bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterisation of a new human epoxide hydrolase capable of metabolizing epoxyeicosatrienoic acids
Authors: Adamska Magdalena , Bekteshi Michaela , Cronin Annette , Decker Martina , di Giallonardo Francesca , Arand Michael
Published: June 2021

Epoxide hydrolases (EHs), initially seen as detoxifying enzymes, proved to be also involved in the metabolism of endogenous signaling molecules and act as physiological regulators. Here, we report the characterization of a novel mammalian epoxide hydrolase (nEH1). We identified gene coding for nEH1 in genomic databases using amino acid motives typical for α/β fold EHs as queries. The corresponding cDNA coding for a protein with the catalytic triad typical for epoxide hydrolases and a membrane‐anchor, was expressed in the baculovirus‐expression system. Differential sedimentation of cellular extracts from infected cells confirmed association of nEH1 with the membrane fraction. Screening for substrates revealed that this enzyme was unable to metabolize the generic EH substrate styrene 7,8‐oxide but efficiently hydrolyzed 9,10‐epoxystearic acid. Consequently we investigate a possible role of nEH1 in the turnover of epoxyeicosatrienoic acids (EETs), known modulators of physiological processes. Using LC‐MS/MS analysis we show that the recombinant nEH1 hydrolyzes three of the four EET regioisomers. Ongoing studies on the tissue and organ specific expression of the mouse nEH1 homolog will provide a better understanding of the nEH1 function. Based on our findings, we hypothesize that this enzyme is involved in the regulation of physiological processes rather than in the detoxification of xenobiotic epoxides.
Identification of a new potential human epoxide hydrolase (ABHD7)
Authors: Decker Martina , Adamska Magdalena , Arand Michael
Published: June 2021

Two human EHs are well‐investigated. The microsomal EH (mEH) hydrolyzes a large variety of structurally different xenobiotic‐derived genotoxic epoxides and therefore is implicated in detoxification. The soluble EH (sEH) converts signalling molecules such as epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs) and thus is involved in the regulation of physiological processes. Using the signature sequence RVIAPDLRGYGDSDKP that is reasonably conserved among epoxide hydrolases and some other structurally related enzymes of the α/β hydrolase fold superfamily, we could recently identify two new genes ABHD9 and ABHD7. All features so far identified to be necessary for a functional EH, including a catalytic triad with an aspartic acid in the position of the catalytic nucleophile, as well as two catalytic tyrosines, are conserved in the primary structures of the proteins predicted from these genes. The amino acid sequences of ABHD9 and ABHD7 share 42% identity and therefore belong to the same family being different from mEH and sEH (sequence similarity below 25%). Our previous studies show an EET converting activity of ABHD9. To elucidate the function of ABHD7 we are presently working on the recombinant expression and biochemical characterization of this new enzyme.

The present work is supported by the Swiss National Fonds.
Protective effect of a novel soluble epoxide hydrolase inhibitor 1471 on brain damage after temporary MCAO in rats
Authors: Poloyac Samuel Mark , Ahmad Muzamil , Mu Ying , Miller Tricia , Hwang Sung Hee , Hammock Bruce D , Graham Steven H
Published: June 2021

Chemical inhibitors of soluble epoxide hydrolase and cytochrome P450 produce neuroprotection upon chronic dosing after focal ischemia. The purpose of this study was to determine the effects of a single dose of the 20‐HETE formation inhibitor (HET0016) or the soluble epoxide hydrolase inhibitor (1471) on lesion volume after focal ischemia. Male, Sprague‐dawley rats received single iv doses of vehicle, HET0016 (0.8 mg/kg), or 1471 (0.8 mg/kg) and were sacrificed at two hours for brain cortical HETE, EET, and DiHETrE measurement. In a separate study, each treatment was administered prior to insult and animals were sacrificed at 24 hours for lesion volume assessment. Our results show that 1471 decreased 14,15‐DiHETrE (controls: 1.89 ± 0.42pmol/gm vs 1471: 1.37 ± 0.51pmol/gm) and 11,12‐DiHETrE (controls: 0.61± 0.23 pmol/gm vs 1471: 0.31 ± 0.13pmol/gm) in the rat brain cortex with no significant alterations in HETE metabolites. Whereas, HET0016 reduced 20‐HETE (control 55.9±11.7pmol/gm vs HET0016 22.5±5.0pmol/gm) in the rat cortex without altering other HETEs or DiHETrEs (mean ±st dev; n=5). We also demonstrated that lesion volumes were significantly reduced after 1471dosing and were 15 ± 3% vs 42 ± 5 % (mean ± SEM; n=9) in vehicle control animals. Single dose administration of HET0016 did not alter lesion volume. These data demonstrate that 1471 is a potent neuroprotective agent after a single iv dose. Supported by NINDS R01NS052315 and NCRR S10RR023461.
Curcumin attenuates leukocyte and platelet adhesion in cerebral microcirculation in sepsis
Authors: Vachharajani Vidula Tushar
Published: June 2021

Sepsis and septic shock are leading causes of death in non‐coronary intensive care units worldwide. Septic encephalopathy, a reversible CNS dysfunction independently increases mortality. We have demonstrated increased leukocyte (LA) and platelets (PA) adhesion in cerebral microcirculation (CM) of mice with experimental sepsis (cecal ligation and puncture: CLP) previously. Curcumin, a yellow pigment in turmeric known for its anti‐inflammatory properties in Ayurveda, has been used successfully in various experimental models of inflammation including sepsis. Although neuroprotective effect of curcumin has been studied in ischemic stroke, its effect in septic ephalopathy is unknown.

