of HelyX‐treated erythrocytes. Our results indicate that HelyX disintegrates mammalian red blood cells by forming pores on their surfaces.

Support or Funding Information

Michigan Technological University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Model for Teaching Bone Biology in a Physical Therapy Curriculum
Authors: Hogg Russell
Published: June 2021

A holistic, up‐to‐date understanding of skeletal biology is a vital component of clinical practice in a variety of fields, including orthopedics, endocrinology, internal medicine, physical medicine and rehabilitation, physical & occupational therapy, and others. However, most entry‐level practitioners in these fields come out of their degree programs with only a cursory knowledge of skeletal biology, which does not adequately incorporate a bounty of research that has been conducted over the last several decades.

One of the great difficulties in educating students in our current understanding of skeletal biology – as is the case for most physiological systems – is the overwhelming complexity and interconnectivity of the skeleton with other body systems, which we are only now fully beginning to appreciate. Since this interconnectivity is also what makes an understanding of skeletal biology so important for the clinic, it is important not to shy away from it in our curricula.

Therefore, the objective of this paper is to present a skeletal homeostasis model developed to educate students in graduate clinical degree programs with a well‐rounded, current understanding of skeletal biology. This model incorporates numerous body systems and strives to adequately incorporate the last three decades of research. The model presented here emphasizes the interaction between different tissues and the skeleton, placing the diversity of humoral and nervous outputs known to affect skeletal biology within tissue‐specific contexts in an attempt to make the interconnectivity of skeletal homeostasis more digestible to students. Each interaction of the skeleton with other systems is approached piece‐by‐piece, incorporating some of the most important neuroendocrine factors sequentially to build up to a broad understanding. While not complete, this model provides the student a good basis for interpreting the influence of muscles, fat, loading, calcium homeostasis, metabolism, and the nervous system upon bone – as well as the reciprocal impact that bone has upon these factors. Is also provides a foundation for understanding how failures in these interconnected networks can lead to pathology such as osteoporosis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Aspects of Glycosylated Protein–Glycan Interactions by NMR
Authors: Prestegard James H , Moremen Kelley W , Amster I Jon , Williams Robert V , Eletsky Alexandre , Chalmers Gordon R , Morris Laura C , Yang Jeong‐Yeh , Chapla Digantkumar
Published: June 2021

Characterization of glycosylated proteins and their response to interaction with glycans is challenging for any method, because of the heterogeneity of glycosylation and the extensive conformational sampling by most glycans. For NMR there are extra challenges because expression in mammalian cell cultures, which can preserve near‐native glycosylation, requires expensive substrates to achieve uniform isotopic labeling with 15N or 13C, and mammalian cells do not tolerate high levels of deuteration (something required to improve spectral resolution). We have developed a sparse‐labeling approach that preserves high resolution without deuteration, proves to be economical in most applications, and returns enough structural data to define geometries of multiple domain proteins and ligand protein complexes. The approach is based on labeling proteins with a single type of amino acid isotopically labeled in methyl groups with 13C (13C‐valine or 13C alanine). NMR signals from methyl groups are special in terms of the sensitivity and resolution that they provide. The addition of paramagnetic tags to specific sites in a protein provides long range distance and angular information on the location of labeled methyl groups. In combination with a newly developed approach to assign methyl resonances to specific sites in a protein, this information proves adequate to define relative domain orientations and facilitate the identification of ligand binding sites. Application to terminal domains from a cell surface signaling molecule, ROBO1, is used to illustrate the approach. It also provides an example of how binding of a heparan sulfate oligomer alters the relative orientation of domains.

Support or Funding Information

This work was supported by grants from the National Institutes of Health, P41GM103390 and R01GM033225.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Engineering and Glycan Optimization Improves Pharmicokinetics of an Enzyme Biologic 10‐fold
Authors: Braddock Demetrios T , Stabach Paul R , Zimmerman Kristin , Kavanagh Dillon , Sauei Chris T , Yarema Kevin J
Published: June 2021

Background and Objectives

We recently demonstrated the efficacy of an enzyme replacement therapy for a lethal neonatal metabolic disorder of vascular calcification called ‘Generalized Arterial Calcification of Infancy’ (GACI). The therapeutic biologic (ENPP1‐Fc) is a recombinant fusion protein consisting of the extracellular domain of Ectonucleotide Pyrophosphatase Phospodiesterase‐1 (ENPP‐1) fused with the Fc domain of IgG1. The fusion protein exhibited exceptional stability and had an in vivo half‐life of 35 hours in rodents. The objective of this study was to investigate protein engineering and bioprocessing methods to further enhance the pharmacokinetic properties of this biotherapeutic enzyme.

