bluuebunny/crossref_metadata_2025_split huggingface dataset:
The apical membrane localization and trafficking of uromodulin (UMOD) is critically dependent on nonmuscle myosin II isoforms Myh9 and Myh10 in mouse thick ascending limb epithelial cells
Authors: Otterpohl Karla , Busselman Brook , Hart Ryan , Shan Kevin , Lin Brendon , Surendran Kameswaran , Chandrasekar Indra 
Published: June 2021

Mutations that alter the trafficking and function of uromodulin, a membrane associated GPI anchored protein expressed in the TAL segment, result in autosomal dominant tubulointerstitial kidney disease (ADTKD)1. Proper localization and function of GPI‐anchored proteins requires intact ER to Golgi to apical membrane transport. Recently Nonmuscle myosin II (NM2) have been linked to vesicular transport2, and we have identified a novel function for NM2 isoforms Myh9 (NM2A) and Myh10 (NM2B) in regulating membrane remodeling in mammalian cells3. Due to the differential expression and unique localization of NM2 isoforms in the murine renal tubules4, we hypothesized that NM2 isoforms are involved in the specialized transport pathways of membrane associated proteins in the TAL epithelial cells. Conditional genetic knockout of Myh9 & Myh10 within the renal tubules of mice results in initial mis‐localization and gradual loss of the TAL‐specific protein: Uromodulin. These mice develop rapidly progressing tubulointerstitial kidney disease and are moribund by 5–6 months of age. In order to understand the mechanisms underlying the mis‐localization of the Uromodulin, we generated an immortalized TAL cell line from the adult kidneys of Pax8‐>rtTA; Tet‐o‐cre; Myh9F/F; Myh10F/F mice crossed to mice harboring Immorto +/Tg transgene. These cells can be maintained at 33C indefinitely due to the expression of the temperature sensitive SV40 T‐antigen transgene. To promote polarization and endogenous expression of uromodulin and other membrane‐associated proteins in these cells, we have modified a recently established culture protocol with cells grown on polyester membranes at 37C on an orbital shaker5. We observe robust expression of endogenous uromodulin detected by immunohistochemistry and western blot analysis. Furthermore, induction of Myh9 and Myh10 inactivation by doxycycline treatment of these cells results in altered levels of uromodulin in the cells possibly indicating changes in N‐glycosylation of uromodulin along the ER‐Golgi transport pathway. Loss of NM2 also decreases the intracellular sodium levels in the knockout cells, observed using sodium green™ indicator. Our studies indicate a novel relationship between NM2 isoforms and uromodulin transport along the TAL segment and are suggestive of a role for MYH9 and MYH10 in tubulointerstitial kidney disease associated with abnormal renal epithelial transport.Support or Funding InformationFunding support from NIH Grant P20GM103620 and Sanford Research Program Funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Accurate segmentation of prostate cancer histomorphometric features using a weakly supervised convolutional neural network
Authors: Bukowy John David , Foss Halle , McGarry Sean D. , Lowman Allison , Hurrell Sarah , Ickzkowski Kenneth A , Banerjee Anjishnu , Barrington Alexander , Dayton Alex , Unteriner Jackson , Jacobsohn Kenneth , See William , Nevalanian Marja , Nencka Andrew , Ethridge Tyler , Jarrard David , LaViolette Peter 
Published: June 2021