This study shows the effects of curcumin on LA and PA in CM of mice with CLP.

Hypothesis: Curcumin attenuates LA and PA in CM in mice with CLP.

Materials and methods: Five to seven weeks old WT (C57Bl/6) mice underwent CLP or sham surgery. A separate group of animals were pre‐treated with 100 mg/kg/d curcumin for 48 hours prior to CLP. Cerebral post‐capillary venules were monitored for LA and PA 4 hours post‐surgery with intravital microscopy.

Results: LA and PA were significantly increased in CLP vs sham groups with significant attenuation of both in curcumin pre‐treated CLP animals (Fig 1).

Conclusion: Curcumin attenuates LA and PA in CM in experimental sepsis, suggesting a potential role for curcumin in sepsis.
Explicit Evidence of Smooth Muscle Cell Transdifferentiation in Calcifying Blood Vessels: an In Vivo Lineage Tracing Study using Matrix Gla Protein Knockout Mice
Authors: Speer Mei Y. , Yang Hsueh‐Ying , Look Amy , Lin Wei‐Ling M , Frutkin Andrew , Dichek David , Giachelli Cecelia M
Published: June 2021

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. In order to develop appropriate prophylactic and therapeutic strategies, it is important to understand the origins of the cells that participate in this process. In this study, we crossed matrix Gla protein null mice (MGP−/−) that spontaneously develop vascular calcification with mice carrying SM22α‐Cre recombinase and Rosa26‐LacZ Cre reporter transgenes. Thus, cells of SM lineage were genetically traced by their ability to express β‐galactosidase irrespective of subsequent SMC lineage protein expression. Co‐localization of within a single cell of β‐galactosidase with the loss of SM lineage markers and the gain of osteochondrogenic and chondrocytic markers identified transdifferentiation of SMCs to osteochondrogenic precursors or chondrocytes. Furthermore, transdifferentiation of SMCs occurred prior to calcium deposition, and was associated with an early onset of Runx2/Cbfa1 expression and the down regulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or BMP2. Osterix, Wnt3a and Wnt7a were not detected. These results are the first to definitively demonstrate transdifferentiation of adult SMCs and indicate that SMC transdifferentiation is an important process driving calcification as well as the appearance of skeletal elements in calcified vascular lesions.
Adipogenesis of Adipose Stromal Cells is Reduced by Endothelial Cell Co‐cultivation: Role for Wnt‐signaling
Authors: Rajashekhar Gangaraju , Roell William , Traktuev Dmitry , Merfeld‐Clauss Stephanie , MacDougald Ormond , March Keith , Clauss Matthias
Published: June 2021

Objective: Adipose stromal cells (ASC) display functional and phenotypic overlap with the pericytic cells of microvessels in adipose tissues. Here we addressed the hypothesis that physical proximity of ASC and endothelial cells (EC) regulates the adipogenic potential of ASC. Methods: We investigated the effects of EC on ASC adipogenesis in a 2D‐coculture model and analyzed gene expression changes by real time RT‐PCR. Adipogenesis was determined by quantification of lipid vesicles or increased gene expression of alpha lipase and PPAR‐gamma. Results: A significant decrease in adipogenic differentiation was observed in ASC when cocultured with EC but not in cocultures of ASC with fibroblasts. The majority of molecules involved in Wnt signal transduction were found to be upregulated in coculture conditions with Wnt1 and Wnt4 being most prominent. Using short interfering duplex RNA (siRNA) against Wnt1 in ASC‐EC‐cocultures promoted the adipogenic differentiation. Conclusion: These data identify the close proximity between EC and ASC as a negative regulator of adipogenesis and suggest that perivascular pericytes may protected against adipogenesis. Expression and functional studies further indicate a major role of Wnt signaling proteins in this activity and suggest these molecules as novel candidates to understand regulation of adipogenesis and angiogenesis in adipose tissues.
Porcine aortic valve interstitial cells are rich in mesenchymal progenitors
Authors: Chen Jan‐Hung , Yin Yip Cindy Ying , Sone Eli D. , Simmons Craig A.
Published: June 2021

Introduction: Advanced valvular lesions often contain mesenchymal tissues that may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitium. Objectives: To identify and characterize a subpopulation of mesenchymal progenitor cells in the aortic valve. Methods: The multi‐lineage potential of freshly isolated and subcultured porcine aortic valve interstitial cells (VICs) was tested in vitro. Progenitor frequencies and self‐renewal capacity were determined by limiting dilution and colony‐forming unit (CFU) assays. Results: VICs were inducible to osteogenic, adipogenic, chondrogenic, and myofibrogenic lineages. Primary VICs had strikingly high frequencies of mesenchymal progenitors (57.1 ± 6.1%) and osteoprogenitors (34.4 ± 0.4%). High frequencies were maintained for up to six population doublings but decreased after nine population doublings to 27.8 ± 8.3% and 5.2± 2.1% for mesenchymal progenitors and osteoprogenitors, respectively. We further identified the putative progenitor subpopulation as morphologically‐distinct cells that are highly enriched for osteoprogenitors, occur at high frequency, self‐renew, and elaborate bone matrix from single cells. These novel findings suggest that the aortic valve is rich in a unique multipotent mesenchymal progenitor cell population that is distinct from other vascular progenitor cells. Support: HSFO.
Extracellular ATP is a pro‐angiogenic factor for systemic microvascular endothelial cells
Authors: Gerasimovskaya Evgenia V. , Woodward Heather N. , Roedersheimer Mark , Tucker Doug A. , Stenmark Kurt R.
Published: June 2021

