bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteolytic shedding by ADAM 17 (TACE) functions as a gatekeeper for leukocyte emigration to inflammatory sites
Authors: Raines Elaine W. , Tang Jingjing , Zarbock Alexander , Gomez Ivan , Bell Bridgit , Ley Klaus , Wilson Carole L.
Published: June 2021

Leukocyte recruitment to inflammatory sites involves sequential adhesive interactions associated with dynamic changes in expression and function of cell surface proteins. We hypothesize that proteolysis of cell surface proteins allows instantaneous uncoupling of adhesive and signaling events, and thus is a key regulator of the inflammatory response. To test this hypothesis, the sterile irritant thioglycollate was injected into the peritoneum of hematopoietic mouse chimeras that lack the sheddase ADAM17 in circulating cells. Thioglycollate injection first initiates neutrophil emigration into the peritoneal cavity, and at 4 hr Ly6G+ neutrophils are increased by 75% in null chimeras. ADAM17‐/‐ chimeras also display a 50% decrease in rolling velocity and a 2‐fold increase in adherent cells visualized by intravital microscopy of the cremaster muscle. In contrast with accelerated neutrophil emigration, monocyte (CD11b+/Ly6G‐) infiltration in null chimeras is reduced by 25% at 48–72 hr, a time of maximal monocyte accumulation. Studies of mixed hematopoietic chimeras (50% wild type, 50% null) further implicate ADAM17 in promoting macrophage exit from the peritoneum. Together these data reveal that ADAM17's function in wild type mice is critical for restriction of neutrophil infiltration, and promotion of monocyte emigration and resolution of the inflammatory response. (Supported by HL67267 & HL18645)
The primary pathogenetic role of vascular hypoperfusion, mitochondria failure and oxidative stress in aging and Alzheimer disease
Authors: Aliev Gjumrakch , Shenk Justin C , Fischbach Kathryn , Perry George , Ames Bruce N
Published: June 2021

We studied the cellular and subcellular features of vascular lesions, immunoreactivity of vasoactive substances, and mitochondria in brain vascular wall cells and hippocampal neurons from human Alzheimer disease (AD) and animal models that mimic AD. Lesioned vessels with immunopositive staining for AbPP and iNOS, and large, lipid‐laden vacuoles in the cytoplasm of endothelial cells were observed. Mitochondrial abnormalities and lesions were pronounced in microvessels in human AD, YAC, ApoE4 and C57B6/SJL Tg (+) mice, 2‐vessel occluded and non‐occluded aged rats, with and without selective mitochondrial antioxidant (lipoic acid and ALCAR) treatment. In situ hybridization revealed damaged mitochondria in vascular endothelium and in perivascular cells of lesioned microvessels in human AD and rodent models proximal to regions of large amyloid deposition. mtDNA deletions were associated with increased amounts of immunoreactive AbPP, 8OHG and COX but not eNOS; these deletions and expression of oxidative stress markers indicate that energy deficiency and oxidative stress in AD selectively affect the brain vascular tree and vulnerable neurons, which can be ameliorated by selective mitochondrial treatment.
Determining the molecular mechanisms of vascular sympathetic innervation
Authors: Long Jennifer B , Segal Steven S , Madri Joseph A
Published: June 2021

