bluuebunny/crossref_metadata_2025_split huggingface dataset:
A gardian of gut epithelial barrier from inflammation: the elastase inhibitor ELAFIN
Authors: Deraison Celine , Jaeger Marie Charlotte , Guiraud Laura , Rolland Claire , Bellot Audrey , Rolland Corinne , Sebbag Mireille , Bermudez Luis , Langella Philippe , Vergnolle Nathalie 
Published: April 2020

In the gastrointestinal tract (GIT), proteolytic activity is tightly regulated to prevent inappropriate and potentially harmful proteolysis. In IBD this regulation is broken and our recent work has demonstrated that elastolytic activity is significantly increased in colonic epithelium from IBD patients. We have shown that the expression of the only elastase released by epithelial cells, ELA2 is up‐regulated; and the selective elastase inhibitor ELAFIN is severely down regulated in colonic epithelium from IBD patients. We showed that oral treatments with ELAFIN‐expressing L. Lactis bacteria restored proteolytic balance in the inflamed gut and was protective against acute and chronic colitis. ELAFIN is a low‐molecular weight molecule (10‐kDa), which specifically inhibits elastases. At N‐terminal terminus, a cationic helix showed anti‐microbial activities and could interact with phospholipids. Moreover ELAFIN is also able to inhibit NFkB and AP‐1 activation but this domain is not localized on protein. Therefore, several mechanisms of action are thus possible to explain ELAFIN’s protective property.ObjectivesIf ELAFIN‐recombinant L. lactis has to be considered as a possible treatment for IBD in human, there is an absolute need to define the mechanisms by which ELAFIN delivery protects against intestinal inflammation.MethodsWe generated and expressed in LAB ELAFIN’s mutants allowing investigation the biological properties of ELAFIN individually. These different forms of ELAFIN‐recombinant LAB were co‐culture with thapsigargin (10mg/ml) and ELAFIN (107 CFU/well). After 6H, cells were additionally exposed to gentamicin (100 mg/ml) to limit bacterial development. The barrier function was measured using FITC‐dextran passage through the epithelial layer. The elastolytic activity and CXCL8 release were quantified from the culture media. Expression of inflammatory mediators was quantified by qPCR.ResultsAddition of thapsigargin for 24H on Caco2 cells induced ER‐stress and disruption of elastolytic balance. ELAFIN expression was decreased with up‐regulation of ELA2, leading to increase of elastolytic activity in medium, disclosed in IBD patients. Concomitantly, barrier function was reduced, and expression of CXCL8, CXCL10 and hBD2 was increased. Co‐culture with ELAFIN expressing‐LAB normalized all these parameters confirming anti‐inflammatory properties of ELAFIN. Neutralization of a‐helix (mutant K44D/K50G) did not modify ELAFIN’s properties. However, disruption of protease inhibitory function (mutant A62G/M63G) led to unability of ELAFIN mutant to modify permeability and inflammatory phenotype induced by exposure of intestinal epithelial cells to thapsigagin.ConclusionThis suggests that anti‐inflammatory activity of ELAFIN is based on the control of elastase activity released by epithelial cells.
Identification of Key Factors in Cardiomyocyte Development by Single‐Cell Transcriptome Analysis
Authors: Zhu Jun , Wu Dingfeng , Gao Wenxing , Chen Wanning , Fu Qianzheng , Jiao Na , Zhu Lixin , Zhu Ruixin 
Published: April 2020