Approach and Methods

To optimize the pharmacokinetic properties of ENPP1‐Fc we embarked on a 4‐prong strategy: First, we increased glycosylation to shield the protein from degradation by the introducing additional N‐glycan consensus sequences onto the exterior surface of the predicted tertiary structure, guided by three‐dimensional models of ENPP1, other ENPP enzymes in the superfamily, and known inactivating mutations in ENPP1 resulting in GACI. Second, we increased pH‐dependent FcRn‐mediated cellular recycling by mutating the Fc domain to enhance the affinity of the fusion protein for the neonatal receptor (FcRn). Third, we enhanced sialyation of the fusion protein by expressing ENPP1‐Fc in CHO cell lines stably transfected with human alpha‐2,6‐sialyltransferase. Finally, we enhanced sialic acid capping by supplementing the cell culture media with N‐acetylmannosamine or 1,3,4‐O‐Bu3ManNAc, a “high‐flux” precursor of sialic acid.

Conclusions

Using a combination of the above methods we collectively increased the in vivo half‐life of ENPP1‐Fc by over 4‐fold and the in vivo exposure of animal to ENPP1‐Fc (i.e., Area Under the Curve) by over 10‐fold. We additionally found that expressing ENPP1‐Fc in CHO cells stably transfected with sialytransferase dramatically increased the bioavailability (Cmax) of ENPP1‐Fc when the biologic was administered as a subcutaneous depot. Our results demonstrate the ability of protein engineering and bioprocessing methods to dramatically improve the pharmacokinetics and bioavailability of enzyme replacement biologics for use in humans.

Support or Funding Information

Inozyme Pharma

ENPP1‐Fc

image

ENPP1‐Fc

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating The Role of TMPRSS1 In Mouse Inner Ear Structure And Function
Authors: Yang Ting‐Hua , Hung Chia‐Jui , Yeh Peng , Tsai Yu‐Fei , Hsu Yu‐Chen , Lin Shu‐Wha
Published: June 2021

Hearing loss is one of the common birth defects in humans. Defects in the type II transmembrane serine proteases (TMPRSS) have been reported to affect organic structures of the inner ears, and thus, hearing function in mice, including loss of TMPRSS1 or any of at least 4 other TMPRSS members. Here, we used the human TMPRSS1 transgenic (hTMPRSS1‐Tg) mice on a mouse TMPRSS1‐null background to investigate the rescue function of exogenously‐expressed TMPRSS1 in the development of hearing and inner ears. The expression of hTMPRSS1 was detected in the organ of Corti and stria vascularis in the inner ear. Phenotype analysis using auditory brainstem evoked response (ABR) test showed that while the TMPRSS1‐null mice exhibited profound hearing loss as literally reported, the deafness of hTMPRSS1‐Tg mice were alleviated partially with 20 dB improvement. The H&E stain showed that the anomaly of the tectorial membrane in hTMPRSS1‐Tg mice was less serious than the TMPRSS1‐null mice. Although the hearing threshold had positive correlation with the area of the tectorial membrane, no significant difference was calculated. To further analyze the components in tectorial membrane, we used immunofluorescence to quantify molecules mainly expressed on tectorial membrane: α‐tectorin, β‐tectorin, type IX collagen, type II collagen of wild‐type and TMPRSS1‐null mice. The result showed that while α‐ and β‐tectorin and type IX collagen were less homogenously expressed in TMPRSS1‐null mice. Similar results were noted in electronic microscopy. Since α‐ and β‐tectorin form the underlying striated‐sheet matrix in the tectorial membrane, our results suggested that suboptimal connection of α‐tectorin with β‐tectorin could lead to the instability of type IX collagen, causing the deformity of tectorial membrane and consequently deafness in TMPRSS1‐null mice. TMPRSS1 indeed plays an important role in hearing. Transgenic/knockout mice established in our lab was a great tool for studying the add‐back effect of TMPRSS1 for hearing improvement, and may help develop therapies on certain patients with congenital hearing loss in the future.

Support or Funding Information

This study is supported by MOST (107‐2319‐B‐002‐001 and 104‐2314‐B‐002‐159)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Evolution of Pancreatitis—from the Clinical Manifestations to Genetic Determinant
Authors: Vasylieva Oksana
Published: June 2021

In recent years the ideas about etiopathogenesis of pancreatitis have significantly changed. It was found that the development of pancreatitis may be one of the forms of gastrointestinal manifestations of hereditary pathology, accompanied by nausea, vomiting, diffuse abdominal pain, malabsorption and maldigestion. But the viewpoint at the lesion of the exocrine function of pancreas hasn't been yet established among health care professionals as a potential genetic problem, so diagnosis of genetically determined forms of pancreatitis remains mostly undeveloped.

In Kharkiv medical‐genetic center we monitored 68 patients (3–46 y.o.) with manifestations of exocrine pancreatic insufficiency and pancreatitis symptoms, who underwent somatic genetic testing, pedigrees' analysis, abdominal ultrasound, biochemical blood and urine tests and confirming molecular diagnostics. According to the results of examination the presence of the following nosologic forms was diagnosed: cystic fibrosis ‐ in 35(4.22%) patients, Kearns‐Sayer syndrome ‐ 4(0.48%), MELAS ‐ 4(0.48%), MNGIE ‐ 3(0.36%), violation of fatty acid metabolism ‐ 5(0,6%), glutaric aciduria ‐ 3(0.36%), methylmalonic aciduria ‐ 2(0.24%), propionic aciduria ‐ 1(0.12%), maple syrup urine disease ‐ 2(0.24%), homocystinuria ‐ 5(0.6%), connective tissue dysplasia (CTD) ‐ 4(0.48%).