Sympathetic nerves regulate blood pressure via peripheral vasoconstriction and redistribution of cardiac output. As some arteries are innervated while others are not, we tested the hypothesis that molecular cues expressed by vessels promote attraction and/or repulsion of sympathetic neurons. In CD1 mice, tyrosine hydroxylase staining shows sympathetic innervation of femoral arteries increases from 52 to 75% of adult innervation densities between birth and 2d postnatal (p<0.01) and was not different from adult by 4d. Using qRT‐PCR, expression profiles of known candidate molecules between innervated femoral and non‐innervated carotid arteries included significant (p<0.01) increases in carotid Sema3a (3X), femoral VEGF (3X) and Sema3f (2X). In addition, their cognate receptors neuropilin‐1, VEGFR2 and neuropilin‐2 are expressed in superior cervical ganglia (SCG). Suppression subtractive hybridization is being used to identify novel candidates. Further, in a 3D collagen gel, average neurite outgrowth from SCG explants towards the femoral is increased by 20% (p<0.01) compared to carotid suggesting that chemorepulsion contributes to lack of carotid innervation. These findings provide evidence of differential expression of axon guidance molecules by innervated and non‐innervated vessels and present an model for investigating selective vascular innervation. R37 HL28373 to JAM, T32 GM07527.
Genetics of progression of pleomorphic xanthoastrocytoma (PXA) in the pediatric population
Authors: Hazrati Lili‐Naz , Solomon Lauren , Lee Eric , Monsalves Eric , Ma Jing , Tabori Uri , Hawkins Cynthia
Published: June 2021

Recent publications suggest that with time, PXAs, rare low grade astrocytomas of children and young adults, may acquire anaplastic features and progress to malignancy with a prognosis similar to high grade astrocytomas. As malignant progression of low grade astrocytomas is rare in children, we sought to uncover the genetic changes that underlie this transformation.

A search of the databases at our institution (1985–2007) identified 7 patients with a primary diagnosis of PXA. Most had multiple recurrences, with two fatalities. Extracted DNA was hybridized onto 500K SNP mapping gene arrays (Affymetrix) and gene copy‐number changes identified (dCHIP).

Analysis of copy‐number changes at first presentation showed few alterations, mainly corresponding to genes within the Wnt/Cadherin pathways. However, with recurrences, the number of genes with copy‐number changes increased and other pathways implicated in cell proliferation, motility and invasiveness became modified, with prominent involvement of the integrin, Ras, and EGFR pathways. These data show that recurring PXAs acquire cumulative and progressive genetic changes that contribute to their rapid growth and increasing invasiveness and thus their less favorable outcome. Further, our experience supports the idea that in the pediatric population, PXAs are prone to recurrences and do not behave as typical low grade astrocytomas. Funding: CIHR.
Mismatch repair defects underlying temozolomide chemoresistance in recurrent glioblastomas
Authors: Yip Stephen , Cahill Daniel P , Louis David N
Published: June 2021

Sequencing of recurrent glioblastomas post temozolomide (TMZ) chemotherapy revealed two cases with somatic inactivating mutations in the mismatch repair gene MSH6, accompanied by loss of protein expression and a unique “hypermutation” phenotype. We subsequently reported that MSH6 inactivation is relatively common and is also associated with rapid tumor growth during TMZ treatment. To address the hypothesis that inactivation of MSH6 is related to TMZ resistance, we have isolated glioblastoma cell cultures (Gli56 and Gli60) from the two index cases and are examining in vitro sensitivity to chemotherapeutic agents including TMZ. We will use fluorescent‐tagged lentiviral constructs to restore MSH6 expression and functions in these cells. We then plan to introduce MSH6 mutants to systematically dissect MSH6 structure and function. Preliminary results showed MSH6 re‐expression restored in vitro TMZ cytotoxicity in glioma cells with MSH6 inactivation. Inactivation of MSH6 in recurrent glioblastoma during alkylator treatment appears frequent and related to rapid tumor regrowth, and re‐introduction of wildtype and mutated MSH6 genes will enable examination of the role of mismatch repair in TMZ cytotoxicity.
Classic Ras and R‐Ras function in neurofibromatosis type 1‐associated malignant peripheral nerve sheath tumors
Authors: Brossier Nicole M. , Carroll Steven L.
Published: June 2021