We recently reported that neovascularization of the vasa vasorum network occurs in the pulmonary artery (PA) adventitia of chronically hypoxic calves. Because extracellular adenine nucleotides are important regulators of vascular functions, we hypothesized that extracellular ATP may have a direct angiogenic effect on vasa vasorum endothelial cells (VVEC). We found that ATP dramatically increases DNA synthesis, migration, and tube formation on Matrigel in isolated VVEC. The PI3K/mTOR and ERK1/2 play a critical role in ATP‐induced mitogenic and migratory responses. However, only ERK1/2 contribute in part, to ATP‐induced tube formation on Matrigel. In contrast to VVEC, mitogenic responses to extracellular ATP were not observed in aortic and PA endothelial cells, indicating that VVEC, isolated from the sites of angiogenesis, exhibit a unique pro‐angiogenic phenotype with an augmented sensitivity to extracellular ATP. Further, using an in vivo Matrigel plug assay, we found subcutaneous injection of Matrigel containing MeSATP, ATPγ, MeSADP, and ADPβS resulted in plug neovessel formation (n=3). However, adenine nucleotides diminished the effect of total platelet lysates (n=11) on plug neovascularization (n=3). Together, our studies demonstrated that a fundamental molecule, ATP could be recognized as a potent regulator and/or modulator of angiogenesis in microvascular endothelial cells.

(Supported by NIH grant: HL‐14985 and AHA grant: 0665464Z)
The Roles of p38α MAP Kinase in the Regulation of Embryonic Stem Cell Differentiation to Endothelial Cells and Cardiomyocytes
Authors: Guo Yan‐Lin , Ye Jianming , Huang Faqing
Published: June 2021

p38 mitogen‐activated protein (MAP) kinase α (p38α) is a broadly expressedsignaling molecule that regulates various cellular processes in mammalian cells. Most studies of p38α have been focusing on adult somatic cells, but little is known about its function in embryonic stem cells (ESCs). We investigated the roles of p38α in mouse ESC differentiation to endothelial cells (ECs) and cardiomyocytes (CMs) using wild type and p38α deficient ESCs. ESC‐derived ECs formed monolayers similar to mature EC in culture. Furthermore, they could assemble into vascular structures that remarkably resembled the process of vasculogenesis. p38α−/−ESCs were able to differentiate into ECs as judged by the expression of EC specific markers PECAM‐1 and VE‐cadherin and expected morphology. Although it is not certain whether or not the vascular structures developed from p38α−/−ESCs are functionally normal, morphologically they do not significantly differ from the vessel structures derived from p38α+/+ ESCs. On the other hand, deletion of p38α may impair the differentiation of ESCs to CMs. CMs differentiated from ESCs were clearly identified in embryobodies (EBs) by their ability of spontaneous and rhythmical contracting (beating cells), which is a characteristic of mature CMs. The number of beating EBs derived from p38α−/−ESCs was significantly less than that obtained from wild type ESCs. Further analysis by real‐time PCR revealed that the mRNA expression of CM markers, MLC‐2v, α‐MHC and β‐MHC, in p38α−/−EBs was down regulated, indicating that p38α is important for CM differentiation. Taken together, our data suggest that p38α may play different roles in vasculogenesis and cardiomyogenesis.

This work is supported by a grant from the National Institutes of Heath (HL082731, YLG).
Foundational advances in RNA engineering applied to control biosynthesis
Authors: Smolke Christina D.
Published: June 2021

Recent progress in developing frameworks for the construction of RNA devices is enabling rapid advances in cellular engineering applications. These devices provide scalable platforms for the construction of molecular communication and control systems for reporting on, responding to, and controlling intracellular components in living systems. Research that has demonstrated the modularity, portability, and specificity inherent in these molecules for cellular control will be highlighted and its implications for synthetic and systems biology research will be discussed. In addition, tools that translate sequence information to device function to enable the forward design and optimization of new devices will be discussed. The flexibility of the specified framework enables these molecules to be integrated as systems that perform higher‐level signal processing based on molecular computation strategies. The application of these molecular devices to studying cellular systems through non‐invasive in vivo monitoring of biomolecule levels and to regulating cellular behavior, in particular in the control and optimization of biosynthesis, will be discussed.
Synthetic Ligands that Disrupt Bacterial Quorum Sensing Pathways and Outcomes
Authors: Blackwell Helen E.
Published: June 2021

We are developing chemical tools that attenuate cell‐cell communication pathways in bacteria. Bacteria communicate using small organic molecules and peptides to monitor their population densities in a process called “quorum sensing.” At high cell densities, bacteria use this signaling network to switch from an isolated, nomadic existence to that of a multicellular community. This lifestyle switch is significant; only in groups will pathogenic bacteria turn on virulence pathways and grow into drug‐impervious communities called biofilms that are the basis of myriad chronic infections. In turn, certain symbiotic bacteria will only colonize their hosts and initiate beneficial behaviors at high population densities. The molecular mechanisms of these processes are only now being delineated. Our research is focused broadly on the design of non‐native ligands that can intercept quorum sensing and provide new insights into its role in host/microbe interactions. We have developed a series of efficient synthetic methods that provide us with straightforward access to these ligands. This talk will introduce our research approach and highlight recent results.
Polymorphic Perversity in Protein Polymers
Authors: Egelman Edward Herschel
Published: June 2021

We ask questions such as “What is the structure of F‐actin”. Since many proteins polymerize into helical filaments, answering these questions is essential to understanding the functional forms of these proteins. Unfortunately, high resolution structural information is available for very few polymers. One of the main reasons for this lack of structural information has to do with the variability of these polymers. Over the past 7 years, we have developed a new approach for reconstructing helical filaments from electron microscopic images. This method surmounts many of the problems present when using traditional approaches, and has now been applied to systems as diverse as the myosin thick filament, filamentous bacteriophage, pili, bacterial actin homologs and recombination filaments.

I will show, using examples from a number of polymers, that these filaments can exist in a multiplicity of states. While we have described this phenomenon for F‐actin in the past, we can now show that it applies also to RecA‐DNA filaments or Rad51‐DNA filaments, or even bacterial Type III secretion system needles. Thus, for many of these polymers there is no single structure. Rather, explicating the function of these polymers requires understanding the multiplicity of states that can exist.
Laboratory near‐peer mentoring of jr/sr high school students by college undergraduates provides experience and incentives to enhance careers in biomedical sciences and STEM
Authors: Jett Marti , Yourick Debra
Published: June 2021

The U.S.A. faces an enormωus shortage of scientists and engineers in the future in part due to a lack of effective introduction of science and mathematics to our students during important formative periods in their educations. Our aim is to make STEM subjects come alive through exciting laboratory experimentation led by undergraduate STEM majors who are nearly the age of the interns with whom they work. Although our aim was to find a means to make science exciting to untapped and underrepresented pre‐college students (DC Public School System), this approach has provided a wealth of experience and enhanced self‐confidence in STEM for the college students as well. The college near‐peer mentor lead a group of ~4 young interns as they carry out age‐appropriate laboratory experiments. They frequently work with their kids designing experiments that caught their own attention. During mentorship, the college interns work in the research labs pre‐ or post their fellowship and spend a day weekly during the 3 month fellowship. They describe increased skill in communication, confidence and inspiration to continue in higher STEM education.
Facing the biotechnology laboratory challenge at a community college
Authors: Schaefer Peter A. , Fronhofer Van , Bradley Laurie M.
Published: June 2021

As the field of biotechnology rapidly expands, research laboratories are faced with a shortage of trained technologists capable of handling the techniques and instrumentation in the field. Traditionally, graduates of four‐year colleges have filled that role and received technical training in the research lab. As high school science programs expanded, opportunities developed in community colleges to train biotechnicians to fill the lab shortages. As a result of this need five years ago, Hudson Valley Community College initiated an Associate in Science degree (A.S.) program in Biotechnology. Enrollment in the program has increased 224% since its inception, and graduation rates have increased from 33% to 75%. Many students transfer to four‐year colleges, but those seeking lab employment are highly successful. To add a higher level training, we are implementing a biotechnology certificate that requires 40 credits of science and mathematics for entrance. The certificate includes advanced courses in cell culture, immunological methods, molecular lab techniques, and biological imaging, as well as an internship in a research lab, over two semesters.
Biomedical engineering and bioscience students address hypoxia‐related research problems in an interdisciplinary physiology laboratory course
Authors: Stanley Jennifer Anne , Clase Kari , Gerber Jonathan , Hein Patrick , Lenzi Elizabeth A , Sinak Kelly Marie , Walls Elwood , Pelaez Nancy J
Published: June 2021

Innovative transformational research is being promoted by the NSF Strategic Plan 2006–2011, with multidisciplinary projects emphasized. To prepare undergraduates for careers that cross disciplinary boundaries, bioscience students need to practice interdisciplinary communication in academic programs that connect students in diverse disciplines. This report describes a physiology laboratory course being piloted to create and study this educational frontier. Measurement and Design Issues for Experimental Physiology is a course where biomedical engineering and biology students apply knowledge from both fields while cooperating in multidisciplinary teams under specified technical constraints. Students use information, tools, techniques, and theories from their disciplines to solve problems that are beyond the scope of any single discipline. Instructional guidelines are provided to promote effective communication, systems thinking, laboratory skills, self‐management, and professional ethics needed for hypoxia‐related research. This project recognized different problem‐solving approaches used by students of engineering and bioscience, broke down stereotypes between the disciplines, and developed an educational environment where interdisciplinary teamwork was used to bridge differences.
Using biomechanics to introduce hypothesis testing in an undergraduate biology course
Authors: Kirkton Scott D. , Rice Steven K.
Published: June 2021

To improve student's quantitative and hypothesis testing skills, we have developed an inquiry‐based lab activity that uses both physiology and biomechanics to compare the jumping performance of humans and grasshoppers. Students begin by measuring the relative jump performance (jump distance per body length) of grasshoppers and themselves. Class data are compiled and students present their data graphically. To explain the observed differences in jump performance, students generate hypotheses that may include differences in muscle mass:body mass ratio, leg length:body length ratio, and muscle performance. Muscle loading and limb length hypotheses are evaluated using morphological measures of muscle mass and limb length on both grasshoppers and humans. To test the muscle performance hypothesis, students use video image analysis (iMovie, Apple) to digitize single jumps of both humans and grasshoppers. Student groups employ image‐processing software (ImageJ, NIH) to compare maximal jumping velocity and acceleration. From the acceleration and body mass measurements, students calculate muscle‐specific force production. Class data are compiled, and students interpret the relative contribution of each data set (muscle loading, limb length, or jump force) to explain the observed differences in grasshopper and human jump performance. Student response to this interactive lab has been extremely positive.
CD133+ subpopulations of pluripotent human embryonic stem cells differentiate into neuroectoderm
Authors: King Frank , Bernstein Harold
Published: June 2021

The directed differentiation of human embryonic stem cells (hESCs) along a desired lineage has generated much promise in the field of regenerative medicine. To determine whether such subpopulations indicate specific tissue fates or represent a stochastic distribution within proliferating hESC cultures, we used FACS to sort CD133‐positive hESCs from proliferating cultures constitutively expressing GFP, and co‐cultured these CD133+/GFP+ cells with unselected, GFP‐negative hESCs. At various timepoints in culture under proliferating conditions, GFP+ hESCs were re‐analyzed by flow cytometry for the persistence of CD133 expression as well as the presence of other ubiquitous and distinct surface antigens (Tra‐1‐60, SSEA‐4, FGFR‐1, CD135). This demonstrated that CD133+GFP+ hESCs continue to express CD133 over serial passage, and are enriched for SSEA‐4 expression as well. Upon differentiation in vitro, CD133+GFP+ hESCs co‐cultured with unselected, GFP‐negative hESCs primarily gave rise to neuroectoderm, as detected by co‐expression of GFP and nestin (determined by immunostaining) in a characteristic fibrillar pattern. Tissues representing endoderm (anti‐alpha‐fetoprotein) and mesoderm (anti‐smooth muscle actin) were not seen among GFP+ tissues. These data establish that CD133 marks a subpopulation of proliferating hESCs whose tissue fate is determined, and brings new relevance to the existence of distinct surface markers found in “pluripotent” populations of proliferating hESCs. This selection and co‐culture approach also will enable similar analysis of other distinct hESC subpopulations for their use in the study of tissue development and potential therapeutic applications. This work was supported by NHLBI and the California Institute for Regenerative Medicine.
In vivo Uptake of BrdU, GFP and Iron Oxide Nanoparticles by Local Tissue Macrophages from Labeled Stem Cells. Implications for Cell Transplantation
Authors: Pawelczyk Edyta , Jordan Eleine Kay , Johnson‐Barelo Janna , Smith Melissa , Chaudhry Aneeka , Frank Joseph Alan
Published: June 2021

There is increasing interest in using exogenous labels (bromodeoxyuridine (BrdU), green fluorescent protein (GFP) or dextran coated iron oxide nanoparticles (SPION) to label cells, to identify transplanted cells and monitor their migration by fluorescent microscopy or in vivo magnetic resonance imaging. Direct implantation of cells into target tissue can result in >70% cell death. Bystander uptake of labeled cells by tissue macrophages (TM) can confound the interpretation of imaging studies. This study investigates the frequency of BrdU, GFP and SPION uptake by TM from labeled cells using a model of angiogenesis and inflammation.

Methods:

BrdU, GFP, SPION labeled, unlabeled human mesenchymal stromal cells (MSC) were mixed with Matrigel? and implanted over shoulders of mice. Animals (n=72) were euthanized on days 7, 10, 14 post‐implantation and Matrigel? plaques (MPs) dissected and digested. Recovered cells were analyzed by fluorescence microscopy and flow cytometry (FACS) with anti‐CD11b, ‐dextran, ‐GFP or ‐BrdU antibodies.

Results:

FACS analysis of MPs with BrdU‐labeled MSC revealed 7% of BrdU positive TM, while MPs containing GFP or SPION –labeled MSC had 15% of GFP or dextran positive TM across time points.

Conclusions:

Direct implantation of labeled stem cells into target tissue can result in about 15 % of label being taken up by macrophages. Care should be taken to validate labeled cells by microscopy.
Fascial Tissue Reconstruction Using Acellular Collagen Matrix
Authors: Eberli Daniel , Yoo James , Atala Anthony
Published: June 2021

Statement of Purpose: Acellular collagen matrices have been used experimentally and clinically for several applications, including urethra and bladder reconstruction. We investigated the feasibility of using a porcine collagen‐based matrix as an off‐the‐shelf biomaterial for fascial tissue repair in 3 different tissue systems.

Methods: Biocompatibility of the acellular matrix derived from porcine bladders was assessed using cell viability, MTT and apoptotic index. The acellular collagen matrices were used as a sling material for the treatment of incontinence, abdominal hernias and penile tunica reconstruction in 24 rabbits. The animals were evaluated up to 3 months after implantation.

Results / Discussion: The acellular matrix did not induce significant changes in cell viability, proliferation and apoptotic activity when compared with the controls. All animals survived without any untoward effects. The matrices remained intact at their respective implantation sites and demonstrated the maintenance of fascial tissue function. Histologically, there was only a minimal inflammatory response, which gradually decreased over time.