The phenotypic characteristics of examined patients: the signs of CTD (hyperstretchability of skin, brittle hair and nails, caries, moderate joint hypermobility) ‐ in 64.7% and small developmental abnormalities (hypertelorism, short filter, dolichocephaly, deformed ears, clinodactyly etc.) ‐ in 58.8%, that probably reflects the teratogenic effect hereditary metabolic disorders in the embryogenesis process. In 23(33.8%) patients generalized aminoacidaemia was revealed; 22(32.4%) patients have primary mitochondrial insufficiency, 12(17.7%) ‐ the secondary.

In accordance with the revealed changes an appropriate pathogenetic therapy has been appointed. The problem of the clinical and genetic characteristics of patients with symptoms of pancreatitis can be a key to understanding the mechanisms of pancreas pathology launch, which will increase the efficiency of diagnosis and treatment of gastrointestinal tract diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Adipose Tissue‐Derived Mesenchymal Stromal Cells for the Verification of Silicone‐Based Cell Culture Systems
Authors: Stock Peggy , Meltke Martin , Hildebrand Janis , Böttcher Gunther , Christ Bruno
Published: June 2021

Conventional two‐dimensional as well as recently established three‐dimensional cell culture systems exhibit a high modulus of elasticity, yet, without representing the stiffness of human organs. Previously, we established a protocol for the culture of primary rat hepatocytes on a silicone of low stiffness, which sustained their physiological functions. This tool may readily serve as validation system for pharmacological and toxicological studies of agents and compounds. In terms of the development of human‐based tests to evaluate the potential risk of novel drugs, the method was adjusted for use of human adipose tissue‐derived mesenchymal stromal cells (hAT‐MSC).

Aim of the study was to optimize culture conditions for hAT‐MSC on fibronectin‐coated silicone layers with various elastic moduli (50 – 500 kPA) mimicking the stiffness of human organs including different surface modifications to increase hydrophilic properties. The metabolic activity (MTT assay) and growth parameters were analysed and compared to the conventional cell culture on polystyrene (E‐modulus 103 MPa).

hAT‐MSC were cultured for 10 days on five different fibronectin‐coated medically approved silicone layers. The impact of surface modifications of the silicone using sol‐gel‐technique, fluorination and plasma treatment on the cells was determined by the development of cell counts, viability and metabolic activity. hAT‐MSC attached to all 5 different silicone layers and formed cell layers after 10 days. Neither the silicones with surface modifications nor the untreated silicone displayed significant differences in cytotoxicity: the percentage of dead cells in cultures were 7% ± 2.3% vs. 7.2% ± 1%. We identified one culture displaying significantly higher cell counts as compared to the standard culture: based on identical initial cell counts, the growth rate was about 2.5‐fold higher on plasma‐treated silicone with an E‐modulus of 50 kPa. Cells cultured on plasma‐treated silicones with E‐moduli of 50 kPa or 500 kPa showed also significantly higher amounts of metabolic activity as compared to the classical culture: 2.76‐fold (50 kPA) and 2.83‐fold (500 kPa).

With these results, two medically approved silicones treated with plasma were identified for improving culture of hAT‐MSC. Thus, the silicone layers represent an important tool for the development of cell cultures featuring organ‐like stiffness and improving physiological functions.

Support or Funding Information

The study was supported by the European Regional Development Fund and the Free State of Saxony (SAB) 100282466 to J.H. and P.S.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combined administration of epigenetic modifiers regulates macrophage polarization by HIF‐2α signaling and protects mice from acute lung injury
Authors: Ponnan Jeyasekhar M , Burkey Kyley K , Rajasingh Sheeja K , Mehta Manav K , Rajasingh Johnson K
Published: June 2021

Objective

Acute lung injury (ALI) is a common pulmonary disease caused by bacterial infection leading to an imbalance between pro‐inflammatory and anti‐inflammatory immune responses. Studies have shown that macrophage polarization (M1 and M2) during ALI plays a key role in regulating these responses. Here, we have examined the use of DNA methyltransferase (DNMT1) inhibitor Procainamide (PRO) and histone deacetylase 6 (HDAC6) inhibitor Tubastatin A (TBA) together in protecting mice from ALI.

Hypothesis

We hypothesized that combined treatment with PRO+TBA would reduce inflammation and promote an anti‐inflammatory M2 macrophage phenotype by regulating the HIF2α signaling pathway.

Methods

To show the effect of PRO+TBA, lipopolysaccharide (LPS)‐induced macrophages (RAW 264.7) were treated with either PRO (500 μM), TBA (5 μM), or together (PRO+TBA) for 24 hours. The mRNA and protein expressions of HIF‐2α, NOS2 (M1), and CD206 (M2) were measured by RT‐PCR and Western analyses in lung tissue and macrophages.