Neurofibromin negatively regulates classic Ras (H, N, and K‐Ras) and R‐Ras (R‐Ras, R‐Ras2, and M‐Ras) proteins. Neurofibromin loss in malignant peripheral nerve sheath tumors (MPNSTs) thus leads to Ras hyperactivation, suggesting that the Ras proteins required for MPNST pathogenesis would be appropriate therapeutic targets. To identify the classic Ras and/or R‐Ras proteins mediating MPNST proliferation and migration, we examined the expression and action of these molecules in MPNST cells. H‐Ras, N‐Ras, K‐Ras, and R‐Ras2 were uniformly present in 8 MPNST cell lines, while R‐Ras and M‐Ras expression was variable. MPNST cells also expressed specific guanine nucleotide exchange factors capable of activating classic Ras and R‐Ras proteins. We found that R‐Ras2 was constitutively active in MPNST cells and that K‐Ras was activated in these cells by neuregulin‐1, a growth factor that promotes MPNST proliferation and migration. Dominant negative mutants of H‐Ras, an inhibitor of classic Ras proteins, and R‐Ras, which inhibits the R‐Ras subfamily, both inhibited MPNST mitogenesis. We conclude that both classic Ras and R‐Ras subfamily members likely contribute to MPNST pathogenesis. We are currently identifying the specific Ras proteins required for MPNST proliferation and migration by ablating their expression with lentiviruses that express tetracycline‐regulated shRNAs. Supported by R01 NS048353.
Abnormal dopamine synapses in HIV‐1 infection drive circuit specific neurocognitive impairment
Authors: Gelman Benjamin B , Soukup Vicki M
Published: June 2021

Dopamine synapses in HIV infected autopsy brains from the NNTC were analyzed neurochemically. DAergic markers in striatum of decedents with HIV encephalitis (HIVE) were abnormal. Presynaptic dopamine reuptake transporter (DAT1) increased; tyrosine hydroxylase decreased. Postsynaptic dopamine type 2 (D2R) receptor mRNA and protein were decreased with parallel decreases in downstream events including dynorphin and enkephalin expression. To determine if DAergic tone is dynamically changed in other circuits, synapses were examined in the dorsolateral prefrontal cortex (DLPFC) because they drive executive function and working memory. D2R receptor expression and downstream changes were increased in DLPFC, precisely opposite to striatum. Performance was examined for a test of working memory and executive function that specifically drives DAergic circuits in DLPFC (WCST). Increased D2R expression in DLPFC was strongly linked with poor performance on WCST, and also with HIV loading in cerebrospinal fluid. This is the first clinicopathological autopsy illustration that abnormal behavior associated with HIV infection is linked with DAergic dysfunction, and that the DAergic disturbances are circuit specific.
MAP Kinase Phosphatase‐1 (MKP‐1) Deficiency Leads to Reduced Atherosclerotic Lesion Formation in ApoE‐null Mice
Authors: Shen Jianzhong , Chandrasekharan Unni M , Liu Yusen , Smith Jonathan D , DiCorleto Paul E
Published: June 2021

Multiple protein kinases have been implicated in cardiovascular disease; however, little is known about the role of their counterparts ‐ the protein phosphatases. We tested the hypothesis that Mitogen‐Activated Protein Kinase Phosphatase‐1 (MKP‐1), a negative regulator of MAPK signaling pathways, is actively involved in atherogenesis. Mice with homozygous deficiency in MKP‐1 (MKP‐1<−/−>) were bred with apoE‐deficient mice (apoE<−/−>) and the three MKP‐1 genotypes were maintained on a normal chow diet for 16 weeks. The three groups of mice exhibited similar body weight and serum lipid profiles; however, both MKP‐1<+/−> and MKP‐1<−/−> mice had significantly less aortic root atherosclerotic lesion formation than MKP‐1<+/+> mice. Protein array analysis and Luminex bead‐based multiplexing assays showed that the reduction in atherosclerosis was accompanied by decreased serum levels of multiple pro‐inflammatory cytokines and chemokines, including IL‐1a and TNFa; whereas, the level of the anti‐inflammatory cytokine IL‐10 was not changed. Immuno‐histochemical analysis revealed that MKP‐1 protein expression was restricted to the macrophage‐rich, versus smooth muscle cell‐rich regions of the atheroma. Our results provide evidence for a pro‐atherosclerotic role of MKP‐1 via mechanisms independent of lipid metabolism and likely involving macrophage‐mediated inflammation. Support: NIH HL29582.
Granzyme B is important in the progression of atherosclerosis
Authors: Cruz Rani Priya , Boivin Wendy , Zhao Hongyan , Choy Jonathan C , McManus Bruce M , Granville David J
Published: June 2021