Prostate cancer (PCa) arises from the glandular epithelium. To confirm the presence of cancer, a pathologist uses stained tissue samples taken either from biopsy or prostatectomy. Due to the relationship between epithelium and cancer, special consideration is given to regions identified as epithelium. Histomophometric techniques have long been used to identify areas of epithelium within the tissue for automated detection and classification pipelines; however, they are often rigid in their implementation and their performance suffers on large datasets where variation in staining, imaging, and preparation is difficult to control. The recent development and popularity of deep learning methods for image processing and segmentation offer great promise for developing robust classification pipelines for such ends; however, they require large labeled datasets for training.The goal of this study was to combine weakly labeled datasets generated using histomophometric techniques, and high‐quality labeled datasets from human observers to train a convolutional neural network. In doing so we developed a pixel‐wise segmentation algorithm for classification of stromal, epithelium, and lumen (SEL) regions for use on both biopsy core and whole‐mount bright‐field H&E stained tissue. We provide evidence that by simply training a deep learning algorithm on weakly labeled data, we can improve the robustness of the classification. Finally, we show that not only does this method carry primary improvement on SEL labeling within tissue, but that the information provided by the deep learning generated labels improves cancer classification in a higher‐order algorithm over the histomorphometric labels that it was trained on.Support or Funding InformationFunding was provided by the State of Wisconsin Tax Check‐off Program for Prostate Cancer Research (RO1CA218144 and R01CA113580) and the National Center for Advancing Translational Sciences (NIH UL1TR001436 and TL1TR001437).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 three‐dimensional model of the pterygopalatine fossa significantly enhances the learning experience
Authors: Tanner Jordan A , Sakaguchi Alan , Bartanuszova Maria , Ruparel Nikita , Jackson Jeffrey , Johnson Linda , Nation Haley , Jethwa Beeran , King Thomas S , Bhattacharya Arunabh , Sharma Ramaswamy 
Published: June 2021

The pterygopalatine fossa (PPF) is a small bilateral space deep within the skull that acts as a major neurovascular junction between the oral cavity, nasopharynx, orbit, infratemporal fossa, and middle cranial fossa. Because of its important crossroads of afferent sympathetic, and parasympathetic fibers, students, radiologists, ophthalmologists, neurosurgeons, and otorhinolaryngologists need to have a firm understanding of the PPF. However, its small volume, poor accessibility and numerous neurovascular connections make it a difficult space to comprehend using two‐dimensional illustrations and a single cadaveric dissection. We hypothesized that a 3D model of the PPF significantly improves the student's understanding of the PPF's boundaries, its communicating channels, and neurovascular structures as compared to traditional forms of pedagogy by promoting visual and kinesthetic learning experiences. We developed a 3D model of the PPF and an instructional manual describing the model. We evaluated our model by analyzing student performance on pre‐ and post‐quizzes and a student user satisfaction survey based on 5‐point Likert scale. A total of 43 undergraduate students enrolled in pre‐medical and dental schools completed the study; 21 students used a human half‐skull (control group) and 22 students used the 3D model (intervention group). The intervention group performed significantly better on the post‐quiz (p=0.01) when compared to the pre‐quiz; however, the control group did not improve their quiz scores (p=0.17). Surveys also indicated that the model enhanced the learning experience. We conclude that anatomy educational tools such as 3D models that would be easily accessible outside of the laboratory promote student learning ability.Support or Funding InformationUT System Shine Academy of Health Science Education Small Grants Program to Drs. Ramaswamy Sharma and Arunabh BhattacharyaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation and characterization of human iPSCs from human fibroblasts in respect to osteochondral regeneration
Authors: Danisovic Lubos , Csobonyeiova Maria , Nicodemou Andreas , Novakova Zuzana Varchulova , Miko Michal , Zamborsky Radoslav , Varga Ivan 
Published: June 2021

Cartilage and bone disorders are a group of varied acute and chronic traumatic, degenerative, malignant or congenital conditions affecting the joints and bones. Recently, cell‐based therapy appears to be a very promising tool of regenerative medicine and it has attracted the attention of many researchers and physicians. The huge attention is given to induced pluripotent stem cells (iPSCs). In the experiment, the human fibroblasts originally derived from subcutaneous tissue of infant due to cytogenetical analysis were used. Fibroblasts from 2nd passage were thawed and were sub‐passaged two more times in D‐MEM low glucose with 10% fetal bovine serum and antibiotics (Sigma Aldrich). Then, fibroblasts were reprogrammed using the StemRNA‐SR Reprogramming Kit (Stemgent) following the manufacturer's protocol with slight modifications. Briefly, 7 × 104 fibroblasts were plated into one well of a 6‐well plate coated with Geltrex (Thermo Fisher) on day 0, and cultured at 37 °C and 5% CO2 overnight in D‐MEM with 10% fetal bovine serum. Then, a medium was replaced by StemMACS™ iPS‐Brew XF medium (Miltenyi Biotec) and 500 μL NM‐RNA transfection complex solution was added to wells. This step was repeated during the next 3 days. Then, cells were cultivated in StemMACS™ iPS‐Brew XF medium. After 13 to 15 days, when the colonies emerged they were identified, mechanically picked and sub‐passaged. Obtained iPSCs were characterized by flow cytometry, real‐time PCR, and by histochemistry. Moreover, their chondrogenic and osteogenic differentiation potential was assessed according to the expression of specific genes. Normal fibroblasts were used as negative control. Obtained results showed that fibroblasts are easily accessible and they are suitable for generation of iPSCs, which have the potential to differentiate into chondroblasts and osteoblasts. It may be emphasized that after performing further studies focused on their bio‐safety they will represent unique tool for osteochondral regenerative medicine.Support or Funding InformationAPVV No. 14‐0032.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Aldh1l1 Knockout On Metabolic Phenotype in Mouse Liver
Authors: Sharma Jaspreet , Krupenko Natalia I. , Krupenko Sergey A. 
Published: June 2021