Results

Our results revealed that LPS significantly decreased the mRNA and protein expressions of HIF‐2α in lung tissue and macrophages. This expression was significantly increased in LPS‐induced macrophages treated with PRO+TBA. Furthermore, LPS induces the expression of M1 macrophages and increased expression of NOS2 and other inflammatory cytokines whereas LPS induced macrophages treated with PRO+TBA considerably suppressed the expression of M1 macrophages and increased the expression of CD206 and anti‐inflammatory cytokines by M2 macrophages. These results suggest that PRO+TBA treatment together generates more M2 macrophages there by reducing the LPS‐induced inflammatory responses.

Conclusion

Overall, these data suggest that the combinatorial treatment with PRO+TBA regulates macrophage cell polarization and protects mice from ALI through HIF‐2α signaling pathway. Thus, epigenetic modifiers may be used as potential therapeutic drugs for ALI.

Support or Funding Information

American heart Association‐16GRNT30950010 to JR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Clinicopathological Study of Breast Cancer in Young Females (Under 40‐Year‐Old)
Authors: Talaat Iman Mamdouh , Elnahas Salwa Moustafa , Awad Ahmed Tarek , Elsaid Amr Abd Elaziz , Fayed Hytham Mahmoud
Published: June 2021

Background

The average age at presentation of breast cancer in Egypt appears to be a decade earlier than in western countries. Breast cancer is the leading cause of cancer‐related deaths in women under 40 years and survival rates for young women with breast cancer are lower than their older counterparts.

Aim

was to assess the clinicopathological characteristics of breast cancer female patients less than 40 years and to compare them with their counterpart above the age of 40 years. In addition, the impact of age as an independent factor on survival was studied.

Materials and methods

This was a retrospective cohort study of an institutional database of all cases of breast carcinoma patients, whom prospectively were followed up during the period from January 2000 to December 2009, performed at Alexandria Main University Hospital, Egypt. After collecting data of breast carcinoma patients; clinicopathological characteristics, chosen management option and disease‐free survival (DFS), as well as overall survival (OS), were compared among the two age groups. Prognostic factors were evaluated for association with DFS and OS.

Results

It has been found that the number of young patients (≤ 40 years) was 180 (25.4%) while the number of older patients was 529 (74.6%). The age of the patients varied between 21 years and 90 years (mean 47.8). The histopathological assessment showed that the incidence of infiltrated ductal carcinoma (IDC) was higher than the other types in both groups; 641 tumors (91.7 % from the total sample) were IDC; 165 (92.7 %) were in young age group and 476 (91.4 %) in old age group. There was no significant association between the two studied groups in relation to the histopathological types of breast carcinoma. Recurrence rates were significantly higher among young women (49.2%) when compared to older women (30.6%) (p=0.000). Five‐year DFS duration in young women was shorter, the mean follow‐up time of young patients was 44.4 months while in old age group, it was 51.6 months with a hazard ratio of 1.67 (95% CI: 1.117–2.499) greater than old patients, although there was no significant difference between the two groups as regard OS. Multivariate analyses identified larger tumor size, the presence of lymphovascular invasion and young age being ≤ 40 as independent factors associated with poor DFS.

Conclusion

Younger age (≤ 40 years) at the diagnosis of breast cancer can be considered an adverse prognostic factor for the development of locoregional recurrence or distant metastases.

Support or Funding Information

The study was not funded

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of FAK Activation in HER2+ Breast Cancer
Authors: Quintana Victoria Gabriela , Byrd Sherell
Published: June 2021

Hormone responsive breast cancer, a type of cancer that grows more aggressively in the presence of certain types of hormones (i.e. estrogen), makes up about 20–30% of all human breast cancers. Trastuzumab (Herceptin) is a common drug treatment that targets the HER receptor and inhibits estrogen from binding. The goal of my research is to understand whether the use of Herceptin to neutralize the HER receptor can be bypassed by the cell by using the cMET pathway. Studies show that drug resistance to Herceptin in HER+ cancers may be the product of the cell using this alternative pathway resulting in FAK activation. FAK activation via the HER and cMET associated signaling pathways is one of the mechanisms for the spread of cancer cells via metastasis. We examined FAK activation and cell movement capacity using MCF‐7 HER2 positive breast cancer cells. Our results show that MCF‐7 breast cancer cells treated with Herceptin still show activation of FAK. Using a migration assay, we see that Herceptin does not inhibit the migration pathway. Treatment significantly (p = 0.01) increased the migration of the MCF7 cells compared to the untreated cells. The cMET specific inhibitor, Tepotinib, significantly reduced migration, indicating that cMET is being used as an alternative pathway to activate migration. Immunoblotting showed that there was no difference in total phosphorylated FAK between the treated and untreated cells, but the ratio of phosphorylated to unphosphorylated FAK is being investigated. These preliminary results suggest that there is the possibility of an alternative pathway that is being used to activate FAK despite the inhibition of the HER receptor.