Granzyme B (GrB) is a serine protease expressed in certain T‐cells, NK cells, mast cells and macrophages. We have shown GrB expression in human atherosclerotic plaques and its levels correspond to disease severity. Hypothesis: During chronic hyperlipidemia and inflammation, GrB contributes to elastin degradation, medial thinning and reduced elasticity. Methods: To study the role of GrB in a chronic inflammatory, hyperlipidemic environment, ApoE−/− x GrB−/− double knockout (ApoE/GrB‐DKO) mice were created, fed a high fat diet for 30 wks and sacrificed. Tissues were stained with H&E, ORO and Movat's pentachrome. GrB co‐localization to elastin was assessed using confocal microscopy. Results: ApoE−/− mice developed xanthomatosis and atherosclerotic lesions by 30 weeks. The absence of the GrB gene in the ApoE/GrB‐DKO mice abolished cutaneous xanthomatosis in addition to both the frequency and size of atherosclerotic lesions. The absence of GrB was associated with a marked reduction of elastin degradation in both the skin and blood vessels. Using confocal microscopy, we observed GrB strongly co‐localized to the shoulder regions of plaques in addition to the medial elastin fibres. Conclusion: GrB plays a key role in atherosclerotic plaque formation. Lipid accumulation on elastin fibres promotes the recruitment of GrB resulting in a slow, chronic degradation of elastin.
Biomimetic electrospun scaffolds for tissue engineering
Authors: Hadjiargyrou Michael
Published: June 2021

During the last few years electrospinning has gain momentum as an integral part of the fast moving field of tissue engineering. Electrospinning can be used to fabricate biomimetic scaffolds for a variety of tissue engineering approaches; DNA, protein and drug delivery, cell transplantation, prevention of post‐surgical adhesions and wound dressing material. The advantages of electrospun scaffolds, nanoscale fibers, high surface‐to‐volume ratio, large porosity, and flexibility to fabricate a variety of 3D conformations, make them superior to those generated by other available techniques. As such, we have utilized electrospinning and biodegradable polyesters such as polyglycolide (PGA), Poly(L‐lactide) (PLLA) and a copolymer, poly(glycolide‐co‐lactide) (PGA‐co‐PLA) to generate various scaffolds. Particularly, we were able to functionalize the nanofibers with either DNA or drugs and show that these bioactive molecules can be safely electrospun, released in a controlled manner, maintain their intact structure and more importantly remain bioactive. We also functionalized nanofibers with bioactive collagen type I and were able to show that osteoblasts adhere and align along the fibers, migrate throughout the scaffold, proliferate and remain viable and are capable of differentiation and mineralization. Undoubtedly in the future, electrospun scaffolds will find their way into the clinic.
Trabecular bone dynamics in mice subjected to unilateral sciatic neurectomy assessed by in vivo μCT using monochromatic synchrotron radiation
Authors: Matsumoto Takeshi , Nishikawa Ken , Naito Hisashi , Tanaka Masao
Published: June 2021