ALDH1L1 (10‐formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in liver. This enzyme metabolizes 10‐formyltetrahydrofolate to produce tetrahydrofolate (THF) and is involved in the regulation of reduced folate pools, de novo purine biosynthesis, and the flux of folate‐bound methyl groups. The goal of this study was to survey the metabolic impact of the Aldh1l1 gene loss in mouse liver. Aldh1l1−/− (KO) mice were generated using gene‐targeted ES cell clone (C57BL/6N background) from KOMP repository. KO mice were viable and did not show obvious phenotypic differences from the wild type animals. Flash frozen hepatic tissues from 12–14 weeks old Aldh1l1+/+and Aldh1l1−/− mice were profiled to identify metabolite changes and define associated pathways. In this study, mice were kept on standard AIN‐93 diet with 2.2 ppm of folic acid. Global metabolomic analysis was performed by Metabolon Inc. (Durham, NC) using Ultrahigh Performance Liquid Chromatography‐Tandem Mass Spectroscopy (UPLC‐MS/MS) for five samples from each group. This analysis has identified 628 known metabolites and principal component analysis demonstrated good segregation between the two genotypes. The difference for 91 metabolites was statistically significant between the two groups, with 31 compounds being decreased and 60 being elevated in Aldh1l1−/− compared to the Aldh1l1+/+ mice. This analysis indicated that KO mice might experience functional folate deficiency. Specifically, the intermediate of the histidine degradation pathway serving as a marker of folate deficiency, formiminoglutamate, was increased more than 15‐fold in KO mice. Furthermore, tissue folic acid and dihydrofolate were strongly depleted (more than 2‐fold and 5‐fold, respectively) in these animals, which would be in agreement with folate deficiency. In addition, a 2‐fold decrease in glycine was detected in KO mice. Strong drop in several glycine conjugates was also observed in the KO genotype, which is apparently associated with the deficiency of glycine. The depletion of glycine was likely caused by the decrease of the THF‐dependent synthesis of glycine from serine, with the underlying basis for this phenomenon being the decrease of the THF pool observed in Aldh1l1−/− mice. The depletion of glycine and/or folate could be the cause of several secondary effects on metabolism including the decrease in glutathione, lower availability of ascorbate, impaired mitochondrial lipid metabolism, oxidative stress, and less efficient remediation of toxic products. Our data also imply a compromised liver function in the KO mice and associated changes in gut microbiome activity. Overall, our study indicated significant metabolic perturbations caused by the Aldh1l1 gene knockout.Support or Funding InformationNIH R01DK 54388This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel NOX2 inhibitor Mito‐Apocynin Protects Against LPS‐induced Endotoxemia Pre‐Clinical Animal Model
Authors: Padhi Piyush , Lou Jie , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Kanthasamy Anumantha 
Published: June 2021