Support or Funding Information

Fort Lewis College Student Research, Scholarly and Creativity Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Non‐Pungent Capsaicin Analogs: Potential Applications in Lung Cancer Therapy
Authors: Merritt Justin C , Gadapalli Rama S , Akers Austin T , Nolan Nicholas A , Brown Kathleen C , Colclough Kate W , Miles Sarah L , Friedman Jamie R , Rimoldi John M , Dasgupta Piyali
Published: June 2021

Capsaicin is the spicy pungent ingredient of chili peppers. Although traditionally associated with analgesic activity, recent studies have shown that capsaicin has profound anti‐neoplastic effects in several types of human cancers. However, the applications of capsaicin as a clinically viable drug are limited by its unpleasant side effects, such as gastric irritation, stomach cramps and burning sensation. This has led to extensive research focused on the identification and rational design of second‐generation capsaicin analogs, which possess greater bioactivity than capsaicin. Previous studies have shown that addition of long‐chain unsaturated groups after the amide group of capsaicin were non‐pungent and retained the bioactivity of capsaicin. These chemical nature of these compounds are unsaturated N‐acylvanillamides (uN‐AVAMs). However, a majority of these uN‐AVAMs have been studied for their pain‐relieving activity. We synthesized a panel of uN‐AVAMs with 0–4 double bonds in the side chain of capsaicin. We investigated the growth‐inhibitory activity of these compounds with an MTT‐based screening assay. We selected our “hit compound” Arvanil for further studies. Next, we compared the apoptotic activity of arvanil and capsaicin in a panel of human small cell lung cancer (SCLC) cells. We observed that the non‐pungent capsaicin‐analog arvanil displayed greater magnitude of apoptosis than capsaicin. Most interestingly, arvanil did not display apoptotic activity in normal lung epithelial cells. The pro‐apoptotic activity of arvanil and capsaicin was mediated by the intracellular calcium pathway. We measured the uN‐AVAM‐induced levels of intracellular calcium in SCLC cells. The pattern of uN‐AVAM‐induced intracellular calcium was analogous to the results obtained in the MTT assay. Therefore, the measurement of intracellular calcium may be used as a screening tool for capsaicin‐mimetics with anti‐cancer activity.

Support or Funding Information

Funding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanical stimulation injures neurons to form Tau complex
Authors: Cheng Pei‐Wen , Sun Gwo‐Ching , Wu Yi‐Chung , Tseng Ching‐Jiunn
Published: June 2021

Introduction

Living systems experience various form of mechanical stimulations in daily life. Some mechanical stimulations are required for cell survival, others are capable of causing injuries. In vivo, the neuronal system is linked closely to the muscular system. As a result, we respond to pain immediately after physical injury. Proliferating neuron cells can be used to study the effect of mechanical stimulation on neurons. However, cell models developed from neurons have been lacking due to the fact that neuron cells do not or are difficult to proliferate by in vitro culture. In this study, we investigated the effect of stretching on the SH‐SY5Y cells, neuroblastoma cells that proliferate in vitro. The cells retain the dopamine‐β‐hydroxylase activity; thus, it is suitable for neuron mechanistic study.

Method

The culture conditions contain non‐stretched (control) and stretched groups. SH‐SY5Y cells were cultured on stretchable membranes, subjected to 6, 24h of cyclic stretching, 25% elongation at 1Hz.

Results

After 6 and 24h stretching, cells showed decreased red fluorescence for JC‐1 probe which stains the mitochondrial membrane, indicating that cells experienced oxidative stress. Results show that stretching increased b‐amyloid precursor, total Tau protein by western blot. Co‐immunoprecipitation showed that Tau formed complex with GSK3b after 6h stretching.

The observations of change in mitochondrial potential, and formation of Tau complex imply that the neuron cells were under a stress. The stress may be a kind of injury suffered from physical stimulation.

Support or Funding Information

This work was supported by funding from the Ministry of Science and Technology (MOST106‐ 2314‐B‐037‐022‐MY2, MOST107‐2320‐B‐075B‐001) and Kaohsiung Veterans General Hospital (VGHKS107‐175) (to P.‐W. C). The authors declare that they have no competing interests.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
dkNET (NIDDK Information Network): Research tools that assist scientists in improving the rigor and reproducibility of their research
Authors: Lin Ko‐Wei , Bandrowski Anita E. , Gillespie Tom , Go James , Hagstrom Stephanie , Hsu Chun‐Nan , Ozyurt Ibrahim B. , Grethe Jeffrey S. , Martone Maryann E.
Published: June 2021

