bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced thrombosis and platelet aggregation in mice lacking alpha(1,3)‐fucosyltransferase‐IV and ‐VII are P‐selectin independent
Authors: Homeister Jonathon W. , Bodary Peter F. , Morales‐Levy Maria , Eitzman Daniel T.
Published: June 2021

P‐selectin (P‐sel) is a leukocyte adhesion molecule with prothrombotic functions. Fucosylation of its ligand, PSGL‐1, by alpha(1,3)‐fucosyltransferases (FucT)‐IV or ‐VII is required for binding activity. FucT‐IV(‐/‐)/VII(‐/‐) mice have an unexpected prothrombotic phenotype. We used a photochemical‐induced injury model of carotid artery thrombosis and whole blood platelet aggregation to test whether the prothrombotic phenotype was dependent on P‐sel/PSGL‐1 binding interactions. The time to occlusive thrombosis was decreased in FucT‐IV(‐/‐)/VII(‐/‐) mice [19.5±3.0 min, n=10, vs WT=34.0±2.8 min, n=12; p=0.05]. In contrast, the time to occlusive thrombosis was not altered in PSGL‐1(‐/‐) mice [25.7±2.9 min, n=6, vs WT=23.1±1.3 min, n=7; p>0.05], or in Psel(‐/‐) mice [63.0±8.6 min, n=4, vs WT=45.5±7.8 min, n=4; p>0.05]. ADP‐stimulated whole blood platelet aggregation in FucT‐IV(‐/‐)/VII(‐/‐) mice had a shorter lag time [22.2±0.5 sec, n=8 vs WT=27.0±0.6 sec, n=8; p=0.05] and a faster rate [21.4±0.9 sec−1, n=8 vs WT=16.4±1.4 sec−1, n=8; p=0.05]. In contrast, platelet aggregation in PSGL‐1(‐/‐) mice did not have an altered lag time [28.5±2.2 sec, n=8 vs WT=26.0±1.2 sec, n=16; p>0.05], or rate [13.4±1.4 sec−1, n=8 vs WT=17.4±1.4 sec−1, n=16; p>0.05]. The prothrombotic phenotype in FucT‐deficient mice is independent of P‐sel/PSGL‐1 binding activity. Supported by AHA FTF Award 0275023N and RO1‐HL090823.
A high fat diet reduces blood pressure, but not cardiac hypertrophy in spontaneously hypertensive rats
Authors: Lin Han‐yi , Soesanto Will S. , Symons J. David , Jalili Thunder
Published: June 2021

High fat diets can attenuate cardiac hypertrophy in Dahl Salt Sensitive rats (DSS), a model of severe hypertension, cardiac hypertrophy & dysfunction, independent of changes in blood pressure. We hypothesized that a high fat diet can prevent hypertrophy in spontaneously hypertensive rats (SHR), a model of gradually increasing hypertension. Five‐week old SHR and Wistar Kyoto (WKY; normotensive control) rats were fed either a low fat (LF) or high fat (HF) diet with 10% and 60% of Calories from lard, respectively, for 10 weeks. In contrast to our hypothesis, cardiac hypertrophy was unchanged in SHR HF vs. SHR LF but, surprisingly, blood pressure was lower in SHR HF. Pooled statistical analysis also indicated lower plasma glucose (2 h fast) in HF vs. LF rats. These data indicate a markedly different response to high fat diets in the SHR vs. those previously reported in the DSS rats.

(Different letters denote significant differences at p<0.05)
P21 activated kinase 1 (PAK1) protects heart from pressure overload induced pathological cardiac remodeling
Authors: Mao Kai , Healy Chastity , Timm Derek , Kobayashi Satoru , Volden Paul , Chernoff Jonathan , O'Connel Timmothy , Gerdes Anthony Martin , Liang Qiangrong
Published: June 2021

P21 activated kinase 1 (PAK1) is a serine/threonine kinase that is activated in the heart by multiple stress signals. To explore the role of PAK1 in the heart, we performed transverse aortic constriction (TAC) surgery on transgenic (TG) mice that overexpress PAK1 and gene disrupted mice that lack PAK1 (KO) in the heart. Although PAK1 overexpression resulted in moderate cardiac hypertrophy, TAC‐induced cardiac growth was dramatically reduced in PAK1 TG mice as compared to wild type (WT) littermates. This was accompanied by improved cardiac function and decreased myocyte apoptosis, suggesting that PAK1 signaling can protect against pressure overload‐induced pathological cardiac remodeling. Conversely, PAK1 KO mice displayed mild cardiac chamber dilation and compromised heart function at baseline. Not surprisingly, TAC induced greater structural and functional damage in PAK1 KO heart than in WT heart. These results demonstrate that PAK1 is required not only for baseline cardiac homeostasis, but also for cardiac adaptation to pathological stress. In summary, PAK1 conveys a vital protective signal that might be beneficial during pathological cardiac hypertrophy and heart failure.
Stem cells homing and vascular differentiation in lung, heart and skeletal muscle in an experimental model of heart failure
Authors: Castellani Chiara , Ravara Barbara , Gobbo Valerio , Piccoli Martina , Franzin Chiara , Pozzobon Michela , Vettor Roberto , Thiene Gaetano , Dalla Libera Luciano , Vescovo Giorgio , Angelini Annalisa
Published: June 2021

Purpose

Aim of our study was to investigate the homing and differentiation of human amniotic stem cell (hAFC) and rat vascular stromal adipocite GFP positive cells (rGFP) in a rat model of heart failure.

Methods and Materials

Male Sprague‐Dowley rats were injected with monocrotaline to develop right heart failure. hAFC or rat GFP cells were injected via tail vein three weeks after MCT injection. Stem cells differentiation were studied by double immunofluorescence technique.

Results

Plasma brain natriuretic peptide (BNP), was elevated in HF compared to controls (2.01±0.12 HF rats;0.56±0.37 ng/ml controls, p< 0.0001),while was similar to controls in HF animals injected with stem cells (0.75±0.06 ng/ml,p<0.001).

HF stem cells animals showed a RVM/Vol value lower than HF and similar to controls (0.34±0.02 treated animals;0.49±0.29 in controls, p<0.001). Both rGFP and hAFC were detected in the lung (1.01±0.6% hAFC and 0.44±0.4% rGFP) and few of them (0.11±0.1% of hAFC) were differentiated into vascular cells. In the heart stem cells were 0.36±0.3% for hAFC and 0.55±0.2 for rGFP positive cells and hAFC showed a 0.01±0.01 % and rGFP 0.16±0.1% of SMA differentiation. In skeletal muscle stem cells were hAFC 0,31±0,09% and rGFP 0,36±0,2%.

Conclusions

These results suggest that hAFC and rGFP cells are both engrafted with a topographical recruitment gradient in the lung, heart and skeletal muscle and seems to reduce heart remodelling and failure.
CD133, a stem cell surface marker in inflammatory breast cancer, functions in tumor cell survival
Authors: Barsky Sanford H , Xiao Yi , Shetuni Brandon B , Yearsley Kurtis , Ye Yin
Published: June 2021

Our previous studies revealed prominent surface CD133 in 90% of the cells of MARY‐X, a human xenograft model of inflammatory breast carcinoma (IBC), as well as in 22/25 IBC cases. CD133 is a stem cell marker expressed in some human cancers. Compared to other stem cell molecules, CD133 has not been well studied and its function and ligand remain unknown. We decided to study CD133 further in both MARY‐X as well as in other breast carcinoma cell lines (HCC70, HTB132, HCC1143) that expressed CD133 at decreasing percentages. We knocked down CD133 by siRNA approaches and obtained a 10 fold knockdown of mRNA as well as protein in all of the lines studied. CD133 knockdown significantly suppressed growth in all of the breast carcinoma cell lines. The suppression of growth was not mediated by cell cycle blockade as determined by Ki‐67 index or G1, S or G2/M. Instead suppression of growth was mediated by a dramatic induction of apoptosis. Even in HCC1143, which expressed CD133 in only 5% of the cells, induction of apoptosis was as prominent as in MARY‐X. In all of the cell lines, CD133 knockdown induced 100% apoptosis within 5 days. These studies suggest that CD133 is a survival molecule which functions in both an autocrine as well as paracrine manner.
Identification of diagnostic and therapeutic targets of pancreatic cancer using proteomic approaches
Authors: Pandey Akhilesh , Jimeno Antonio , Molina Henrik , Hruban Ralph H , Maitra Anirban , Hidalgo Manuel , Harsha HC
Published: June 2021

Pancreatic cancer remains one of the most lethal of all the solid malignancies. Using a quantitative proteomic strategy, we have carried out profiling studies to identify novel biomarkers and therapeutic targets in pancreatic cancer. Phosphotyrosine profiling revealed aberrant activation of epidermal growth factor receptor (EGFR) in a subset of pancreatic cancers. This correlated with phospho‐EGFR staining and response to the EGFR inhibitor, erlotinib, in mice carrying pancreatic cancer xenografts demonstrating the utility of activated EGFR as a biomarker that could be used to guide therapeutic decisions. Proteomic profiling of membrane fractions identified several proteins that are differentially expressed in pancreatic cancers. Immunohistochemical validation of one of the candidate markers, myeloid associated differentiation marker (MYADM), confirmed overexpression of this molecule in greater than 80% of pancreatic adenocarcinomas. Our studies emphasize the utility of advanced proteomic approaches in identifying novel biomarkers and therapeutic targets.

This work was supported by Sol Goldman Trust for Pancreatic Cancer Research.
A comparative analysis of the cat claw, horse hoof, and human fingernail
Authors: Bragulla Hermann H. , Homberger Dominique G.
Published: June 2021

Earlier authors (e.g., Boas, 1931) have tried to homologize the skin modifications in mammalian cornified digital end organs (claws, hooves, and nails), but have considered only major parts, such as the wall and sole. A recent analysis of the cat claw (Homberger et al., in press) and horse hoof (Bragulla & Homberger, 2007) revealed that the subcutis, dermis, and epidermis are regionally modified to meet functional demands. The structure of digital end organs is, hence, more complex than anticipated. For example, the subcutis can form a hydrostatic cushion or ossify to strengthen the attachment of the dermis to the bony distal phalanx. The dermis forms a papillary body, which increases the dermo‐epidermal interface, enhances the supply of the avascular epidermis via diffusion, and strengthens the dermo‐epidermal bond. In addition, the papillae, ridges, or lamellae of the dermal papillary body often influence the architecture of the modified epidermis that forms the cornified digital end organs. Furthermore, several hard‐ and soft‐cornified segments can be identified and distinguished within the cornified digital end organs. The local and integrated mechanical properties of the digital end organs result, therefore, from different combinations of comparable building blocks of modified skin. Hence, the traditional homologizations between claws, hooves, and nails need to be re‐evaluated at a more detailed level.
Involvement of Fibroblast growth factor 10 (Fgf10) in the anterior‐posterior patterning and specification of muscle and tendon progenitors in the developing mouse limbs
Authors: Hajihosseini Mohammad K , Ramasamy Suresh , De Langhe Stijn , Lana‐Elola Eva , Rice David , Quintana Rio Laura , Kelly Robert , Sharpe James , Bellusci Saverio
Published: June 2021

Fgf10 regulates the specification and early outgrowth of vertebrate limbs. However, the arrested growth of limb buds and neonatal death in Fgf10‐deficient embryos has precluded an understanding of its putative roles in later stages of development. To obtain clues about these roles, we have studied the localization and immediate fate of Fgf10‐expressing cells from Embryonic days 9.0 to 18.5, by subjecting Fgf10‐lacZ reporter embryos to immunocytochemistry with cell type‐specific markers, optical‐tomography scanning and in situ hybridization analysis. These studies have revealed an asymmetric expression of Fgf10 along the anterior‐posterior axes suggestive of critical roles in anterior‐posterior patterning of the limb. Moreover, we have discovered a discreet population of Fgf10‐expressing muscle progenitors that invade the limbs from the body trunk and a specific expression of Fgf10 in select tendon progenitors. Together, these novel findings implicate Fgf10 in multiple and critical roles that underlie the proper formation and sculpturing of vertebrate limb. Conditional loss and gain‐of‐function experiments are in progress to investigate the regulatory roles of Fgf10 in distal mouse limb development.
CNS changes after chronic sleep deprivation: roles in both food intake and metabolism
Authors: Hoffman Gloria
Published: June 2021

In humans and animal models, chronic sleep loss stimulates feeding and leads to metabolic interactions associated with signs of the metabolic syndrome and progression to diabetes II. Understanding how sleep deprivation might produce these effects is more complicated and involves dissection of central nervous system pathways that normally regulate food intake and those which control peripheral metabolism. For the former, hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) are key systems governing feeding. Sleep deprivation up‐regulates NPY and down‐regulates POMC in the hypothalamus that accounts for the hyperphagia. For the changes in metabolism, usually hyperphagia is accompanied by a slowing of metabolic rate but the opposite is seen in chronic sleep deprivation. The CNS's role in regulating metabolic rate is less well understood, although histamine may be involved. Central histamine neurons not only affect the maintenance of wakefulness, but are also regulators of peripheral metabolism via actions at the preganglionic sympathetic neurons. The rate‐limiting enzyme for histamine synthesis, histidine decarboxylase, is up‐regulated during sleep deprivation, and these changes predict the attenuation of body weight (prompted by hypermetabolism). The relationship of changes in histamine synthesis to those of glucose suggests that the amine might also be responsible for changes which lead to metabolic syndrome. Elevations in glucose:insulin ratios in sleep deprived rats were positively correlated with the extent of increased histidine decarboxylase gene expression. Together, these data raise the possibility that a dysregulation of histamine function when sleep is impaired might serve to trigger increased sympathetic tone and metabolic changes leading to diabetes.

Supported by a Pilot and Feasibility grant from the Clinical Nutrition Research Unit of Maryland, P30DK072488.
Developmental biology paradigms: lessons from spiders
Authors: Damen Wim G.M.
Published: June 2021

The segmentation gene cascade as known from the fruit fly Drosophila forms one of the paradigms in developmental biology. One outstanding question in animal evolution is whether the last common ancestor of the three segmented phyla, chordates, annelids and arthropods, was itself segmented. We use the spider Achaearanea tepidariorum, which is an emerging model organism, to study the evolution of body segmentation. We showed that spider segmentation involves two signalling pathways, Delta‐Notch signalling and Wnt8 signalling, which is similar to vertebrate somitogenesis, but is in contrast to Drosophila segmentation. Delta/Notch signalling is required for dynamic gene expression preceding segmentation in both spiders and vertebrates. Furthermore, knockdown of Wnt8 suggests that Wnt8 has a similar role in vertebrates and spiders in establishing and maintaining a posterior segmentation zone. Both Delta‐Notch and Wnt8 signalling may be part of an ancient regulatory network for segmentation that could have been present in the common ancestor of segmented animals. Our work is supported by the German Research Foundation (SFB572) and the European Union via the Marie Curie Research and Training Network ZOONET.
The Genomic Code for Nucleosome Positioning
Authors: Widom Jonathan
Published: June 2021

Eukaryotic genomes encode an additional layer of genetic information that controls the organization of the genomic DNA into arrays of nucleosomes. This information is superimposed (multiplexed) on top of the regulatory and coding information that was previously understood. We have developed an ability to read this nucleosome positioning code and predict the in vivo locations of nucleosomes. In our most recent studies, we developed a "pure" statistical profile of nucleosome DNA sequence preferences by competitive reconstitution of nucleosomes onto genomic DNA in vitro, using a system comprising only purified genomic DNA and purified histones (the protein components of nucleosomes). Using this statistical profile, we find that genome's intrinsically encoded nucleosome positioning information accounts for the overwhelming majority of nucleosome‐occupied versus nucleosome‐depleted regions across the entire genome. Analyses of nucleosome occupancy in different biological conditions reveal that the condition‐specific action of gene regulatory proteins and chromatin remodeling factors lead to specific localized changes in nucleosome occupancy and positioning, leaving much of the chromatin organization unaffected. Our results suggest that genomes utilize the nucleosome positioning code to facilitate specific chromosome functions including to delineate functional versus nonfunctional binding sites for key gene regulatory proteins, and to define the next higher level of chromosome structure itself.
Deep sequencing of mRNA to understand function of splicing regulatory factors
Authors: Burge Chris
Published: June 2021

Splicing regulatory elements (SREs) and the associated splicing factors play central roles in the control of pre‐mRNA splicing and in exon evolution. We are studying in depth the regulatory properties of representative splicing factors, including the widely expressed factor hnRNP H, and the muscle‐ and brain‐specific factors of the CELF and Muscleblind families, using knockout or knockdown followed by deep sequencing of mRNAs (KO‐Seq or KD‐Seq), cross‐linking/immunoprecipitation/sequencing (CLIP‐Seq), and other approaches. In this talk I will discuss our progress towards understanding the functions of these factors, including the finding that the activity of G‐run intronic splicing enhancers (ISEs) and hnRNP H is highly dependent on the strength of the adjacent 5' splice site sequence, and the functional and evolutionary implications of this finding.

Our work is supported by the NHGRI and the NIGMS.
Importance of Recruitment and Retention of Minority Scientists to the Future of Biomedical Research
Authors: Alderete John F
Published: June 2021

Research universities have developed plans to diversify the faculty, paying particular attention to U.S. racial minorities. Unfortunately, efforts by university academic units to recruit minority scientists have largely been ineffective. The importance of recruiting and retaining minority scientists in the biomedical research enterprise cannot be overstated. The absence of minority scientists means that the very health of significant communities and of our country is at risk. Having educated minority scientists at research universities and government translates to efforts to improve our nation's situation in terms of education of the growing low‐income populations. Visible minority scientists who are respected at the highest levels for their contributions to science are those who can express views to others who do not understand minority communities. Active, respected minority scientists are also competitive for membership in the National Academy, for NIH Boards and Advisory Councils, and for faculty positions at the top‐tier research universities. Successful recruitment and retention of minority scientists is complex. This involves buy‐in by all of the administration, oversight of hiring practices, a priority in the strategic plan, sustained investment of resources, a respectful environment and culture, and an acknowledgment that diversity is valued greater than the money to be generated.
DNA damage signaling and DNA repair
Authors: Chen Junjie
Published: June 2021

The Breast Cancer Susceptibility Gene 1, BRCA1, plays a crucial role in DNA damage response through its interactions with many proteins including brca1‐associated C‐terminal helicase (BACH1) and CtBP‐interacting protein (CtIP). We recently identified an ubiquitin‐binding protein RAP80 and a coiled‐coil domain‐containing protein CCDC98 as new BRCA1‐interacting proteins. RAP80 and CCDC98 form a stable complex. This complex colocalizes with BRCA1 and is required for BRCA1 foci formation in response to ionizing radiation (IR). Moreover, like BRCA1, RAP80 and CCDC98 also play a role in radiation sensitivity and damage‐induced G2/M checkpoint control. Together, these results suggest that RAP80 and CCDC98 are new mediators involved in the mammalian DNA damage response. Interestingly, the focus localization of RAP80/CCDC98 and BRCA1 depends on RAP80 ubiquitin‐interacting motifs, raising the possibility that there may be proteins that are ubqiutinated at the sites of DNA damage and serve to recruit RAP80/CCDC98 and BRCA1. We and others have identified an upstream E3 ligase RNF8 that is critically important for protein ubiquitination at sites of DNA damage. Our subsequent studies have revealed how RNF8 acts in the DNA damage‐signaling cascade and regulates DNA damage checkpoint and DNA repair in mammals.
Role of the RNA polymerase trigger loop in transcript elongation, cleavage, and pausing
Authors: Landick Robert
Published: June 2021

Multisubunit RNA polymerases (RNAPs) mediate gene expression in all cellular organisms. However, the structural bases of nucleotide addition, of regulatory mechanisms for RNA chain elongation such as transcriptional pausing, and of transcript cleavage by RNAP remain poorly understood. The RNAP active center possesses two distinct types of activities: a nucleotidyl transferase activity that catalyzes nucleotide addition and its reverse reaction, pyrophosphorolysis; and a polynucleotide hydrolytic activity that cleaves the RNA transcript, a reaction that can be accelerated by transcript cleavage factors GreA and GreB. We find that the ability of the trigger loop (a conserved mobile structure located near the secondary channel of RNAP) to fold into two alpha‐helices immediately adjacent to the catalytic center, is required for its nucleotidyl transferase activity (nucleotide addition and pyrophosphorolysis) but not for its polynucleotide hydrolytic activity (intrinsic transcript cleavage and GreB‐stimulated cleavage). In addition, we find that transcriptional pauses, which regulate transcript elongation by temporarily interrupting the nucleotide addition cycle, are accompanied by a catalytic‐center rearrangement that appears to allow fraying of the RNA 3́ nucleotide away from the DNA template and to constrain the trigger‐loop conformation in a way that interferes with nucleotide addition.
Structural Basis for Proteasome Activation
Authors: Hill Christopher Peter
Published: June 2021

Proteasomes are compartmentalized proteases of ~700 kDa and 14 subunits. They are organized as barrel shaped structures that sequester proteolytic sites in a central chamber that is accessed via axial pores through the center of the end rings of alpha subunits. Unliganded proteasome are by default in a closed conformation, whereas binding of activators serves to open the axial pores to allow substrate access and product egress. Structural and biochemical data will be presented that gives insight to the mechanisms by which the three known classes of proteasome activator, 11S/PA28/PA26/REG, PA200/Blm10, and 19S/PAN bind and induce gate opening.
Structure and Mechanism of Phytochrome
Authors: Forest Katrina T.
Published: June 2021

The strongly conserved phytochrome photoreceptor has been studied for more than 50 years because of its central role in plant adaptations to varying light quality, with important implications in agriculture. More recently, microbial phytochromes have been discovered and have allowed us to make great strides in understanding the molecular nature of these tetrapyrrole‐containing light sensors. Several x‐ray crystal structures combined with molecular biology, biochemistry, and spectroscopy have revealed the knotted fold of the phytochrome protein and its interactions with its buried chromophore, as well as the critical nature of many of the conserved amino acids within the phytochrome family in the photocycle.
Blue light photosensors: Examples of environmentally‐regulated protein/protein interactions
Authors: Gardner Kevin H. , Nash Abigail I. , Ko Wen‐huang , Correa Fernando , Wu Qiong
Published: June 2021

Many biological processes are regulated by controlling protein conformation in response to specific environmental cues. This is often mediated by modular protein domains that use environmentally‐triggered changes in bound cofactor conformation or configuration to regulate protein/protein interactions. Photoreceptors exemplify these principles, as they depend on photochemistry mediated by organic chromophores to detect light. In particular, several classes of blue light sensors use flavin chromophores that alter their interactions with the surrounding protein to initiate conformational changes and signaling. We have used solution NMR spectroscopy and other biophysical methods to examine these processes in two classes of photosensory domains.

The first of these are Light‐Oxygen‐Voltage (LOV) domains, members of the PAS family of environmental sensors. Illumination generates a covalent protein‐flavin bond within the α/β core of LOV domains, leading to conformational changes in the β‐sheet adjacent to the chromophore. These changes are propagated to non‐canonical protein elements on the other side of this sheet, modifying their function. We will show the generality of this process with data for several classes of LOV‐containing proteins, including serine/threonine kinases, histidine kinases and DNA binding proteins.

We will compare these results with parallel studies of BLUF domains (sensors of Blue Light Utilizing FAD), which use different protein topology and photochemistry than LOV domains, but appear to harness light‐induced conformational changes in a similar manner despite these fundamental differences.

This work is supported by grants from the NIH (R01 GM081875) and the Robert A. Welch Foundation (I‐1424).
Mutations in NPC1 and cholesterol metabolism in the brain
Authors: Dietschy John M.
Published: June 2021

This presentation reviews the processes involved in cholesterol (C) homeostasis in the brain. The amount of C in an animal equals about 2100 mg/kg bw, and 15% and 23% of this sterol in the mouse and human, respectively, is found in the CNS. No uptake of C carried in plasma lipoproteins across the blood‐brain barrier can be detected. High rates of synthesis in glia and neurons provide the sterol necessary for early development. Sterol synthesized in oligodendrocytes (250 µg/day) in the newborn mouse supplies sufficient C for myelin formation. Cholesterol, along with apoE, is synthesized in astrocytes, released into the pericellular fluid, and taken up by neurons through receptor‐mediated endocytosis. Mutation of NPC1 leads to accumulation of this C in the late endosomal/lysosomal compartment. In the mature brain, the rate of excretion of C from the CNS must equal the rate of synthesis, and this excretion is accomplished by movement of both C and 24(S)‐hydroxycholesterol across the blood‐brain barrier. This excretion is partially controlled by a target gene of the LXR nuclear receptor. The neurodegeneration and glial activation that is seen with a mutation in NPC1 is ameliorated by agents that either facilitate C transport across the lysosomal membrane or that divert C away from neurons across the blood‐brain barrier. Cholesterol turnover in myelin is very slow while turnover in neurons may be very rapid. (NIH 5R01 HL09610).
Lipid Droplet Associated Proteins and Control of Triacylglycerol Metabolism
Authors: Brasaemle Dawn L , Montero‐Moran Gabriela , McMahon Derek , Caviglia Jorge M , Liang Xiaofang , Sullivan Sean , Golem Devon , Russell Deanna , Marcinkiewicz Amy
Published: June 2021

Perilipin A controls lipolysis in adipocytes and is phosphorylated by protein kinase A (PKA) on six serine residues, promoting hormone‐sensitive lipase activity, and triggering remodeling of lipid droplets, whereby large droplets fragment into myriad micro‐droplets. Phosphorylation of perilipin Ser492 initiates this process and is required for maximal lipolysis. We hypothesize that perilipin A forms a dynamic scaffold on lipid droplets that organizes lipid metabolic enzymes; perilipin conformation responds to the metabolic status of the adipocyte. CGI‐58 binds to the carboxyl terminus of perilipin A in adipocytes under basal conditions. Humans with neutral lipid storage disorder (NLSD) have mutations in CGI‐58 and store excessive triacylglycerol (TAG) in many cells and tissues; hence, CGI‐58 plays a role in cellular TAG homeostasis. Recombinant CGI‐58 shows Coenzyme A‐dependent lysophosphatidic acid acyltransferase activity, and is important for channeling fatty acids released from triacylglycerol hydrolysis to phospholipid synthesis. CGI‐58 is a substrate for PKA and is phosphorylated on Ser239, yet mutation of Ser239 to Ala does not alter the function of CGI‐58 in reducing TAG levels of NLSD fibroblasts. This PKA‐mediated phosphorylation facilitates release of CGI‐58 from the perilipin scaffold. Supported by DK54797 from NIH and an American Heart Association Established Investigator Award.
The altered redistribution and accumulation of E‐cadherin as a dominant‐acting oncogene in inflammatory breast cancer (IBC)
Authors: Barsky Sanford H , Ye Yin , Shetuni Brandon B , Yearsley Kurtis , Xiao Yi
Published: June 2021

E‐cadherin, an adhesion protein within lateral junctions, is thought to function as a tumor suppressor in breast carcinomas and promote metastasis when its expression is lost by mutation, promoter methylation or repression by mediators of EMT. However in IBC, E‐cadherin is overexpressed circumferentially which accounts for the formation of the lymphovascular embolus, a structure efficient at metastasis and drug resistance. Our studies with a human IBC xenograft, MARY‐X, revealed that E‐cadherin transcripts were less than MCF‐7 and MCF‐10A (E‐cadherin positive lines) but the protein levels were 100 fold greater. We found E‐cadherin overexpressed because of altered degradation, redistribution and trafficking. Since previous studies had shown that E‐cadherin lysosomal targeting was mediated by the hepatocyte GF‐tyrosine kinase substrate (Hrs) and that v‐src activation of Rab7 depleted E‐cadherin in EMT, we investigated whether these genes might be decreased in MARY‐X and found this so. Since other Drosophila proteins, eg., sec10 mediated E‐cadherin trafficking from recycling endosomes to plasma membranes, we observed the human equivalent, exoC5, to be increased in MARY‐X. siRNA Rab 7 knockdown in MCF‐7 produced an increase in the 120 KD band of E‐cadherin as well as E‐cadherin intermediates. This suggested that altered E‐cadherin redistribution may be contributing to its oncogenic role in IBC.
The effects of beta‐glucan treatment on endotoxin and sepsis‐induced cytokine production
Authors: Newsome Courtni Takiyah , LeBlanc Brian , Ayala Alfred , Reichner Jonathan
Published: June 2021

β‐glucans, a class of glucose polymers found in the fungal cell wall, stimulate innate immune cells. Stimulating immune cell function can be of clinical benefit; however, it often causes the overproduction of inflammatory cytokines. The soluble β‐glucan, PGG‐β ‐glucan, primes antimicrobial functions without inducing cytokines. In vitro studies have tested whether the lack of cytokine production is due to a suppressor mechanism. These studies indicated that β‐glucan pretreatment reduces LPS‐induced TNF production. The aims of this project were 1) to evaluate the effect of β‐glucan pretreatment on the LPS‐induced TNF production in vivo and 2) to evaluate the effect of β‐glucan on IL‐6 production in septic mice induced by cecal ligation and puncture when given after the onset of infection. During this study, the age and sex of the mice and the time of β‐glucan administration were varied. After the administration of LPS or the onset of sepsis, serum TNF and IL‐6 levels were measured by ELISA. The results indicate that β‐glucan pretreatment reduces LPS‐induced TNF production and that there is a possible age‐response. The results also indicate that β‐glucan given after the onset of sepsis reduces IL‐6 and that the effect may be sex‐specific. The development of β‐glucan as a therapeutic agent with negligible side effects could aid patients with uncontrolled inflammation. NIH R01‐GM066194 (JR) and NIH F31‐GM086069 (CN)
Rotaviral components induce a type 1 IFN response via an apical receptor in intestinal epithelia
Authors: Frias Amena Husain , Vijay‐Kumar Matam , Jones Rheinallt , Gewirtz Andrew T.
Published: June 2021

Rotaviruses (RV) are the leading cause of diarrhea in children. RV primarily infects intestinal epithelial cells, which typically clear the infection within 7 days via pathways not strictly dependent on adaptive immunity. We hypothesize such innate immune clearance of RV is mediated by epithelial antiviral gene expression involving type 1 interferon (IFN), and that these antiviral responses are induced upon detection of RV components. Thus, we sought to define anti‐viral signaling induced in epithelia by RV infection and determine the extent to which this response was recapitulated by non‐infectious RV components. Model human intestinal epithelia (HT29 cells grown on permeable supports) were treated with RV (MOI 1‐3) or UV‐irradiated RV (UV‐RV) which was non‐infectious but structurally intact. Viral protein expression and activation of antiviral markers such as IRF (3/7), STAT1, and PKR was detected by western blotting. Type 1 IFN‐α / β and IL‐8 (inflammatory cytokine) induction was measured by qRT‐PCR or ELISA. Apical, but not basolateral, treatment of model epithelia with RV resulted in productive infection and robust activation of these pathways including IFN‐ β but not IFN‐α. This pattern of anti‐viral signaling was largely mimicked by UV‐RV. Our results suggest intestinal epithelia detect rotaviral components via an apical receptor and mount type 1 IFN responses as part of a cellular antiviral program.
Visual aids and analogies for adding spice to and aiding recall of anatomy and physiology lectures
Authors: Fitch Greg Kent
Published: June 2021

Students listen more actively when they occasionally hear a vivid analogy or see a visual aid or manipulative. These tactics can be used whether the instructor is lecturing or leading a discussion. Over many years, I have accumulated analogies and visual aids that help students understand lectures on anatomy and physiology. For example, the function of cerebrospinal fluid (CSF) in cushioning the brain can be illustrated by placing two raw eggs into two glass jars, one filled with water and one lacking water. The egg, water, and jar represent the brain, CSF, and skull, respectively. Vigorously shake both jars to show the benefit of the CSF surrounding the brain. As an example of an analogy, the decremental travel of graded potentials along neuronal membranes is like a bowling ball that's been pushed to make it roll along a carpet, whereas the travel of action potentials along membranes without decay is like the lit fuse of a firecracker, in which the fire continually burns into the adjacent segment of the fuse and re‐ignites it to burn at the same temperature as in the prior segment. This poster outlines visual aids and analogies for illustrating all mammalian organ systems.
Foundational Physiochemical Concepts in the Biological Sciences
Authors: Klymkowsky Michael , Cooper Melanie
Published: June 2021

When presenting complex ideas to students, it is critical that we make explicit the conceptual foundations upon which we intend to build; what needs to be understood before new words, ideas, or skills can be effectively integrated into their existing thought processes. Understanding of biological/biochemical systems, their behavior and evolution, builds on a number of foundational physiochemical concepts. These include the conservation of energy/matter, free energy, entropy, and entropy‐driven reactions, stochastic and random processes, the behavior of interacting networks (ranging from the molecular genetic and physiological to the ecological), chemical bonding, intermolecular interactions and the determinants of bond/interaction stability and specificity, and an appreciation of statistical mechanics. Once our analysis of conceptual foundations is complete, we need to determine our student's "preexisting condition"; what ideas have been mastered and what confusions or lacunae remain. While important within the context of a specific course, these same considerations also apply to curricular design. They are of particular import when we consider how K12 science teachers are trained, since "fragile understanding" in this key population can cascade, and undermine the level of understanding achieved by their students.
Leptin promotes mesenchymal cells of the hematopoietic microenvironment to differentiate to osteoblasts
Authors: Tuljapurkar Sonal R , Jackson John D , Sharp J G
Published: June 2021

Multipotential mesenchymal stromal cells (MMSCs) serve as a common precursor to osteoblasts and adipocytes. During aging, osteoblasts decrease and bone marrow (BM) fat increases, thereby converting the BM from red to yellow. Alcohol hastens this process of conversion and likely contributes to osteoporosis, anemia, aplasias and other hematopoietic deficiencies of aging. Leptin, a hormone secreted by adipocytes, increases bone density and mass, including in alcoholics. However, information on role of leptin in hematopoiesis is limited. This study was aimed at determining whether leptin affects MMSCs and opposes the effects of ethanol. Varied concentrations (10,000, 1,000, 100 and 10 pg) of leptin were added to MMSC cultures containing growth supporting medium or osteoblast‐stimulating medium. Controls were grown in medium without leptin. At various time points (days 3, 6&10), MMSC survival and proliferation was measured by alamar blue and BrdU assays, respectively. Osteoblast differentiation was quantified by alkaline phosphatase assay. Results demonstrated that leptin induced differentiation of MMSCs into osteoblasts, but did not have significant effects on MMSC survival and proliferation. We have previously demonstrated that ethanol induced proliferation and adipocytic differentiation of MMSCs. Since osteoblasts are the critical cells of the hematopoietic microenvironment, leptin may oppose adipose‐inducing effects and inhibit hematopoietic deficiencies associated with alcohol. (Supported by NIA grant AG024912)
Primitive stem cells in adult feline, canine, ovine, caprine, bovine, and equine peripheral blood
Authors: Lochner F , Lochner D , Lochner D , Samples O , McCommon G , Alena C , Hixson D , Bowyer F P , Black A C , Young H E
Published: June 2021

Stout et al. (Amer Surg. 73:1106‐1110, 2007) reported primitive endogenous stem cells circulating within adult porcine peripheral blood. The current study was undertaken to determine whether similar primitive stem cells could be isolated from the peripheral blood of adult felines, canines, ovines, caprines, bovines, and equines. Adult cats, dogs, sheep, goats, cows and horses had their blood withdrawn following the guidelines of FVSU‐IACUC. The blood was obtained by venipuncture and four ml withdrawn. After gravity separation, the plasma fraction was withdrawn and 15 microliters of the plasma from each animal was mixed with 15 microliters of 0.4% Trypan blue. The solution was mixed, placed onto a hemocytometer and the isolated cells counted: blastomere‐like stem cells (BLSCs) are trypan blue positive and < 2.0 microns in size; transitional‐BLSCs (Tr‐BLSCs) are both trypan blue positive & negative and 2‐5 microns in size; and epiblast‐like stem cells (ELSCs) are trypan blue negative and 6‐8 microns in size (Young and Black, Minerva Biotech 17:55‐63, 2005). The results show that BLSCs, Tr‐BLSCs, and ELSCs are circulating within the peripheral blood of all species examined.
The Role of Hepatocyte Growth Factor in Satellite Cell Mediated Angiogenesis
Authors: Flann Kyle L. , Rathbone Chris R. , Allen Ron E. , Rhoads Robert P.
Published: June 2021

Skeletal muscle regeneration is a multifaceted process involving the spatial and temporal coordination of myogenic and angiogenic events. Satellite cells are well‐established myogenic precursor cells responsible for skeletal muscle growth and repair. We have recently demonstrated that satellite cells also promote angiogenesis, in part, through the secretion of soluble‐acting factors. Satellite cells produce a number of growth factors, some with pro‐angiogenic properties, although it not clear which of these growth factors participate in satellite cell‐mediated angiogenesis. One likely candidate protein is hepatocyte growth factor (HGF). To identify a role for HGF in satellite cell mediated angiogenesis, we employed an in vitro model consisting of rat microvascular fragments (MVF) embedded in a three‐dimensional collagen matrix. In an effort to block HGF action, various levels of anti‐HGF antibody were added to rat satellite cell conditioned medium and fed to MVF cultures. Following 5 days of culture, MVF were analyzed for indices of angiogenesis. Sprout number and length were decreased in a dose‐dependent manner by increasing levels of HGF inhibition. Addition of recombinant HGF during HGF inhibition sufficiently rescued angiogenesis in the MVF cultures. This data indicates that satellite cell‐derived HGF plays an important role in satellite cell mediated angiogenic processes.

Grant Funding Source

NIH
Serum‐dependent epithelial cell sheet shrinkage upon detachment from temperature‐responsive culture surfaces
Authors: Kondo Makoto , Murakami Daisuke , Yamato Masayuki , Takagi Ryo , Namiki Hideo , Okano Teruo
Published: June 2021

Primary oral mucosal epithelial cells show cell stratification similarly to those in vivo under the optimum culture conditions supplemented with serum. By temperature reduction, all the cells on temperature‐responsive surfaces are spontaneously detached as a single contiguous cell sheet since the surfaces change hydrophilic. We have utilized these cell sheets in regenerative medicine of cornea and esophagus. Interestingly, harvested cell sheets shrank to 50% of the original size. By utilizing porous membrane, stratified epithelium‐like cell sheets were also harvested even without serum. However, the harvested cell sheets didn't show any shrinkage, while cell proliferation and colony‐forming efficiency were the same with or without serum. In addition, cell adhesion on culture substrata was significantly weak under the serum‐free culture condition. Electron microscopy revealed that the basal surfaces of cell sheets cultured in the serum‐free condition had less minute processes participating in cell adhesion onto substrata. DNA array analyses showed some inflammatory‐related genes including MMP‐9, IL‐1α, IL‐1α receptor, and calpain 5 were significantly upregulated by serum. These findings would prompt the further investigations to understand underlying molecular mechanisms to achieve reproducible, safe, and effective clinical applications of epithelial cell sheets.

Grant Funding Source

Grant‐in‐Aid for Scientific Research
Endoplasmic reticulum stress response is induced by sulfur mustard in the mouse ear vesicant model
Authors: Chang Yoke‐Chen , Soriano Melannie , Nguyen Michael , Casillas Robert P , Sabourin Carol L. , Gordon Marion K. , Gerecke Donald R.
Published: June 2021

Time‐related changes in the histopathology of mouse skin occur after topical exposure to the vesicant agent, bis(2‐chloroethyl) sulfide (SM) using the mouse ear vesicant model (MEVM). Some of these changes include increasing edema, detachment of the epidermis from the dermis, and upregulation of laminin‐332. Endoplasmic reticulum stress response (ESR) was observed by confocal microscopy. Microarray analysis and RT‐PCR experiments indicated there was upregulation of several heat shock proteins and folding chaperones such glucose‐regulated protein 78 and GRP94 in the ER. It confirmed the confocal observations and suggested that ER stress occurs very quickly in the MEVM. The accumulation of laminin γ2 protein, one of the chains of laminin‐332, in the ER was visualized by confocal microscopy and co localized with the ER chaperones. This accumulation appeared specifically in the migrating, but not proliferating cells. These observations are consistent with recent reports that laminin‐332 is found in migrating, transformed epithelial cells that have left the cell cycle, but not in proliferating cells. These results suggest that laminin γ2 is misfolded after SM treatment, resulting in decreased secretion and reduced overall amounts of laminin‐332 in the extracellular matrix. This would explain the observation that there is a delayed wound healing response evident in this wound model.

Grant Funding Source

ES005022, ES004738, EY09056, and the NIH CounterACT Program through NIAMS U54AR055073
HEALING OF CORNEAL WOUNDS INDUCED BY VESICANTS
Authors: DeSantis Andrea , Hahn Rita A , Beloni John , Schultz Gregory , Heindel Ned , Svoboda Kathy KH , Babin Michael C , Gerecke Donald R. , Gordon Marion K
Published: June 2021

Vesicants cause injury to the corneal epithelial‐stromal junction. Matrix metalloproteinase (MMP) activity delays proper wound healing. We followed healing of organ cultured rabbit corneas exposed to half mustard (CEES) and nitrogen mustard (NM) with subsequent addition of MMP inhibitors or a steroid. Corneas were cultured adapting the method of Foreman et al, Exp. Eye Res. 62:555, 1996. Vesicants were applied, and after 2 hr, were rinsed off. Over the next 22 hr medium alone or medium containing doxycycline, sancycline, Ilomastat, or Tobradex was added. Samples were analyzed by light and electron microscopy (EM). Half mustard (CEES) caused mild injury affecting basal cells. These rarely separated from the stromal layer. EM showed focal degradation of the basement membrane and dissolution of hemidesmosomes. Nitrogen mustard injury was more severe. Regions between the anchoring complexes showed epithelial‐stromal separation. EM showed adhesion complex remnamts prevented total separation. For CEES injury doxycycline, sancycline and Ilomostat ameliorated effects. For nitrogen mustard injuries, all 4 agents improved the histological appearance of the epithelial‐stromal border as compared to vesicant treated samples. However these agents did not totally eliminate separations between the layers.

Grant Funding Source

NIH EY009056 and U54AR055073
The Cystatin S Promoter Drives Expression in the Spleen, Brain and Lung
Authors: Shaw Phyllis A. , Li Jinhua , Luo Jian
Published: June 2021

The cystatin S gene has 3 types of transcription factor binding "modules" clustered in its 1.9kb 5′‐flanking region, including a hormone module containing 3 regions found in the 5′‐flanking sequence of all known salivary gland specific genes, an immune module and 2 SRY (male sex determining modules). To determine which of the cis‐elements regulate tissue specific expression of the cystatin S gene, we generated 2 transgenic mouse models. The 1.9kb/GFP expressing transgene, contains all 4 modules and salivary gland‐specific elements; the 1kb/GFP expressing transgene has the hormone, an SRY and immune modules, and salivary gland‐specific regions. We examined 1.9kb and 1kb promoter driven expression of GFP mRNA and GFP protein levels during development of the submandibular, sublingual, and parotid glands, liver, heart, kidney, muscle, spleen, brain, and lung. 1.9kb of the cystatin S promoter regulates GFP mRNA in all tissues examined during development. Submandibular, sublingual and parotid glands exhibit the highest levels of GFP mRNA at days 10 and 20; the liver, heart, muscle, kidney, brain and lung have the highest level of GFP mRNA at day 10 of development. Spleen shows the highest level of GFP mRNA at day 25. Spleen, kidney, brain and lung have high levels of GFP protein in the 1.9kb/GFP developing transgenic mice. GFP protein remains high in spleen, brain and lung, but is not detected in kidneys of 1kb/GFP transgenics.