bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ontogeny of mRNA expression of the genes from the SHFM3 critical region in chicken
Authors: Chryst‐Ladd Megan , Maze Jennifer , Schwartz Charles E 
Published: June 2021

The split‐hand/split‐foot malformation (SHFM) is a congenital limb malformation that is characterized by absence or reduction of the central digital rays of the hands and feet. At one of the six well established loci, SHFM3 at chromosome 10q24, tandem duplications have been associated with the SHFM phenotype in humans; the smallest reported includes the genes POLL, BTRC, DPCD, and a portion of FBXW4. SUFU, a gene near the duplicated region, has been shown to be overexpressed in SHFM3 patients. RNA collected from chicken embryo limb buds (E2‐6), limbs (E7‐13), and wings (E14‐15) was analyzed for expression of the homologs of these genes by RT‐PCR. Poll expression was present in limb RNA at stages E2‐E15, although barely detectable. Expression of fbxw4 was detected in stages E3 through E15. Both sufu and dpcd expression was detected at E2, with no expression at E3, and the intensity of the bands varied for E4‐15. These results suggest that these four genes are expressed in the limb during development.To better understand how the expression of these genes correlates to the stages of limb development, E3‐15 cDNA was analyzed using quantitative PCR (qPCR) by comparing their expression to that of a control gene. Early results indicate the timing of the expression of these genes correlates to the end of digit formation on the chicken limb. Future studies will investigate the localization of these genes in the developing chicken limb.
Evidence that Farnesoid X Receptor ligand through SF‐1 responsive element inhibits aromatase expression in Leydig tumor cells.
Authors: Andò Sebastiano , Malivindi Rocco , Rizza Pietro , Gu Guowei , Panza Salvatore , Catalano Stefania 
Published: June 2021

The Farnesoid X Receptor (FXR) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors, that heterodimerizes with the Retinoid X Receptor (RXR).FXR, normally present in the liver and in the gastrointestinal tract, has been characterized as a bile acid receptor, but it is also expressed in nonenterohepatic tissues, including kidneys, adrenal gland, heart, vascular tissue, thymus, ovary, spleen, breast and testis.In this study, we investigated how transactivation of FXR by a specific ligand, chenodeoxycholic acid (CDCA), may modulate aromatase expression in R2C Leydig tumor cells.Upon prolonged exposure to CDCA we observed a strong decrease of aromatase expression, in terms of protein content, as revealed by immunocitochemistry and western‐blotting analysis, mRNA and aromatase enzymatic activity.Transient transfection experiment, using vector containing rat aromatase promoter PII, evidenced that CDCA reduced basal aromatase promoter activity. Specific mutagenesis studies, EMSA and ChIP assay, revealed that these inhibitory effects were due to FXR binding to SF‐1 responsive element present in the aromatase promoter region.Our findings address how FXR ligands may perspectively represent potential pharmacological tools to inhibit local estrogen production sustaining Leydig cell tumor growth and progression
Isoflurane opens mitochondrial KATP channel and enhances ROS production in human embryonic stem cells derived cardiomyocytes
Authors: Sepac Ana , Sedlic Filip , Si‐Tayeb Karim , Bienengraeber Martin , Park Frank , Lough John W , Duncan Stephen A , Bosnjak Zeljko J 
Published: June 2021

Transplantation of cardiomyocytes derived from human embryonic stem cells (CM‐hESCs) is promising tool for recovery of impaired cardiac function. We investigated whether cardioprotective agent isoflurane, elicits cardioprotective mechanisms in CM‐hESCs, which may suggest its role in protection of CM‐hESCs. CM‐hESCs were obtained by differentiation from H1 hESC line. Beating areas indicated the presence of CM‐hESCs which was confirmed by immunostaining for cardiac specific proteins, cardiac troponin I and titin. Living CM‐hESCs were labeled with eGFP under transcriptional control of the ventricular myosine light chain promoter, introduced into the cells using the lentiviral vectors. Once dissociated, eGFP‐positive cells were detected using confocal microscopy. Isoflurane depolarized mitochondria, as measured by an increase in TMRE fluorescence (110±1% of baseline) via opening of mitochondrial K channels (mitoKATP), since the channel inhibitor, 5‐hydroxydecanoate, attenuated the effect (103±1% of baseline), and diazoxide, mitoKATP opener mimicked the effect of isoflurane (109±1% of baseline). Isoflurane enhanced reactive oxygen species (ROS) production, measured by increase in DHE fluorescence (112±3% of baseline). In conclusion, isoflurane enhances ROS production and opens mitoKATP, two important mediators of the preconditioning signaling cascade.Supported by HL034708
Comparative expression and transcriptional networks of human‐mouse syntenic genes during osteoblastic differentiation of bone marrow mesenchymal stem cells
Authors: Dernowsek Janaina Andréa , Fontana Vanessa , Evangelista Adriane F , Junta Cristina M , Marques Márcia M C , Macedo Claudia , Simões Belinda P , Prado Karina F B , Passos Geraldo A S 
Published: June 2021

Osteoblasts are cells differentiate from mesenchymal precursors in a complex process regulated by transcription factors that regulate the expression of key genes. Many of these genes present human‐mouse synteny, i.e, they share sequence identity and the same physical distribution in chromosomal regions. Our aim in this study was to compare the transcriptional profiling and network interactions of syntenic genes during the osteogenesis in vitro. The mesenchymal stem cell (MSC) were collected from of the bone marrow of adult healthy donors or of 4 week‐old mice. SC cells were cultured in α‐MEM medium with fetal bovine serum. After expansion, cells were transferred to differentiation medium containing. Total RNA was prepared from undifferentiated and differentiated MSC after 24 and 48 h and 7 days. Fluorescent Cy3/Cy5 cDNA probes from total RNA of these cells were generated and hybridized with a cDNA microarray. The gene expression profiling was analyzed by hierarchical clustering and to reconstruct the regulatory transcriptional networks, we used the GeneNetwork program. A set of 227 syntenic genes were identified from which 108 were comodulated, i.e, genes presenting the same expression profiling. Gene profiling, although useful for characterizing the osteogenesis and identifying the expression signatures of syntenic genes. The GeneNetwork program was a useful tool allowing the detection of seven syntenic gene nodes (SHMT1, CACNB3, CASP8, GFM2, XPC, SYNGR2 and SDF2), which establish interactions with other genes. These are then considered as candidate genes controlling the osteogenesis.Grant Funding SourceFAPESP, CNPQ, FAEPA
The effect of early Bone Morphogenetic Protein (BMP) activation on craniofacial development
Authors: Chong H. Jonathan , Marcucio Ralph 
Published: June 2021

Bone morphogenetic proteins (BMPs) are a family of growth factors that play important roles in craniofacial development. Inhibition of BMP signaling in the head mesenchyme of early stage chick embryos leads to craniofacial defects. The aim of this study was to activate the BMP pathway and investigate effects on craniofacial development. Activation of the BMP pathway was achieved via placement of BMP‐2‐soaked beads in the head mesenchyme or injection of DF‐1 fibroblasts transfected with an avian retrovirus expressing BMP2 or BMP4 (RCAS‐BMP2 and RCAS‐BMP4, respectively). Placement of BMP‐2‐soaked beads led to significant defects affecting the brain, eyes, face, and jaw structures. Phenotype severity appeared to be dose‐dependent, and TUNEL analysis revealed an increase in cell death compared to controls. Injection of DF‐1 cells transfected with RCAS‐BMP2 or RCAS‐BMP4 yielded similar results. In conjunction with previous studies examining BMP inhibition in the head mesenchyme, these data suggest that a delicate balance in the degree of BMP signaling regulates normal craniofacial morphogenesis.Grant Funding SourceNIH/NIDCR R01DE018234 to R.M. and HHMI Medical Research Training Fellowship to H.J.C.
Structuring a structures course around core competencies
Authors: Gregory Jeremy K , Camp Christopher L , Jacobs Sarah M , Chen Laura P , Alexander Cara J , Lachman Nirusha , Pawlina Wojciech 
Published: June 2021

Recent curricular redesign at Mayo Medical School integrated radiology into first‐year gross and developmental anatomy. The resulting six‐week (120 contact hours) Human Structure block teaches gross anatomy through dissection, radiologic imaging, and correlation with embryology. To further promote holistic physician‐development, refinement of the Human Structure course has been driven by the six core competencies of the Accreditation Council for Graduate Medical Education (ACGME): patient care, medical knowledge, practice‐based learning, interpersonal skills, professionalism, and systems‐based practice. We report here more than 20 educational interventions built into the Human Structure block that may serve as a model for incorporating the ACGME core competencies into early medical education. The block emphasizes clinically oriented anatomy, invites self‐ and peer‐evaluation, provides daily formative feedback through an audience response system, and employs team‐based learning. The course curriculum includes didactic briefing sessions and roles for students as teachers, leaders, and collaborators. Third‐year medical students serve as teaching assistants, enhancing their own knowledge of anatomy, bettering their skills as medical educators, and facilitating group communication and critiques. This work was supported by the Mayo Clinic Department of Anatomy.Grant Funding SourceNone
Learning to lead: Student perceptions of team leaders in medical school
Authors: Chen Laura PeiHan , Jacobs Sarah M , Gregory Jeremy K , Alexander Cara J , Camp Christopher L , Lachman Nirusha , Pawlina Wojciech 
Published: June 2021