The use of problematic resources such as contaminated or misidentified cell lines and cross‐reactive antibodies lead to reproducibility problems. Research has shown that about twenty percent of published studies use contaminated cell lines [1]. Many factors, including the ability to easily retrieve alert information, result in the continued use of these resources wasting both time and money. Research Resource Identifiers (RRIDs) are unique identifiers for resources that assist finding, identifying, and tracking antibodies, cell lines, biological samples, organisms, and digital resources in the published literature [2]. RRIDs currently appear in over 8000 papers across 600 biomedical journals. Because authors have to supply RRIDs in their manuscripts ahead of publication, RRIDs provide a unique means for disseminating information about resource performance. For example, a search in the RRID portal (Nov 14, 2018) found 802 problematic cell lines (contaminated and/or misidentified cell lines) out of 101528 cell lines, as reported by the International Cell Line Authentication Committee (ICLAC). Analysis of cell lines in published papers using RRIDs vs those that didn't use RRIDs, showed the reported use of contaminated cell lines is significantly lower in papers with RRIDs than in the average paper [3], suggesting that making researchers aware of problematic resources may decrease their usage. To make it easier for researchers to find complete information, the NIDDK Information Network (dkNET.org) used RRIDs to develop several tools to help scientists enhance research rigor and reproducibility. dkNET introduced Resource Reports, a detailed overview of each resource identified by an RRID, including the characteristics of the resource, any warning and alert information, citation metrics from the biomedical literature and validation and rating information. Resource Reports let a researcher “follow” a resource, receiving up to date information on the usage of the resources they used (or created) in the literature. dkNET also offers a Rigor and Reproducibility tool that can generate an authentication report for grant applications in support of the NIH mandate on authentication of key biological resources. RRIDs and the associated tools and services make it easier for information on resource performance to be tracked and disseminated.

Support or Funding Information

NIH NIDDK Grant U24DK097771

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adolescent in Stress, Depression and Violence in NigeriaGertrude Bassey, George P. Einstein, Orien L. Tulp College of Medicine, Einstein Medical Institute, Montserrat, B.W.I
Authors: Bassey Gertrude
Published: June 2021

Background

The studies have examined how multiple settings and experiences of violence are associated with depression and how these influence teens to be violence perpetrators. This research sets out to trace how the experience of violence among teens can lead to depression and also lead to teens being violent themselves.

Methods

Youths were asked several items to measure their levels of family conflict, amount of violence they have observed, and violent victimization. Self‐report questionnaires were administered to the 500 youths. Types of violence explored included: Psychological abuse, Physical violence in home, Intimate partner violence, School witnessed violence, School violence victimization, and Neighborhood witnessed violence.

Multiple latent class analyses were performed to identify adolescents with common patterns of violence exposure and to find classes with the best fit.

Results

Prevention towards adolescents by school clinic, and individual counseling need to be followed. Law enforcement agencies need to respond to cases of violence. Symptoms of post‐traumatic stress disorder and depression is related to violence. Anxiety symptoms only to exposure to violence perpetrated by someone known to the child or in the home.

Conclusion

Exposure to a high magnitude of violence within a single domain or multiple domains portrays significant risk for Depression and Anxiety. Violence is associated with the highest risk for psychopathology.

The results suggest that it may be beneficial to direct efforts at prevention and treatment towards adolescents at high risk for violence within multiple domains of life, as these individuals may face greatest risk for depressive and anxiety disorders and also become perpetrators of violence.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cyclooxygenase‐2 Regulates the Renal Expression of Indoleamine 2,3‐dioxygenase
Authors: Mutsaers Henricus A.M. , Jensen Michael Schou , Olinga Peter , Nørregaard Rikke
Published: June 2021

Chronic kidney disease (CKD) affects approximately 10% of the population. In CKD patients, the majority of metabolic pathways are in disarray, which most likely contributes to the wide variety of comorbidities observed in these patients. However, few studies have focused on the unique regulatory aspects of enzyme systems during CKD development and progression. The cyclooxygenase‐2 (COX‐2)/prostaglandin (PG) system plays a dominant role in the progression of renal injury. Interestingly, it has been shown that the COX‐2/PG system modulates the activity of indoleamine 2,3‐dioxygenase (IDO) in tumors. Catabolism of tryptophan by IDO gives rise to numerous biologically active metabolites implicated in CKD progression. Therefore, the aim of this project is to explore the interplay between the COX‐2 and IDO enzyme systems during renal injury.

We studied the gene expression of IDO, COX‐2 as well as several fibrosis and inflammatory markers in human and murine precision‐cut kidney slices (PCKS) and COX‐2 KO mice. Furthermore, we evaluated the gene expression profile after treatment with butaprost, an EP2 receptor agonist, in hPCKS and following treatment with SC‐236, a selective COX‐2 inhibitor, in mPCKS.

Our results demonstrated that IDO gene expression was markedly increased in both human and mouse PCKS during the first 24h of culturing, after which levels dropped again. This expression profile was similar to the gene expression of various cytokines (e.g. IL‐6 and CXCL1), suggesting a potential pro‐inflammatory role of IDO. In addition, in COX‐2 KO animals, constitutive gene expression of IDO was increased as compared to wild‐type mice, and the knockout animals appeared to be more prone to renal fibrosis. Moreover, treatment with butaprost suppressed IDO gene expression, while exposure to SC‐236 induced IDO mRNA expression as well as the gene levels of IL‐6 and IL‐8.

Taken together, it appears that COX‐2 suppresses IDO gene expression. In addition, our findings suggest a potential pro‐inflammatory role of IDO. However, the precise role of IDO in CDK development and progression requires further study.