Sepsis is a serious medical condition caused by the body's response to an infectious or noninfectious insult. Various experimental data suggests that systemic inflammation resulting from the release of pro‐inflammatory cytokines and the chronic activation of the innate immune system is a key pathophysiological contributor to sepsis. The existing therapy for sepsis has largely been ineffective due to the heterogeneity in clinical severity and presentation among individuals. Commonly, Lipopolysaccharide (LPS) is used as an endotoxin to generate a pre‐clinical mouse model of sepsis model. Intraperitoneal injection of purified LPS has been shown to trigger systemic activation of the innate immune response followed by organ injury and mortality in mice. Recently, we demonstrated a newly‐synthesized and orally available derivative of apocynin that targets mitochondria, mito‐apocynin (Mito‐Apo), to be efficacious as an anti‐neuroinflammatory agent to inhibit NOX2 activity, oxidative damage and improve mitochondrial function in both cellular and pre‐clinical animal models of Parkinson's disease. Herein, we further evaluated the pre‐clinical efficacy of Mito‐Apo in the LPS‐induced endotoxemic mouse model of sepsis. C57BL6 mice were administered LPS (5 mg/kg, i.p. once) and Mito‐Apo (30 mg/kg, oral gavage, four times every 6 h at 0, 6, 12 and 18 h), and blood was collected at 1, 3 and 24 h post‐LPS treatment. Mice were sacrificed at 24 h post‐LPS injection. Initial results showed that oral interval administration of Mito‐Apo significantly attenuates LPS‐induced systemic inflammation evidenced by reduced circulating pro‐inflammatory cytokines interleukin‐β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) in plasma and decreased proIL‐1β/TNF‐α mRNA expression levels in mouse liver. Importantly, Mito‐Apo was also shown to significantly reduce LPS‐induced mortality and protect against LPS‐induced motor deficits. Collectively, these findings demonstrate that Mito‐Apo could be used as a novel plausible therapeutic agent for treating sepsis.Support or Funding InformationNS088206, ES026892 and NS100090, Lloyd Endowed ChairThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Formononetin attenuates isoproterenol‐induced cardiac toxicity in rats owing to its antioxidant, anti‐inflammatory and anti‐apoptotic activity
Authors: Suchal Kapil , Arya D S , Singh Sunita 
Published: June 2021

IntroductionA high rate of mortality and morbidity is associated with myocardial infarction (MI), indicating a need to hunt for newer treatment modalities. Formononetin (FMN) is a dietary flavonoid with remarkable antioxidant and anti‐apoptotic properties. Therefore, the aim of this study was to evaluate the ability of FMN in attenuating isoproterenol (ISO) induced oxidative stress and myocardial infarction.MethodsA total of 48 Male Wistar rats were administered either FMN (5, 10 or 20 mg/kg/day, i.p.) or vehicle for 15 days along with subcutaneous ISO, 85 mg/kg for 2 consecutive days i.e. 13th and 14th days. On the 15th day, rats were anaesthetized followed by cannulation of right coronary artery to record hemodynamic parameters. Blood sample was collected and heart was excised to evaluate for biochemical, histopathological, ultrastructural and immuohistochemical studies.ResultsISO‐treated rats demonstrated disturbed hemodynamic. There was also a significant decrease in antioxidant enzyme levels and increase in the level, of malondialdehyde, serum TNF‐α and IL‐6. The cardiac injury markers like creatine kinase‐MB and lactate dehydrogenase were increased in the serum. Additionally, immunohistochemistry displayed an increased Bax/Bcl‐2 ratio in the myocardium. Histopathological and ultrastructural studies support the protective effect of FMN in ISO‐induced myocardial infarcted rats.ConclusionFMN alleviates myocardial damage in ISO induced cardiac injury by preserving hemodynamic and biochemical function and reducing inflammation due to its anti‐apoptotic, anti‐inflammatory and antioxidant activities.Support or Funding InformationThe Project has been funded by the AIIMS, DelhiThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phoca vitulina swallowing physiology using videofluoroscopy: Creating a comparative rehabilitation model
Authors: Skoretz Stacey A , Haulena Martin , Akhurst Lindsaye , Johnson Emily , Dawson Camilla 
Published: June 2021

BackgroundUp to 150 wild harbor seal pups (Phoca vitulina) are admitted annually to our rescue centre. All require medical management including gavage feeding until weaned to whole fish prior to release back to the wild. The rehabilitation model is personnel intensive requiring animal restraint and force feeding. To the best of our knowledge, no systematic imaging has been conducted on the upper aerodigestive tract of wild seal pups. As a result, the information regarding their swallowing physiology is limited as are ways in which to optimize their feeding and swallowing rehabilitation. Our objectives were to develop a videofluoroscopic swallowing study (VFSS) protocol and to determine the feasibility of conducting a VFSS with a wild seal.MethodsFeasibility parameters included the determination of: contrast delivery methods, image capture and analyses, and the operational and infrastructure requirements. A Siemens mobile c‐arm fluoroscopy unit and retrofitted tub were utilized for imaging. After thawing, we injected whole flash‐frozen herring with liquid barium suspension (105% w/v). Recently weaned seals (N = 2) were imaged at 30 frames per second with loss less capture. Two blinded raters evaluated swallow physiology across 6 metrics with disagreements resolved by consensus. Results were summarized descriptively.ResultsWe conducted a freely‐behaving videofluoroscopic swallowing study on two infant seals (1 male: 8 wks, 3 d; 1 female: 5 wks, 3 d). Personnel included veterinarian, veterinary technicians (2), imaging technician and VFSS raters (2). Five boluses were administered to each seal (67–90 g each) with a total exposure of 2 min, 54 sec. We identified three distinct swallow phases: preparatory (preswallow), oropharyngeal and esophageal. The preparatory phase was characterized by airway closure and base of tongue (BOT) positioning. The oropharyngeal phase included bolus transport via BOT retraction, repeated anterior‐superior hyolaryngeal excursion, and posterior pharyngeal wall contraction. Bolus propulsion to the stomach occurred during the esophageal phase.ConclusionsWe have successfully conducted the first‐ever VFSS on wild seal pups. With this protocol, we will gather the first normative swallowing data, enhance diagnostic capabilities and potentially improve feeding and swallowing rehabilitation for this population. Ultimately, this will inform our development of the first cross‐species conceptual swallowing framework between seals and humans facilitating a synergistic knowledge translation model.Support or Funding InformationDr. Skoretz’ startup funding from the Faculty of Medicine and the School of Audiology and Speech Sciences at UBC provided support for this project.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inverse relationship between very low‐density lipoprotein (VLDL) ceramides, diacylglycerols, and triacylglycerols in human hepatic lipid accumulation
Authors: Mucinski Justine M , Syed‐Abdul Majid Mufaqam , Garrett Timothy J , Parks Elizabeth J 
Published: June 2021

Ceramides (CER), along with other lipotoxic lipids (diacylglycerols, DAG and triacylglycerols, TG), have been implicated in the development and progression of nonalcoholic fatty liver disease (NAFLD). No study to date has measured CER, DAG, and TG concentrations in VLDL with relation to liver fat content. Twelve male subjects with characteristics that put them at risk for NAFLD (mean±SD, age 42.1±5.4y, BMI 37.8±3.5 kg/m2, ALT 45±21 U/L) were studied. VLDL particles were isolated in the fasted state and lipid species measured via UHPLC‐MS/MS analysis. VLDL‐apoB100 was measured by ELISA and intrahepatic triglycerides (IHTG) by MRS. Total VLDL‐CER concentrations were positively correlated with both VLDL‐TG (r = 0.572, P = 0.004) and VLDL‐apoB100 (r = 0.557, P = 0.005). Total VLDL‐DAG concentrations were significantly positively correlated with VLDL‐apoB100 (r = 0.724, P < 0.001), tended to correlate with VLDL‐TG (r = 0.263, P = 0.088) but were most strongly correlated with VLDL‐CER (figure, r = 0.860, P < 0.001). With regard to IHTG, both plasma VLDL‐CER and VLDL‐TG were negatively correlated with liver fat (r = −0.447, P = 0.018 and r = −0.551, P = 0.006, respectively), while the relationship between IHTG and VLDL‐DAG was more modest (r = −0.290, P = 0.071). These data suggest that CER, DAG, and TG are packaged together in the liver and secreted simultaneously on VLDL particles. A less likely scenario is that CER or DAG are added to VLDL after the particles have been secreted into the plasma. These data support the concept that hepatic secretion rates of CER and TG, and to some extent, DAG, are potentially beneficial physiological processes that lower excess hepatic lipid accumulation in metabolic syndrome to support overall liver health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.