Conclusions: These results show that the acellular collagen matrix derived from porcine bladders is biocompatible, durable and safe for use in vivo.
Autosomal Dominant Subcortical Gliosis Presenting as Frontotemporal Dementia: Clinical and Histopathological Descriptions
Authors: Miller Bradley Bryan , Lopes Beatrice , Wooten Frederick , Damgaard Paula , Manning Carol , Brashear H. Robert , Swerdlow Russell
Published: June 2021

We describe a multigenerational kindred with a frontotemporal dementia clinical syndrome (FTDS), extensive subcortical gliosis neuropathology and autosomal dominant genetics. Members of the unique kindred now reported were evaluated at the University of Virginia. Studies were performed in order to establish the likely presence of an underlying neurodegenerative pathogenesis. An extended family tree was assembled. Results from five prospective brain autopsy evaluations and one retrospective brain autopsy were considered. Kindred members affected with this FTDS syndrome have extensive subcortical gliosis capping the frontal horns of the lateral ventricles, evident by neuroradiologic examination. Histopathologic analyses show florid subcortical gliosis, and reveal neither tau nor ubiquitin inclusions. The presence of both maternal and paternal transmission indicate this is an autosomal dominant disorder. Subcortical gliosis in this kindred is either due to mutation of a novel gene, or else represents a unique, previously unrecognized FTDS variant caused by mutation of an already known gene. The kindred is extensive enough to facilitate linkage analysis. Determining the basis of this kindred's autosomal dominant subcortical gliosis could potentially yield mechanistic insight into vascular‐attributed cognitive impairment/dementia syndromes.
An alternative in vitro model of neurodegeneration in FTLD‐U with PGRN mutation
Authors: Gitcho Michael , Mukherjee Odity , Wang Jun , Chakraverty Sumi , Strider Jeffrey , Norton Joanne , Goate Alison , Cairns Nigel J.
Published: June 2021

Recently, the gene for familial frontotemporal lobar degeneration with ubiquitin‐positive, tau‐negative inclusions (FTLD‐U) linked to chromosome 17 was cloned. In the present study, 42 unrelated patients from the Washington University Alzheimer's Disease Research Center (WUADRC) with clinically diagnosed FTLD and/or neuropathologically characterized cases of FTLD‐U with or without MND were screened for mutations in PGRN. Bidirectional sequencing was performed for mutational screening. Mechanistically, most mutations in PGRN described thus far have been reported to display a haploinsufficiency through nonsense mediated decay (NMD). We describe a novel missense mutation in exon 1 (c.4068 C>A) introducing a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (A9D) which displayed no significant difference in the total postmortem brain PGRN mRNA between cases and controls. In SHSY‐5Y neuroblastoma cells overexpressed with mutant or wild‐type PGRN, subcellular fractionation, biochemistry, and confocal microscopy indicate that mutant protein is expressed, but is not processed, through the ER for secretion, and appears to be misslocalized in the cytosol possibly resulting in a functional haploinsufficiency. This defect in trafficking results in a decrease in cell viability: an alternative in vitro model of FTLD‐U with PGRN mutation neurodegeneration. Support for this work was provided by grants from the National Institute on Aging of the National Institutes of Health (P01‐AG03991, P50‐AG05681) and the Buchanan Fund.
Blood Vessel‐Like Tissue Spheroids as Modules for the Assembly of Blood Vessels
Authors: Gentile Carmine , Fleming Paul , Mironov Vladimir , Argraves W. Scott , Drake Christopher J.
Published: June 2021

Blood vessel‐like tissue spheroids (BVLTS) were generated in hanging drop culture from either murine allantoic tissue or mixtures of human EC and SMC. Essential to the generation of BVLTS was the vascular fusion‐promoting activity of VEGF, which allowed the inner network of small caliber endothelial tubes initially formed within the spheroids to be transformed into a single, large endothelial‐lined central cavity. Our designation of these spheroids as blood vessel‐like was based on their morphological and immunohistochemical properties. Specifically, BVLTS were characterized by the presence of an inner PECAM/VE‐cadherin positive endothelium defining a central lumen (diameter >300μm) that is surrounded by an outer layer of (SMA‐positive cells. That BVLTS possessed a true lumen was demonstrated by the finding that Qdots microinjected into the central space were both confined to and retained within the EC‐lined cavity. Further, the blood vessel‐like properties of BVLTS were demonstrated by our finding that murine BVLTS responded to vasoactive compounds in a manner consistent with that observed in vivo. That BVLTS have utility as modules for the assembly of intermediate sized blood vessels is supported by our finding that individual BVLTS can fuse to form larger vascular structures. Future efforts will be directed at assembling and perfusion testing vascular tubes generated from BVLTSs.
Evidence that Increased Transmural Pressure is the Main Stimulus for Vascular Remodeling
Authors: Watson Ralph Edward , Pereira‐Hicks Cristiane Neves , Wang Hui , Galligan James Joseph , Furspan Philip B , Fink Gregory
Published: June 2021

Remodeling of arteries occurs in hypertension (HTN). Intraluminal pressure stimulates growth. Venous pressure is not increased in systemic (HTN), and evidence for remodeling of small veins is less clear. This study is the first to compare the structure of small resistance arteries and small veins in the Dahl salt‐sensitive (SS) rat model of HTN. Both the SS and salt‐resistant (SR) rats were fed regular rat chow for 8 weeks. The average systolic blood pressure for the SS 182 + 2 mm Hg, for the SR 137 + 2 mm Hg. We studied 10 SS and 9 SR mesenteric veins and 9 SS and 9 SR mesenteric arteries. The arteries and veins were mounted on glass microcannulae in calcium free Kreb's solution and measurements were made using a pressure myograph. Results: In the arteries, the SS had higher wall thickness (WT) (p=0.003), media/lumen ratio (M/L) (p=0.006), and cross‐sectional area (CSA) (p=0.005). There was no difference in lumen diameter (LD) or incremental distensibility (ID). There were no differences betweent the SS and SR veins in WT, M/L, CSA, LD, or ID. Conclusions: In Dahl rat HTN, we found that arteries of the hypertensive (SS) rats showed remodeling, while the veins had no remodeling. Although pressure is not increased in the veins in HTN the neurohumoral milieu is similar to that in arteries. This suggests that intraluminal pressure is the major cause of the remodeling seen in hypertensive arterial walls.
Enhancement of proteasome‐linked TonEBP/NFAT5 degradation in cardiomyocytes exposed to doxorubicin
Authors: Ito Takashy , Fujio Yasushi , Azuma Junichi
Published: June 2021

TonEBP is a transcriptional factor that regulates the induction of transporters and enzymes involved in organic osmolyte accumulation by hypertonic stimulation. Although TonEBP is ubiquitously expressed and plays an important role during osmotic stress, little is known about its role and regulation in pathological states that are associated with no osmotic imbalance. To determine whether TonEBP might respond to pathological stimuli in the absence of osmotic stress, levels of the taurine transporter (TauT), a target of TonEBP, were examined in cardiomyocytes exposed for 24 h to the cardiotoxic agent, doxorubicin (Dox). The Dox treated cells exhibited reduced TauT and nuclear TonEBP protein content. Since the decline in TonEBP protein content was prevented by proteasome inhibitors but was not associated with a change in TonEBP mRNA content, Dox appears to accelerate the degradation of TonEBP. This decrease in TonEBP content may have significant consequences, as downregulation of TonEBP using either siRNA or overexpression of dominant‐negative TonEBP leads to reduced myocyte viability. In conclusion, Dox mediates the degradation of TonEBP via a proteasome‐related proteolysis pathway, a reaction that plays is an important role in Dox‐induced cardiotoxicity.
Thalamic Damage in Periventricular Leukomalacia (PVL)
Authors: Ligam Poonam , Folkerth Rebecca D , Haynes Robin L , Liu Lena , Volpe Joseph J , Kinney Hannah C
Published: June 2021

The thalamus is an integral structure in the processing and relaying of cognitive, emotional, and sensory information, and thalamic damage in the premature period could contribute to long‐term neurological disabilities in survivors. A recent study by us identified that premature infants with PVL had thalamic injury at autopsy, characterized by neuronal loss (38%) and/or gliosis (56%) (Pierson et al. Acta Neuropathol 2007). Here we sought to establish the spectrum and regional pattern of thalamic injury, as well as the role of axonal injury. We studied 21 thalami in PVL cases ranging from 24 to 41 gestational weeks (mean, 33.3 ± 1.0 weeks) and 25 to 56 postconceptional weeks (mean, 37.8 ± 1.5 weeks) from the pathology archives of our hospital. There was a spectrum of injury in 71% of the thalami, from mild (10%) to severe (24%), with profound neuronal loss, gliosis, and mineralization. Parenchymal axonal spheroids were present in 19% of thalami with acute/subacute injury; one case had microinfarction. The mediodorsal nucleus (limbic relay) was involved in 66% of the cases. These data suggest that the incidence of thalamic injury is even more prevalent in PVL then originally reported by us, as determined by more extensive analysis of individual cases, and that the underlying substrate of emotional as well as cognitive disorders in survivors may reflect, at least in part, damage to relevant thalamic subnuclei.
Molecular Basis of dTTP Specificity by Gene 4 DNA Helicase of Bacteriophage T7
Authors: Lee Seung‐Joo , Richardson Charles C
Published: June 2021

Translocation on ssDNA and the unwinding of dsDNA into ssDNA is mediated by DNA helicases at the expense of hydrolysis of nucleotide triphosphate (NTP). While most of helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To understand the molecular basis of this specificity we have examined the efficiency of unwinding of dsDNA by T7 helicase using a variety of NTPs and their analogs. The 5‐methyl group of thymidine is critical for the efficient unwinding of DNA although the presence of a 3′‐ribosyl hydroxyl group partially overcomes this requirement. In order to identify specific interaction between the NTP and residues within the NTP binding pocket of the protein, several amino acid residues, selected from structural data, were replaced with random amino acid residues. The resulting library of altered gene 4 proteins led to the identification of those residues (T320, R504, Y535, and L542) that are critical for NTP utilization by T7 helicase. Interestingly, some altered proteins containing a single amino acid replacement exhibit NTP utilization profiles distinct from the wild‐type helicase. One of these examples is gp4‐R504G that utilizes dATP more efficiently than dTTP. This altered protein can also utilize dCTP which is barely used by the wild‐type protein. We suggest that the NTP must be stabilized by specific interactions within the NTP binding site of the protein to achieve efficient hydrolysis. These interactions dictate the NTP specificity.

This work was supported by Public Health Service Grant GM 54397‐4.
Analysis of the mechanism by which telomerase impacts chromosomal stability and cell viability in Ku mutants
Authors: Holland Cory Lee , Lewis Lysle Kevin
Published: June 2021

The Ku complex is a heterodimer comprised of two subunits, Ku70 and Ku80, which binds double‐stranded DNA (dsDNA) ends. Ku has multiple roles in DNA metabolism. The presence of Ku at the ends of the chromosomes protects the telomeres from degradation by nucleases and may also aid in recruitment of telomerase to DNA. Interestingly, Ku has also been shown to play a critical role in the non‐homologous end‐joining (NHEJ) pathway of repair of induced double‐strand breaks in DNA.