Support or Funding Information

This work was kindly supported by Lundbeckfonden, grant number R231‐2016‐2344.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Therapeutic potential of commercially‐available cannabidiol oil for Alzheimer's disease using Caenorhabditis elegans as a model
Authors: Flores‐Narvaez Molishka A , Ramos‐Ortolaza Dinah Lee
Published: June 2021

Alzheimer's disease (AD) is a neurodegenerative disease linked to loss of cognitive and motor abilities. The neurodegeneration is thought to be caused, in part, by the accumulation of Aβ peptides in the brain. Currently, there is no cure for AD, and the available treatments are rather limited in scope. Most of them are aimed at treating the symptoms, not preventing or reversing the progression of the disease. Recent studies have suggested a therapeutic potential for cannabidiol (CBD), a component of the Cannabis sativa plant. Both, in vitro and in vivo studies have shown improvement in some of the physiological and cognitive signs of the disease, suggesting it may be a better therapeutic strategy compared to current treatments. Given CBD's therapeutic potential and the increase in availability of commercial over‐the‐counter CBD‐based oils, this study is aimed at assessing the therapeutic potential of these products using an invertebrate model of AD. To achieve our aim, we decided to use a transgenic Caenorhabditis elegans worm strain that expresses human Aβ in neurons (CL2355). This is an inducible strain that shows behavioral deficits that make it a suitable model for AD. Prior to treating the animals, a toxicity assay was performed with several concentrations of a commercially available CBD oil. Although more animals need to be tested, preliminary results showed no significant effect of the CBD oil on the worms' mortality. Once we determine the optimal concentration of CBD, Aβ expression will be induced in synchronized animals from the CL2355 strain by a temperature upshift. This will be followed by a 24‐hour exposure to the CBD oil or the vehicle. By the time the CBD exposure ends, the animals will be adults, and behavioral testing will be performed. Specifically, we will perform a thrashing and a short‐term associative learning assay to test for locomotor and memory deficits respectively. Results from this study will allow us to determine whether commercially‐available CBD oils can either prevent or alleviate some of the deficits associated with AD and will support the use of invertebrate models to further explore new possibilities for the development of therapeutic strategies for AD and other neurodegenerative disorders.

Support or Funding Information

PUCPR‐PR Louis Stokes Alliances for Minority Participation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMB‐FUBINACA (FUB‐AMB) and related "spice" compounds induce gene‐dose effects in mice lacking cannabinoid‐1 receptor (CB1R) alleles
Authors: Baumann Michael H. , Fedorko Julie M. , Prisinzano Thomas E. , Garibay Nancy
Published: June 2021

Synthetic cannabinoids (SCs), commonly known as ‘K2' or 'spice’, are a constantly evolving class of new psychoactive substances that pose public health risks. Little information is available about novel SCs when they first emerge into the recreational drug marketplace. In this study, we investigated the pharmacology of AMB‐FUBINACA (FUB‐AMB) and 5F‐AMB, two recently encountered indazole‐based SCs responsible for mass intoxications and fatalities. The effects of known cannabinoid‐1 receptor (CB1R) agonists AM‐2201 and Δ9‐THC were examined for comparison. Groups of genetically‐engineered mice lacking CB1R alleles (CB1R −/− and CB1R +/−), and their C57Bl/6J wild‐type (WT) counterparts, received surgically‐implanted subcutaneous (s.c.) temperature transponders under isoflurane anesthesia. Beginning one week after surgery, mice received s.c. injection of either SCs or vehicle and were subjected to a “triad” test battery consisting of sequential assessment of: 1] body temperature, 2] catalepsy bar latency and 3] hot‐plate analgesia latency. All SCs induced significant dose‐ and time‐dependent hypothermia, catalepsy and analgesia in WT mice, which lasted up to 120 min. The rank order of potency was FUB‐AMB>5F‐AMB=AM‐2201>Δ9‐THC. Importantly, ED50 values in the triad test battery for FUB‐AMB (0.09–0.22 mg/kg, s.c.) were at least 50‐fold more potent than those associated with Δ9‐THC (12.72–30.37 mg/kg, s.c.), and SCs were more efficacious in their ability to produce hypothermia. Homozygous CB1R −/− mice were not affected by any dose of the compounds tested, whereas heterozygous CB1R +/− mice displayed responses intermediate between homozygous and WT. CB1R binding assays in WT mouse brain revealed that IC50 values for inhibition of [3H]SR141716 binding were 1.2, 2.2, 12.6 and 50.6 nM for FUB‐AMB, AM‐2201, 5F‐AMB and Δ9‐THC, respectively. Our findings demonstrate that newer indazole‐based SCs exert their effects via potent agonist actions at CB1R, and do not support the notion that SCs induce in vivo effects by non‐CB receptor mechanisms. Given the widespread abuse of synthetic cannabinoids, CB1R antagonists should be made available for overdose rescue, and medical marijuana could be deployed as an agonist treatment for dependence on these substances.

Support or Funding Information

This research was generously supported by IRP, NIDA, NIH (DA00523)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Corticotropin Releasing Factor Invigorates Cocaine‐Seeking via CRF‐R2 in the Nucleus Accumbens Core
Authors: Leonard Michael Z. , Miczek Klaus A.
Published: June 2021

Adverse life events often precipitate the initiation of– and relapse to– cocaine use in drug‐dependent individuals. For example, acute stressors can potently exacerbate the extent to which drug‐related stimuli elicit craving to seek and procure drug, perhaps via peptidergic modulation of mesolimbic dopamine transmission. Indeed, dopaminergic signaling within nucleus accumbens core (NAcc) is critical to guiding motivated behavior in response to rewarding or aversive stimuli. The present studies, therefore, examine the site‐specific actions of the stress peptide corticotropin releasing factor (CRF) as a putative substrate for stress‐potentiated arousal. First, we assessed extent to which pharmacological manipulation of CRF within the NAcc alters instrumental responding for cocaine; and, in a second set of experiments, we begin to characterize the conditions under which CRF modulates local dopamine transmission. To that end, male Long‐Evans rats were trained to self‐administer cocaine intravenously under a chained schedule of reinforcement (FI‐FR) in order to dissociate appetitive (‘drug‐seeking’) from consummatory (‘drug‐taking’) behavior. Completion of a fixed interval (5min.) was followed by 10 min of continuously reinforced responding (0.4mg/kg cocaine; FR1) on another lever. After training, rats were microinjected with CRF (50 or 500ng/side) into the NAcc prior to self‐administration sessions. Microinfusion of CRF dose‐dependently increased responding during the fixed‐interval link of the chain schedule, but did not affect subsequent cocaine intake. These effects were CRF‐R2‐dependent, as CRF‐potentiated responding was prevented by pretreatment with Astressin‐2B (1000ng/side), but not CP376395 (500ng/side). Astressin‐2B was also sufficient to attenuate cocaine‐seeking responses when administered alone. Parallel microdialysis studies revealed CRF is endogenously released into the NAcc during acute stress exposure (i.e. social defeat) and, moreover, that perfusion of CRF into the NAcc is sufficient to elicit a robust increase in extracellular dopamine. Ongoing studies aim to clarify the role for CRF‐R2 in moderating this function in cocaine‐experienced rats. Taken together, these data suggest a role for CRF‐R2 in modulating appetitive drug‐seeking behavior, perhaps via interactions with NAcc dopamine, which may offer insight into the substrates of stress‐potentiated relapse. CRF‐R2 may therefore represent a worthy therapeutic target for cocaine use disorder, despite the initial clinical shortcomings of CRF‐R1‐related compounds.

Support or Funding Information

NIDA grant DA031734 to KAM

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing the cognitive‐enhancing effect of desformylflustrabromine in rat model of Aβ‐induced cognitive impairment
Authors: Deba Farah , Munoz Kemburli , Peterson Steven L , Hamouda Ayman K
Published: June 2021

Accumulation of β‐Amyloid peptides (Aβ)‐containing plaques, perturbation of the neocortical cholinergic pathways, and loss of brain acetylcholine receptors (AChR) are hallmark characteristics of Alzheimer's disease (AD). Several AChR subtypes are expressed in the cortex and the hippocampus and play essential role in learning, memory, and neuronal survival during ageing. Indeed, three out of the only four FDA‐approved drugs for AD are inhibitors of acetylcholinesterase, the enzyme responsible for ACh degradation. In addition, stimulation of α4β2 nAChRs have been shown to protect against Aβ1‐42‐induced cell death in cultured cortical neurons and to improve working memory and cognition in abstinent smokers. Because drugs that enhance the α4β2 nAChR would be beneficial for the treatment of the cognitive decline associated with many neuropathologies including AD, we focus on selective targeting of the α4β2 nAChR using positive allosteric modulators (PAM). PAMs of nAChR enhance ACh signaling without alteration of cholinergic transmission due to the prolonged activation and desensitization of nAChRs seen with agonists. In this study, we examine the cognitive‐enhancing effect of the α4β2 nAChRs PAM, desformylflustrabromine (dFBr), in a rat model of cognitive impairment. dFBr isolated originally from the marine bryozoan Flustra foliacea then its chemical synthesis and interaction with nAChRs in vitro were illustrated. In two‐electrode voltage clamp recording from Xenopus oocyte, dFBr potentiated ACh‐induced currents of low‐sensitivity (α4)3(β2)2 and high‐sensitivity (α4)2(β2)3 nAChRs maximally by ~300 and ~400% and with IC50s of ~0.4 and ~2 μM, respectively. In preliminary study, we tested the effect of two daily treatments of 10 mg/kg dFBr on rats' performance in passive avoidance test measured 10 days after intracerebroventricular (ICV) injection of vehicle (sham) or Aβ1‐42. dFBr treatment increased retention time (the time spent in the light compartment before entering the dark compartment that is associated with mild foot shock) in both naive (sham injected) and rats with AD‐like cognitive impairment (Aβ1‐42 injected). Experiments are in progress to evaluate the effects of dFBr on rats' performance in other learning and memory paradigms such as the 8‐arm radial maze.

Support or Funding Information

University of Texas System Rising STARs Program Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.