BackgroundA promising intervention for heart diseases is the application of cardiomyocytes differentiated from pluripotent stem cells. However, the driving factors of the differentiation process still remain unclear. This study aims to map the transcriptional landscapes of directional differentiation of pluripotent stem cells at multiple stages, using single‐cell sequencing data, in order to identify the key drivers of the differentiation process.MethodSingle‐cell sequencing data from pluripotent stem cell‐derived cardiomyocytes at age day 0, day 2, day 5, day 15 and day 30 were included and normalized. UMAP algorithm was then used to reduce the dimensionality of the transcriptome profiles, followed by community detection to group the cells and cell type identification using marker genes. Graph‐autocorrelation analysis was implemented to identify differentially expressed genes among cell groups with Gene Ontology term enrichment. Next, we calculated the pseudotimes of each cardiomyocyte to construct the pseudotime trajectories. Finally, knowledge based dynamic changes in gene expression were analyzed to identify the key factors driving the cardiomyocyte development.ResultWe identified 9 different cell types in the process of cardiomyocyte development, including cardiomyocytes, induced pluripotent stem cells, cardiogenic mesoderm cells, cardiovascular progenitor cells, embryonic stem cells, endoderm cells, epithelial progenitor cells, cardiomyocyte progenitor cells and multipotent progenitor cells. Identification of cardiomyocyte was confirmed with specific gene expression profiles such as significant enrichment in the biological processes of cell adhesion and supramolecular fiber organization. The expression of NKX2‐5, which encodes a homeobox‐containing transcription factor, was found to be at a lower level in the early stages and continually increased as cardiomyocyte develop, suggesting that NKX2‐5 was one of the driving factors of cardiac development, acting through promoting myocardial maturation and development.ConclusionSingle cell sequencing data revealed the dynamic changes of gene expression during cardiomyocyte development differentiation. NKX2‐5 was identified as a key factor in cardiac development. Our finding contributes to the understanding of the mechanism behind the cardiomyocyte development, and may serve as the basis for stem cell based therapies.Support or Funding InformationThis work was supported by National Natural Science Foundation of China 81774152 (to RZ) and 81770571 (to LZ).
Secreted insect transferrin‐1 with strong and reversible iron‐binding has potentially tissue specific roles in immunity and iron transport
Authors: Weber Jacob J. , Park Yoonseong J. , Gorman Maureen 
Published: April 2020

Iron is biologically essential and potentially toxic. To maintain healthy physiological systems, iron must be properly sequestered, transported and stored in animals. The transferrin family of proteins plays a major role in maintaining this homeostasis in vertebrate animals. However, little is understood about iron homeostasis and transferrin’s role in invertebrates. In mammals there are two well understood secreted iron‐binding transferrins: (1) serum transferrin (STF) binds two ferric‐iron ions in the blood and transports them into cells by receptor mediate endocytosis; (2) lactoferrin (LTF) functions as an immune protein by sequestering two ferric‐iron ions in secreted fluids to inhibit bacterial growth. Insect Transferrin‐1 (Tsf1) is found in insect hemolymph and secreted fluids and is homologous to STF and LTF; however, its function in the iron transport and immunity of invertebrates is not clear. As a first step towards elucidating its function, I have purified recombinant Drosophila melanogaster Tsf1 (DmTsf1) and hemolymph derived Manduca sexta Tsf1 (MsTsf1). Comparing the spectroscopic and kinetic data of DmTsf1 and MsTsf1 to STF and LTF shows that these Tsf1s coordinate iron differently and have a relatively lower but still potent iron binding affinity at pH 7.4 (DmTsf1 log K′= 17, MsTsf1 log K′= 17 and STF log K′ = 20). The Tsf1s release iron over the pH range of approximately 6 to 4, which is nearly identical to STF and indicates the possibility of iron release in the endosome. Current work is aimed toward determining how iron from Tsf1 is transported into cells. One hypothesis is that Tsf1 is endocytosed and iron is released in the endosome—similar to the iron transport mechanism of STF in mammals. I tested this hypothesis using immunohistochemistry and confocal imaging of Drosophila tissues. Immunostaining shows that DmTsf1 colocalizes with an early endosome marker (Rab5) in the developing oocyte, supporting my hypothesis of iron delivery. DmTsf1 was also found in the tracheal tissue, but instead of clearly colocalizing with Rab5, it appears that Tsf1 is secreted into the tracheal lumen and not taken back up. Overall, these results indicate that Tsf1s may have tissue specific roles: iron delivery to the oocyte and iron sequestration in the tracheal lumen. Future work will be completed to test if Tsf1 is endocytosed in other tissues and if it plays an important role in supplying iron to these tissues.Support or Funding InformationNSF: 1656388
Distinct projections from the prelimbic cortex modulate active avoidance
Authors: Valentín-Valentín Viviana P. , Diehl María M. , Iravedra-García Jorge M. , Morán-Sierra Jonathan , Rojas-Bowe Gabriel , Gonzalez-Díaz Fabiola N. , Quirk Gregory J. 
Published: April 2020