In budding yeast (Saccharomyces cerevisiae), inactivation of either of the Ku subunit genes, YKU70 or YKU80, abolishes NHEJ DNA repair and makes cells incapable of growing at elevated temperatures (37°C) due to telomere instability. The precise cause of telomere instability in yku70 mutants at high temperatures is unknown, but cell death can be alleviated by increasing levels of TLC1, the RNA subunit of telomerase, as well as the polymerase subunit Est2. We have shown that overexpression of the Exo1 exonuclease accelerates the death of Ku mutants at 37°C and that TLC1 RNA and Est2 are able to rescue the accelerated death of Ku mutants. A series of yku70 double mutants that were constructed demonstrated that other telomere associated proteins such as Mlp2, Rif2, and Rad50 are not required for rescue. Funding for this project was provided in part by the Department of Chemistry and Biochemistry, Texas State University – San Marcos and Research Corporation.
MAMMALIAN TETHERED CATALYSIS (MTeC): A NOVEL METHOD TO IDENTIFY MODIFICATION‐SPECIFIC BINDING PROTEINS IN VIVO
Authors: Spektor Tanya Magazinnik , Rice Judd Christopher
Published: June 2021

Protein:protein interactions within the cell are critical for most biological processes. How proteins bind each other and how they regulate this interaction are fundamental questions in biology. One of the best examples of this phenomenon in the nucleus is the interaction of post‐translationally modified histone proteins with key transcriptional regulatory proteins that control gene expression.

In order to identify modification‐specific binding proteins in vivo, we have created a novel method called Mammalian Tethered Catalysis, or MTeC. First, a tandem epitope‐tagged eukaryotic expression plasmid is created that contains the portion of the target protein that will be modified (“bait”) fused to the catalytic portion of an enzyme that will post‐translationally modify the target residue within the bait. A negative control is also created by mutating this target residue. Each construct is transfected into a cell line of choice and purified by chromatography and/or immunoprecipitation(s). The resultant proteins within the purified material are identified by mass spectrometry and proteins unique to the experimental sample are analyzed further. Using histone modifications as a proof‐of‐principle, we have successfully employed this method to identify a novel interaction between monomethyled lysine 9 of histone H3 and chromatin‐modifying protein, G9a.
Expression of cell fate‐specific genes are regulated by monomethylated Histone H4 Lysine 20
Authors: Sims Jennifer Kae , Rice Judd Christopher
Published: June 2021

Post translational modifications of N terminal histone tails play an important role in regulating gene expression. In this study, we show that one of these modifications, the monomethylation of Histone H4 Lysine 20 (H4K20) is found within lineage specific genes and that loss of H4K20 monomethylation causes upregulation of these genes. Runx1, a regulator of hematopoeisis, is an example of a gene that is upregulated in the absence of H4K20 monomethylation. Chromatin immunoprecipitation assays (ChIP) confirmed the enrichment of H4K20 monomethylation at Runx1 promoter region but not at an upstream region. The H4K20 monomethyl binding protein, L3MBT, was also enriched at the promoter regions. This is consistent with our previous data and shows that L3MBT binds to H4K20 monomethyation to cause gene repression. Knockdown of H4K20 monomethylation also caused misexpression of Runx1 in HeLa cells. To further investigate the biological relevance of H4K20 monomethylation at Runx1, we dfferentiated the pre‐erythroid cell line, K562, into megakaryocytes, which are Runx1 positive, and found that as Runx1 protein levels increased, global levels of H4K20 monomethylation decreased. Taken together, these data define a novel repression pathway in which H4K20 monomethylation and L3MBT cooperate to repress lineage specific genes such as Runx1.
The GAIT (interferon‐gamma‐activated inhibitor of translation) system defines an auto‐regulatory, negative‐feedback circuit that controls inflammatory gene expression
Authors: Fox Paul L , Mukhopadhyay Rupak , Ray Partho S , Arif Abul
Published: June 2021

Ribosomal protein L13a is an integral component of the heterotetrameric GAIT complex that represses translation of inflammatory genes, including vascular endothelial growth factor (VEGF)‐A and ceruloplasmin, in monocytic cells. L13a phosphorylation by interferon‐gamma (IFN) is rate‐limiting for GAIT activation and it inhibits target mRNA translation by binding mRNA‐bound eIF4G and preventing recruitment of the 43S preinitiation complex. We now show IFN activates a kinase cascade in which death‐associated protein (DAP) kinase activates zipper‐interacting protein (ZIP) kinase, which phosphorylates Ser77 on L13a to initiate translational silencing. Remarkably, the kinase cascade itself is subject to GAIT‐mediated inhibition; DAP and ZIP kinase mRNAs contain functional 3’UTR GAIT elements and are translationally repressed by the GAIT complex. The negative‐feedback system prevents complete shut down of translation, instead permitting continuous, low‐level translation of target transcripts, e.g., VEGF‐A, despite the robust presence of its mRNA. Thus, the GAIT system defines a post‐transcriptional operon in which expression of the activating kinase cascade is subject to autoregulatory, negative‐feedback which stringently controls expression of target transcripts. Supported by an AHA Postdoctoral Fellowship, Ohio Valley Affiliate (to AA) and NIH grants P01 HL29582 and P01 HL76491 (to PLF).