Early medical education courses provide opportunities for promoting leadership in future physicians. Our research sought to integrate explicit activities of teamwork and leadership into the first‐year gross anatomy course and assess student leaders through self reflection and team evaluation. Fifty first‐year medical students were assigned to three‐ or four‐member dissection teams with group leaders designated on a rotating basis. Teamwork was emphasized through daily group quizzes, oral patient presentations relating clinical diagnoses to anatomy, and extensive embryology presentations. Group leaders were responsible for leading the dissections after attending daily orientation sessions on the teaching cadaver. Team and self evaluations of group leaders were gathered with weekly electronic surveys. Preliminary results suggest that team leaders rate themselves lower than their teammates do with respect to altruism, accountability and responsibility. One explanation for this discrepancy is decreased self‐awareness of student leaders regarding their own performance. Further research may demonstrate whether increased communication between leaders and team members can improve leaders' awareness of their capabilities. Research support came from the Mayo Clinic Department of Anatomy.Grant Funding SourceN/A
Determining optimal learning conditions for acquiring spatial 3D information using computer‐based anatomical reconstructions
Authors: Nguyen Ngan , Nelson Andrew J , Wilson Timothy D 
Published: June 2021

Interactive stereoscopic 3D digital models of head and neck structures, generated from CT scans of a male cadaver, have been developed. The display and interactive features of the models will be used to determine optimal conditions for acquiring 3D information for individuals with different spatial abilities. A pre‐mental rotation test will be used to determine participants' spatial ability. Based on pre‐test results, participants will be assigned to either the high or low spatial ability group. Members of each group will be further divided into four subgroups based on four learning conditions. A stereo/high‐interactive group will study anatomy using stereoscopic models and will have active control over the pace and direction of the model. A non‐stereo/low‐interactive group will examine anatomy with 2D images and restricted interaction. A stereo/low‐interactive and non‐stereo/high‐interactive group will study the same anatomy in corresponding conditions. Learning will be assessed by 50 multiple‐choice questions that require mental manipulation of internalized 3D anatomical representations. The results of this study will aid the design and implementation of effective 3D computer visualizations to help students comprehend the spatial 3D organization of the human body, while accounting for individual differences in spatial abilities.Grant Funding SourceInternal
Effects of diminished protein synthesis on bone anabolic response to load in RPL29‐deficient mice
Authors: Sloofman Laura Gail , Chen David , Zhou Xiaozhou , Price Christopher , Novotny John E. , Wang Liyun , Kirn‐Safran Catherine B. 
Published: June 2021

Deletion of a single ribosomal protein, RPL29, increases bone fragility due to diminished protein synthesis and poor tissue quality, suggesting an important link between skeletal tissue growth and efficient protein production. The goal of this study is to determine if reduced capacity to synthesize large volumes of proteins modifies bone anabolic response to mechanical load. To test this idea, tibiae of RPL29‐null mice and age‐matched wild type (WT) controls were loaded at 20% maximal load, 0.5 Hz for 50 cycles/day, with a five‐second rest period inserted between two cycles for two weeks.The cortical bone microstructure and mechanical properties of the non‐loaded and loaded tibiae of both RPL29‐null and WT mice were analyzed using micro‐computed tomography (microCT) and three‐point‐bending tests, respectively. Results indicate that following loading minor changes occur at the structural and material property levels in both control and null tibiae. Ongoing studies are using dynamic histomorphometry to quantify differences in bone formation capacity between loaded and non‐loaded conditions. These studies will establish the importance of high volume protein synthesis for the regulation of bone formation. This work is supported by NIH P20 RR016458‐06 and an HHMI Undergraduate Science Education Grant.
The BTI1 Azoreductase Colorimetric and Fluorometric Reporter System
Authors: Johansson Hans E. , Johansson Mary K. , Wong Albert , Peterson Erik J. , Armstrong Eliana S. , Grant Richard E. , Reddington Mark V. , Cook Ronald M. 
Published: June 2021

Reliable duplex and multiplex assays remain a challenge for current fluorescence reporter enzyme technology. To this end we have developed a system that takes advantage of pro‐fluorescent probes and a novel azoreductase.The BTI1 azoreductase gene was cloned from a soil sample, optimized and used to bacterially express the protein. Biochemical characterization of the purified enzyme in solution revealed a tetramer carrying a non‐covalently bound flavin mononucleotide cofactor. BTI1 readily reduced an array of carboxylated and sulfonated azo dyes including methyl orange (DABSYL) and the Black Hole Quencher (BHQ(tm)) dyes. Enzyme kinetic parameters of the four‐hydride transfer reaction were consistent with the bi‐bi ping‐pong mechanism of related oxidoreductases. In our optimized colorimetric assay, the detection limit of the BTI1 azoreductase was >10 fold lower than that of beta‐galactosidase.Reduction of the azo‐quenchers in pro‐fluorescent substrates containing BHQs and a range of fluorophores readily released fluorescence in an NADPH‐dependent manner with robust signal to noise ratios.In short, by matching BTI azoreductase with pro‐fluorescent substrates we have created a simple, yet adaptable, reporter system that generates diverse fluorescence signals.This research was supported in part by a grant from NIH to HEJ (R43/R44 GM076843).
Connexin 43 is present in subsarcolemmal but not in interfibrillar mitochondria of rat cardiac myocytes
Authors: Boengler Kerstin , Stahlhofen Sabine , van de Sand Anita , Gres Petra , Buechert Astrid , Garcia‐Dorado David , Heusch Gerd , Schulz Rainer 
Published: June 2021

In cardiac myocytes, connexin 43 (Cx43) is present in mitochondria, and mitochondrial Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). Cardiac myocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ functionally. Variations in the protein distribution between SSM and IFM have not yet been identified. The aim of the present study was to characterize the function of SSM and IFM, and to investigate whether Cx43 is present in SSM or IFM. The ADP‐stimulated respiration was greater in IFM than in SSM from rat ventricles using substrates for complex I and II. The calcium‐induced loss of membrane potential occurred at lower concentrations in SSM than in IFM. Cx43 was exclusively detected in SSM by Western blot analysis (n=4).Taken together, our data confirm that SSM and IFM differ in respiration and calcium handling. Cx43 is localized in SSM. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM might serve different functions in the signal transduction of IP.
The cholesterol homeostatic protein Niemann‐Pick Type C‐2 (NPC2) is required for HIV‐1 assembly and release
Authors: Coleman Ebony Michelle , Hildreth James E.K. 
Published: June 2021

HIV‐1 utilizes host proteins for productive viral replication. It is well established that HIV‐1 buds from cholesterol‐rich microdomains called lipid rafts, and that cholesterol is required for HIV‐1 entry, infectivity, and structural integrity. NPC2 protein binds cholesterol with high affinity and regulates cholesterol efflux from the late endosomal/ lysosomal (LE/L) compartments. NPC2 mutations result in NPC disease (NPCD), a cholesterol storage disorder characterized by an accumulation of cholesterol in LE/L compartments. We hypothesize that NPC2 is required for HIV‐1 assembly and release. Our findings show that in the absence of functional NPC2, HIV‐1 release is decreased. In fibroblasts from NPC2‐deficient patients infected with VSVG pseudotyped HIV‐1, virus release is reduced by 80%. Moreover, HIV‐1 Gag accumulated in the cholesterol laden LE/L compartments of the fibroblasts. Lysosomal targeting of NPC2 is mannose receptor dependent. Addition of mannose 6‐phosphate to chronically‐infected Jurkat cells disrupts both lysosomal targeting of NPC2 and HIV‐1 egress. Similarly, siRNA‐mediated silencing of NPC2 in these cells inhibited virus release. Collectively, these data indicate that NPC2 is required for HIV‐1 assembly and release.Supported by NIH grant 5732HL‐007737‐15
A novel fluorimetric assay for detection of aggrecanase‐1 (ADAMTS‐4) activity using a long wavelength FRET peptide substrate
Authors: Rakhmanova Vera A , Lui Siu Kei , Fan Weizhen , Bai Yuehong , He Jianjun , Hong Anita , Tong Xiaohe 
Published: June 2021

Aggrecanase‐1, also called ADAMTS‐4, is a member of the ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) family of proteases. Aggrecanase cleaves specific peptide bonds in aggrecan, the main proteoglycan of articular cartilage. The high activity of aggrecanase results in cartilage degradation in diseases such as arthritis. To develop homogeneous high throughput screening of ADAMTS‐4 inhibitors, we synthesized a novel internally quenched peptide substrate for aggrecanase‐1 consisting of 5‐FAM/TAMRA as the FRET pair. Active ADAMTS‐4 cleaves this FRET substrate into two separate fragments resulting in an increase of 5‐FAM fluorescence, monitored at excitation/emission = 490 nm/520 nm. The new FRET substrate is more sensitive to aggrecanase‐1 cleavage than the previously described Abz/Dnp substrate and can detect subnanogram amounts of ADAMTS‐4. Its cleavage by other members of aggrecanase family, such as ADAMTS‐1 and ADAMTS‐5, is negligible. Inhibitor screening assays with several known metalloprotease inhibitors were validated using this substrate.This novel FRET substrate provides a convenient homogeneous format for aggrecanase‐1 activity assay where signal is less interfered by the autofluorescence of test compounds due to the long wavelength excitation and emission of 5‐FAM, resulting in an overall increase of signal to background ratio.
Nuclear factor erythroid derived‐2 (NF‐E2)‐mediated regulation of NF‐kB activation in neutrophils: A potential mechanism of NF‐E2‐mediated apoptosis
Authors: Johnson Paul R , Cummins Timothy D , Manning Terri R , Wu Rui , Sachleben Leroy R , Gozal Evelyne , Rane Madhavi J 
Published: June 2021

Neutrophils (PMNs) are short‐lived cells that constitutively undergo apoptosis to prevent excessive tissue damage. We previously identified NF‐E2 as an Akt substrate in human PMNs, and showed that antibody depletion of NF‐E2 delayed PMN apoptosis at 24 hours as assessed by propidium iodide flow cytometry. Additionally, NF‐E2 depletion of PMNs increased IκB phosphorylation, a marker of NFκB activation with concomitant increases in pro‐survival Mcl‐1 expression. These studies led us to hypothesize that NF‐E2 promotes constitutive PMN apoptosis by inhibiting NFκB activation. Preliminary EMSA revealed increased PMN NFκB DNA binding after NF‐E2 depletion. Furthermore, co‐transfection of HEK‐293 cells with NF‐E2 and NFκB luciferase reporter resulted in significantly lower NFκB luciferase promoter activity after TNF‐α stimulation as compared to vector and NFκB luciferase reporter transfected cells. These results collectively demonstrate that NF‐E2 regulates PMN apoptosis by inhibiting NFκB activity. In our future studies, we will utilize NFκB inhibitors to further delineate a role for NFκB in promoting PMN survival and Mcl‐1 expression as a consequence of NF‐E2 depletion. Taken together, our results suggest that NF‐E2 is a pro‐apoptotic protein that promotes constitutive PMN apoptosis by inhibiting NFκB‐mediated transcription of pro‐survival genes.Support for this work was provided by AHA #0815433D (PRJ) and AHA #0335278N, NIH/NIAID #1R56AI059165‐01A2, RO1AI075212‐01A1 (MJR).ASBMMB abstract, EB2009, Deadline November 5, 2008
Pharmacologic evidence for stabilization of a nucleotide‐bound, stimulatory conformation of SUR1Q1178R, an ABCC8 neonatal diabetes mutation
Authors: Ortiz David Javier , Rüdiger Benjamin , Bryan Joseph , Quast Ulrich 
Published: June 2021

Mutations in ABCC8/SUR1 that generate hyperactive neuroendocrine‐type Kir6.2/SUR1 KATP channels are a cause of neonatal diabetes. Analysis of one mutant, SUR1Q1178R, is consistent with the MgATP‐dependent accumulation of a post‐hydrolytic, ADP‐bound enzymatic intermediate that stimulates channel opening. SUR1 is the target for KATP channel inhibitors, e.g., glibenclamide, and openers, e.g., diazoxide and NCC. In the Kir6.2/SUR1 channel, the mutation potentiated the negative allosteric effect of MgATP on binding of [3H]‐glibenclamide reducing the number of specific channel binding sites by ~50% whereas in wild‐type, the KD increased 4.5x. This is consistent with a greater depopulation of antagonist binding sites in the R1178 channels at steady‐state. MgATP is required for opener function and high‐affinity agonist binding. SUR1R1178 increased the apparent number of agonist binding sites, without affecting their Ki, implying these agonists interact with the stimulatory, post‐hydrolytic conformation stabilized by the R1178 substitution. The data suggest the stimulatory, post‐hydrolytic conformation could accumulate either because R1178 increases the rate of ATP hydrolysis or reduces the rate of product dissociation, and rationalize the need for a higher glibenclamide dose to reach metabolic control in ABCC8 neonatal diabetes patients.Supported by NIH DK44311(JB); DFG QU_100/4‐1(UQ).