Avoiding threatening stimuli is a survival mechanism that can become maladaptive when avoidance interferes with other goal‐directed behaviors. For example, when an individual is seeking food, a threatening stimulus may be encountered, leading to an avoidance response, which comes at a cost of obtaining food. To model this behavior, we used an active avoidance task in which food‐restricted rats learn to step onto a platform to avoid a tone‐signaled shock, at the cost of obtaining a sucrose pellet. Activity in prelimbic cortex (PL), basolateral amygdala (BLA), and ventral striatum (VS) are necessary for the expression of platform‐mediated active avoidance (PMA) (Bravo‐Rivera, et. al., 2014), suggesting that these structures form a neural circuit to regulate PMA. We recently reported that inhibitory tone responses in PL signal the avoidability of shock, and opposing these responses using 4Hz photoactivation impaired avoidance (Diehl, et. al., 2018). Here, we want to know if PL inhibition signals downstream structures that are necessary for avoidance (i.e. VS and BLA), using an optogenetic approach. Photoactivating PL‐VS projections using Channelrhodopsin (ChR2) impaired avoidance (eYFP 88% (n=12) vs. ChR2 22% (n=8), p<0.0001), whereas photosilencing PL‐VS projections using Archaerhodopsin (ArchT) had no effect, suggesting that inhibition of this pathway promotes avoidance. Additionally, photoactivating PL‐BLA projections accelerated avoidance (eYFP 75% (n=9) vs. ChR2 88% (n=8), p=0.017), and photosilencing PL‐BLA projections impaired avoidance (eYFP 77% (n=9) vs. ArchT 40% (n=7), p=0.026), suggesting that excitation of this pathway promotes avoidance. Overall, these findings suggest that distinct PL projections have opposing roles in controlling avoidance by driving or suppressing avoidance.Support or Funding InformationThis work was supported by NIH grants R37‐MH058883 and P50‐MH086400 (Conte Center) to GJQ; F32‐MH105185 to MMD; 5T34‐GM00782140 to GRB, 5R25‐GM097635 to JIG and VVV; 5R25‐GM061151 to GRB, FDG and JMS.
The Lecture is Not Dead: Combined Peer Assisted Flipped Class, an Alternative to Classical Lecture in Basic Medical Education
Authors: Waly Nermien , Shawky Noha Osama , El-Bab Mohamed Fath 
Published: April 2020

PURPOSEStudent engagement at lectures is declining especially at those of of basic sciences. The main method of Physiology teaching, in our settings, is the lecture. It has been increasingly challenging to employ new educational methodologies in teaching physiology for several economical and technical reasons. Our objective was to enhance lecture attendance and increasing student engagement in physiology lectures.METHODScombined peer assisted learning (PAL) and flipped classroom (FC) strategies (PAFC) was employed in the teaching of neurophysiology course for second year medical students. Course Material and reading assignments were provided ahead of class on instructor and departmental websites. Twenty Students participated in lectures preparation and presentation under instructor’s supervision. Class members participation was conducted via pre‐class reading assignments, in class discussions and instructor guided questions. Method evaluation was conducted using both course surveys, quizzes, end of course evaluations and lecture attendance.RESULTSlecture attendance showed about 30 % increase compared to anatomy class conducted in classical lecture format. Course surveys revealed a 77% of students were satisfied with the new methodology while 23% preferred the traditional method. All PAFC participants expressed their positive feedback when interviewed after the course had ended and their comments was documented. End of course evaluations showed that class results were Comparable to anatomy course taught in traditional method (same class and conducted at the same time).CONCLUSIONIn conclusion, PAFC can be beneficial in teaching basic science courses for medical students. It can enhance lecture attendance and student satisfaction. Also, it can provide an affordable alternative method to improve medical education in institutes with limited resources.
The Compatibility of Varied Rib Cartilage Shapes with the Inferior Rim of the Orbit: A Gross Anatomical Study
Authors: Hobeika Natalie A. , Bolden Alexys M. , Burkett Jesse T. , Zdilla Matthew J. , Lambert H. Wayne 
Published: April 2020