Monochromatic synchrotron radiation μCT (SRCT) allows quantitative imaging of bone microstructure with high spatial resolutions. Using an SRCT system developed at Japan large‐scale synchrotron radiation facility (Spring‐8), we evaluated the structural changes of tibial metaphyseal trabecular network in ICR mice (n=6) subjected to unloading in one hindlimb induced by sciatic neurectomy at 13 weeks of age. Both hindlimbs were scanned for five minutes at 10 days and 20 days after surgery. The three‐dimensional image was reconstructed with a two‐dimensional filtered backward projection algorithm based on radiographic images acquired with 25‐keV X‐ray over an angular range of 0°–180° with 0.2° steps, providing contiguous images comprised of 1000 × 1000 cubic voxels of 11.7 μm in size. Two reconstructed images at the two different time points were matched by a registration method based on maximizing mutual information. After bone segmentation by setting a threshold value to the hydroxyapatite density of 0.82 g/cm3, local trabecular resorption and formation were quantitated and compared between intact (INT) and sciatic‐neurectomized (SN) hindlimbs. Mechanical unloading reduces the rate of trabecular bone formation but has no effect on the rate of bone resorption as shown in the figure. In both hindlimbs, the number densities of segments and nodes in the trabecular network decreased whereas the trabecular bone thickeness increased, possibly in compensation for the regression of trabecular network. However, the increase of trabecular thickness decreased by 13% in SN hindlimbs.
A multi‐method, formative assessment process aided the development an effective, but shortened, clinical anatomy course
Authors: Rizzolo Lawrence J. , Rando William , O’Brien Michael , Stewart William B.
Published: June 2021

Essential concepts of clinical anatomy have to be conveyed to medical, physician assistant (PA) and nurse‐practitioner (NP) students in courses that are reduced in hours. We employed five assessment methods to guide the design and implementation of new anatomy courses for these career paths. 1) Focus groups produced qualitative data on students’ experience and learning behaviors; 2) Written surveys produced data on how students used course resources to achieve course objectives; 3) User logs kept data on students’ use of course‐specific web sites; 4) Faculty observations reported on students’ participation in lab and discussion; and 5) Traditional exams tested students’ mastery of course content. Among our core findings: Surveys and focus groups revealed how the course created unanticipated pressures for students and faculty. Web log, survey and focus group results showed that websites were used heavily, but inefficiently. Focus groups and faculty observations confirmed high levels of preparation by students. In response to our assessments we: 1) instituted programs for faculty development and new course materials to help faculty and students cope with new demands; and 2) provided greater guidance to students about how to make use of course resources. Aside from preliminary results of summative assessments, unanticipated events provided early indications of the course's success: increased clinical faculty participation; the NP program converted anatomy from an elective to a required course; the PA program requested we develop a version for them; and residency programs use elements of it.

FIPSE grant P116B031017, US Dept of Education
Phenotypic and functional characterization of proangiogenic monocytes
Authors: Duda Dan G , Jones Rosemary , Dawson Michelle , Capen Diane , Tyrrell Alex , Fukumura Dai , Jain Rakesh K
Published: June 2021

The function of proangiogenic monocytes in blood vessel assembly is currently unclear. We characterized myeloid progenitors among bone marrow‐derived cells (BMDCs) from 3 mouse strains by flow cytometry, and established their function in tumors by intravital and confocal microscopy. These cells expanded ex vivo, expressed PDGFRβ, c‐Kit and Sca1, and were distinct from mesenchymal and endothelial BMDCs. Implanted with tumor cells, they persisted and integrated into stroma but not tumor vessel walls, although they significantly promoted tumor growth and metastasis. We also studied myeloid progenitors in a model of capillary repair after hyperoxic acute lung injury. By immunogold labeling and flow cytometry, we detected a population of CD11b+CD45+ cells positive for VEGFR2 and PDGFRβ. We used high‐resolution imaging to capture sequential changes in these cells as they formed capillary walls. By transition to an endothelial or pericytic phenotype, these myeloid progenitor cells contributed to capillary repair, engrafting into the endothelial surface and aligning as perivascular cells. In summary, we show that a previously unrecognized population of myeloid cells can both promote tumor growth and function as common endothelial and perivascular cell precursors.

Supported by R01‐HL070866, R01‐CA115767 and AACR.
Molecular mechanisms of mRNA degradation
Authors: Conti Elena , Bono Fulvia , Glavan Filip , Lorentzen Esben
Published: June 2021

Eukaryotes have evolved quality‐control mechanisms that allow cells to remove defective messenger RNAs (mRNAs). Aberrant mRNA with premature stop codons (PTCs) are recognized and degraded by a process known as nonsense‐mediated mRNA decay (NMD). The NMD pathway is conserved from lower to higher eukaryotes. In humans, the recognition of a PTC‐containing mRNA depends on both splicing and translation.

In the nucleus, a complex of proteins known as the exon junction complex (EJC) is deposited on mRNA upon splicing and stays stably bound until the nucleic acid is exported to the cytoplasm. Here, the crosstalk between a ribosome stalled at a PTC and a downstream EJC elicits a cascade of interactions that ultimately lead to the recruitment of ribonucleases and mRNA degradation. The talk will focus on our current understanding of the molecular mechanisms of NMD as elucidated by structural biology.
New Strategies for Carbohydrate‐Based Drug Discovery
Authors: Wong Chi‐Huey
Published: June 2021

This lecture will present our new development of methods and strategies to target the enzymes associated with carbohydrate synthesis and processing, with particular focus on the development of inhibitors and molecular probes associated with infectious diseases and cancer progression.
Inactivation of Ribonucleotide Reductase by gemcitabine‐5′‐diphosphate
Authors: Stubbe JoAnne , Wang Jun
Published: June 2021

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms supplying the monomeric precursors required for DNA replication and repair. The relative ratio of the deoxynucleotide (dNTP) pools and the absolute amounts of dNTPs govern the fidelity of these processes. RNRs are composed of two types of subunits: α and β. α contains the binding site for nucleoside diphosphates (NDPs) that are reduced and the allosteric effectors (ATP and dNTPs) that control the specificity and rate of NDP reduction. β houses the essential diferric‐tyrosyl radical (Y•) cofactor. The central role of RNRs in nucleic acid metabolism has made them the successful target of drugs that are used clinically. Gemzar, 2′, 2″‐difluorodeoxycytidine (F2C) is used clinically in the treatment of non small cell lung carcinomas and advanced pancreatic cancer. While the mode of cytotoxicity is complex, one its targets is RNR. Synthesis of [5′‐3H]‐F2C CDP has shown that 1 equivalent per α2 is sufficient to inactivate the E. coli and human RNRs. Studies on the mechanism of inactivation will be presented.
Harnessing actin dynamics for endocytic trafficking
Authors: Drubin David
Published: June 2021

Until recently, the actin cytoskeleton and the endocytic machinery were thought to operate independently. However, the actin cytoskeleton is an integral part of the cell cortex and there is growing evidence in diverse eukaryotes that F‐actin plays a direct role during endocytic internalization. Genetic studies in Saccharomyces cerevisiae have demonstrated that Arp2/3‐mediated F‐actin assembly is required specifically for the internalization step of endocytosis. Using real‐time image analysis, we recently defined a pathway for receptor‐mediated endocytosis in budding yeast. We have extended this analysis to the functions of some 60 proteins, and have analyzed the dynamic appearance, movement and disappearance of about 30 proteins at endocytic sites. A major conclusion of this work is that the proteins analyzed to date can be grouped into four functional modules involved in endocytic internalization. We are now analyzing in depth the molecular mechanisms responsible for actin's endocytic role. For example, we are performing experiments to distinguish the contributions of actin assembly versus myosin motor activity to endocytic internalization forces, and the specific roles of different activators of the Arp2/3 complex.
Molecular mechanisms in endocytosis at neuronal synapses
Authors: De Camilli Pietro
Published: June 2021

Release of neurotransmitters at neuronal synapses relies on a highly specialized secretory system based on synaptic vesicles. Following fusion with the plasma membrane, the membranes of these organelles are rapidly reinternalized by endocytosis and reutilized for new cycles of secretion. Given their high specialization for this endocytic recycling, synapses are a powerful model system to study fundamental mechanisms in endocytosis. The talk will focus on some recent advances that we have made in this field using a variety of complementary approaches that range from reconstitution experiments involving purified endocytic proteins and lipid membranes, to studies of genetically modified mice. I will focus on the role of isoforms of the GTPase dynamin, a protein implicated in the fission reaction, as well as on the function of dynamin binding partners with curvature generating and curvature sensing properties (BAR and F‐BAR domains). I will also discuss progress in the elucidation of the coupling between endocytosis and the dephosphorylation of phosphoinositides at synapses an in other systems.
Optical probing of neuronal membrane proteins
Authors: isacoff ehud
Published: June 2021

With the explosive advance of structural biology, a major challenge for biology is shifting from identifying the protein players, and defining their individual properties, to developing new ways of determining how they operate coherently in cells. This requires molecularly focused methods for dynamic interrogation and manipulation of protein structure and function. An attractive approach is to use light as both input and output to probe molecular machines in cells. Methods will be described by which small chemical fluorophores and photoswitches can be conjugated to proteins in a site‐directed manner in live cells and organisms and used to monitor the structural rearrangements that underlie functional transitions or to trigger these functional transitions in rapid and reversible manners.
The Digital Fish Project‐ In toto imaging and FlipTraps for Digitizing Development
Authors: Megason Sean G , Gouaillard Alexandre , Trinh Le , Bronner‐Fraser Marianne , Fraser Scott
Published: June 2021

The genome provides a program for creating an organism. Understanding how this program works will require moving from the one‐dimensional string of letters that has now been determined for the genomic code to understanding how this code is executed in the 4‐dimensions of space and time of a developing embryo. We are developing imaging‐based techniques that allow genome function to be probed quantitatively and systematically across the dimensions of space and time. Specifically, in toto imaging uses time‐lapse, laser‐scanning microscopy to track all the cells in a developing tissue and custom developed software to extract quantitative, cell‐based data. We are using in toto imaging to determine complete lineages of developing tissues and to quantitate gene/protein expression patterns. FlipTraps, a novel gene‐trapping approach, allow a variety of data to be read‐out in vivo including protein expression patterns, transcriptional expression patterns, protein subcellular localizations, and conditional mutant phenotypes. As part of our Center of Excellence in Genomic Science, we are scaling‐up and integrating these approaches to generate quantitative, systematic data suitable for a systems biology based approach to development.
Mechanism of Auxin Perception by the SCF‐TIR1 Ubiquitin Ligase
Authors: Zheng Ning , Tan Xu , Caldeon‐Villalobos Luz Irina A. , Estelle Mark
Published: June 2021

The plant hormone auxin regulates almost every aspect of plant physiology and has fascinated biologists since Darwin's time. How plants sense and respond to auxin, however, remained mysterious until recently. In 2005, the substrate receptor subunit of the plant SCF‐TIR1 ubiquitin ligase complex was identified as the long‐sought auxin receptor. By promoting the SCF‐mediated ubiquitination and degradation of a family of plant transcription repressors, auxin regulates the expression of numerous auxin response genes that control plant growth. By determining the high resolution X‐ray crystal structures of the auxin receptor TIR1 alone and in complexes with auxin and substrate polypeptides, we have revealed how auxin is sensed by and in turn activates its receptor, which functions as a ubiquitin ligase. Our results unravel a surprising role of the plant hormone as a “molecular glue” to enhance substrate recruitment by SCF‐TIR1, thereby, accelerating protein polyubiquitination and degradation. Unexpectedly, an inositol hexakisphosphate molecule was also discovered by our crystallographic analysis as a potential co‐factor of the auxin receptor TIR1. The mechanism by which auxin regulates the plant SCF‐TIR1 complex strongly suggests that developing small molecule compounds as ubiquitin ligase agonists represents a new direction in drug discovery targeting the ubiquitin‐proteasome system.
Structure and Function of Rhomboid Intramembrane Proteases
Authors: Urban Sinisa
Published: June 2021

Rhomboid proteins are integral membrane enzymes with the unusual ability to cleave transmembrane protein segments within the membrane. This form of intramembrane proteolysis plays key roles in diverse cell communication events including EGF signalling during animal development, and quorum sensing during bacterial growth. In these contexts, rhomboid proteins act in the signal‐sending cell to activate signal precursor proteins and initiate the signalling event. Our recent biochemical advances have culminated in a pure enzyme reconstitution system for studying rhomboid activity, and the first high‐resolution crystal structures of any intramembrane protease. This work revealed that intramembrane proteolysis is catalyzed within a ‘sink’, a cavity that opens to the outside of the cell and allows water to enter, but is protected laterally from membrane lipids by a ring of protein segments. Importantly, our recent structure‐function analysis has implicated one transmembrane helix that provides a rate‐limiting movement to gate substrate entry laterally from the membrane. Finally, our recent functional studies have also expanded the cellular role of rhomboid proteins to broad biological processes, including breakdown of host‐parasite junctions during invasion by malaria parasites and tissue lysis by parasitic ameba. These data implicate rhomboid enzymes as possible therapeutic targets in human disease.
TOR signalling and control of cell growth in yeast and mammals
Authors: Hall Michael N
Published: June 2021

TOR (target of rapamycin) is a highly conserved serine/threonine kinase that controls cell growth and aging. The discovery of TOR led to a fundamental change in how one thinks of cell growth. It is not a spontaneous process that simply happens when building blocks (nutrients) are available, but rather is a highly regulated, plastic process that is controlled by TOR‐dependent signalling pathways. TOR is found in two functionally distinct multiprotein complexes, TORC1 and TORC2. The two TOR complexes, like TOR itself, are highly conserved. TORC1 in yeast and mammals mediates temporal control of cell growth by regulating several cellular processes including translation, transcription, ribosome biogenesis, nutrient transport and autophagy – all processes implicated also in aging, suggesting that TORC1 may control aging as an extension of its control of cell growth. TORC2 in yeast and mammals mediates spatial control of cell growth by regulating the actin cytoskeleton. Thus, the two TOR complexes constitute an ancestral signalling network conserved throughout eukaryotic evolution to control the fundamental process of cell growth. The physiological consequences of mTORC1 dysregulation suggest that inhibitors of mTOR may be useful in the treatment of age‐related diseases such as cancer, cardiovascular disease, and metabolic disorders. Data on the role of mTORC1 and mTORC2 in specific tissues will be presented.
Target based Antibacterial Drug Discovery. Zero for the 20th Century
Authors: Projan Steven Jay
Published: June 2021

Armed with whole genome sequences of multiple pathogenic bacteria, large collections of synthetic small molecules and natural product extracts, increasingly sophisticated laboratory automation, high throughput genetics, long lists of essential genes, high throughput screening, crystal structures, virtual (in silico) screening and phalanxes of medicinal chemists not a single antibacterial compound, derived by target based drug discovery, has entered clinical development. Despite a considerable industrial (and to a lesser extent academic) investment the futility in discovering new antibiotics has been palpable, especially during a time of increasingly widespread multidrug resistant strains of bacteria mainly in hospitals but apparently now spreading into the community. These failures may be more apparent than real but two things are becoming clear: we do not really understand bacterial resistance, especially as the ecological problem it is; nor do we appreciate what properties a truly “valid” molecular target should possess.
Effect of energy balance on gene expression in bovine mammary tissue
Authors: Moyes Kasey M , Drackley J K , