bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 ObjectivesWe 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 MethodsTo 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.ConclusionsUsing 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 InformationInozyme Pharma
ENPP1‐FcimageENPP1‐FcThis 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 InformationThis 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 InformationThe 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

ObjectiveAcute 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.HypothesisWe hypothesized that combined treatment with PRO+TBA would reduce inflammation and promote an anti‐inflammatory M2 macrophage phenotype by regulating the HIF2α signaling pathway.MethodsTo 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.ResultsOur 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.ConclusionOverall, 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 InformationAmerican heart Association‐16GRNT30950010 to JRThis 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

BackgroundThe 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.Aimwas 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 methodsThis 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.ResultsIt 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.ConclusionYounger 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 InformationThe study was not fundedThis 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 InformationFort Lewis College Student Research, Scholarly and Creativity GrantThis 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 InformationFunding 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

IntroductionLiving 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.MethodThe 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.ResultsAfter 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.SummaryThe 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 InformationThis 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.