Recently, reports have documented the reconstruction of the infraorbital rim with costal cartilage grafts. In order to determine the costal cartilage with the shape that is most compatible to that of the infraorbital rim, a geometric morphometric analysis was performed. Cadaveric costal cartilages 5–7 and infraorbital rims were skeletonized and photographed. Photographs were utilized to perform shape analysis. The report highlights the pros and cons of utilizing costal cartilage from each level and side of the body. This information will help improve pre‐operative decision making in the reconstruction of the infraorbital rim.Support or Funding InformationWVU INTRO (Initiation to Research Opportunities) Summer Research program; WV Research Challenge Fund [HEPC.dsr.17.06]
Proteolytic Activation of ENaC Mediates Fructose‐Induced Salt‐Sensitive Hypertension
Authors: Shukla Rashi , Hong Nancy , Henderson Ryan , Gonzalez-Vicente Agustin , Hopfer Ulrich , Garvin Jeffrey 
Published: April 2020

Proteolytic cleavage of the amiloride‐sensitive epithelial sodium channel (ENaC) by serine proteases such as trypsin leads to channel activation. In nephrotic syndrome, glomerular injury allows filtration of such enzymes causing ENaC‐dependent Na retention and hypertension(PMID: 23216619). Dietary fructose also causes Na retention and salt‐sensitive hypertension at least, in part, via altered hormonal signaling and metabolism in the proximal nephron. Proximal tubules normally secrete proteases into the forming urine but whether they are a source of proteases that cleave and activate ENaC in fructose‐induced salt‐sensitive hypertension is unknown. We hypothesized that dietary fructose increases expression and release of proteases from the proximal nephron, and that these enzymes activate ENaC thereby causing Na retention and salt‐sensitive hypertension. To test this hypothesis we first measured proximal nephron protease expression and urinary protease excretion in rats on either a high‐salt diet containing 4% NaCl without fructose (high salt) or one containing high salt plus 20% fructose in the drinking water (high salt/fructose) for 7 days. High salt/fructose treatment increased proximal nephron expression of trypsin I, an enzyme known to cleave and activate ENaC, by 70% (p < 0.01) compared to high salt alone. Urinary excretion of trypsin I was 8.4±1.3 arbitrary units/μg protein in rats fed high salt while it was 20.3±4.6 arbitrary units/μg protein in those on the high salt/fructose diet, 142% greater (p < 0.02). There was no difference in total urinary protein excretion between groups. Finally, we examined the effect of high salt/fructose and high salt plus 20% glucose (high salt/glucose) on blood pressure before and after oral amiloride. After 7 days, the systolic blood pressure of rats on high salt/fructose was 142±6 mm Hg while it was only 121±6 mm Hg in those on high salt/glucose (p < 0.05). After 10 days, the systolic blood pressure of rats on high salt/fructose was 148±6 mm Hg while it was only 124±5 in those on high salt/glucose (p < 0.02). Amiloride treatment for 24 hrs reduced systolic blood pressure in rats on the high salt/fructose diet from 148±6 to 134±5 mm Hg but did not decrease it in the high salt/glucose group. We conclude that proteolytic cleavage of ENaC contributes to fructose‐induced salt‐sensitive hypertension and that the source of the protease(s) is likely the proximal tubule rather than glomerular filtration.Support or Funding InformationNLHBI HL128053
Molecular Landscape of Canine Osteosarcoma
Authors: Das Sunetra , Idate Rupa , Fowles Jared S. , Lana Susan E. , Regan Daniel P. , Gustafson Daniel L. , Duval Dawn L. 
Published: April 2020

Osteosarcoma affects about 2.8% of dogs with spontaneous cancer and its occurrence is significantly higher than observed in humans. The prognosis of this disease is relatively poor, where the survival rate of ~45% of dogs with bone cancer is one year. With the recent advancement in characterizing mutational landscapes of various cancer types, personalized medicine has become increasingly important for cancer treatment. The purpose of this study is to characterize the mutation and expression profile of canine osteosarcoma. Using whole exome capture (Agilent Sure Select Canine V2) we sequenced 26 primary tumors and matched normal samples, with coupled treatment and outcome data. These samples were previously analyzed for gene expression using Affymetrix Canine 2.0 microarrays.The BWA tool was used to map Illumina reads to the CanFam3.1 genome and short variants were called and annotated using Mutect2 and SnpEff tools, respectively. Additionally, copy number variations were called and annotated using Sequenza and GISTIC tools. The total number of somatic variants identified across 26 samples ranged from 148 to 3,785 The protein coding mutations per Megabase ranged from 0.25 to 11.92. Among the protein coding somatic variants identified as SNVs and INDELS, an average of 80% were missense mutations (range 64% to 92%). The top recurrent mutations were observed in TP53 (82% of the samples), SETD2 (22%). We have also identified 56 known cancer genes with copy number variations (amplification or deletion) in at least 58% of the samples. When compared to pediatric osteosarcoma, there was an overlap of 38 genes (eg: TFEB, MYC) that were recurrently amplified and 69 genes (eg: RB1, MSH3) were recurrently deleted in both canine and human osteosarcoma.Using the microarray data, we have identified 585 differentially expressed genes between normal bone and osteosarcoma samples. Using GeneSet Expression Analysis (GSEA) we observed down‐regulation of myogenesis genes and up‐regulation of extracellular matrix genes in tumor samples. Additionally, we have quantified T‐cell and macrophage infiltration in the tumors via immunohistochemistry. Across 20 samples, the median percentage of tumor area with CD3+ and MAC387 positive cells was 0.14% and 0.21%, respectively. There was no significant correlation between disease‐free interval (DFI) and median dichotomized T‐cell or macrophage infiltration. However, Kaplan‐Meier analysis revealed that dogs with TP53 missense mutations had significantly longer DFIs than those with TP53 wildtype/null mutations. Further, correlation of gene expression and DFI can provide us with prognostic biomarkers for osteosarcoma in dogs.Support or Funding InformationAnschutz Foundation and Morris Animal Foundation
Development of a micro‐plastination protocol for morphological research
Authors: Ottone Nicolas Ernesto , Baptista Carlos , Fuentes Ramon , del Sol Mariano 
Published: April 2020

IntroductionUltrathin‐sheet plastination technique has been used to study microscopic details in anatomy. The aim of this study was to develop an ultra‐thin sheet plastination protocol in an induced model of osteoarthritis in rat humeral joint.Material and methodRat humeral joints, with osteoatrhitis, were used for this research. Sixteen weeks after osteoatrhitis induction, the animals were euthanized and injected with red epoxy resin through toracic aortha. Humeral joints and surrounded tissue were fixed in 10 % formalin. Samples were submitted to the plastination technique. The first step was dehydration during ten days, with acetone (100 %) at −25 °C. Later, for degreased, samples were inmerse in methylene chloride at room temperature during one week. Forced impregnation, the most important step in plastination, was peformed inside a stove with vacuum chamber. The plastinated blocks were cutted with a low speed band saw, with a diamond saw blade. Slices were placed in curing chambers to achieve polymerization and final transparentation.ResultsWe obtained slices of humeral joint of 230 μm thick, in which articular cartilage characteristic osteoarthritis morphological changes, including neovascularization, were observed.DiscussionUltra‐thin sheet Plastination is generally not used as a methodology for the identification of morphological changes caused by pathologies. However, we have applied this technique to identify the changes caused by osteoarthritis in the humeral joint of rats, as tjhe invasion of blood vessels in subchondral bone.ConclusionUltra‐thin sheet plastination technique was a methodology that can be used for the identification, measurement and comparison of morphological modifications caused by various pathologies, including osteoarthritis.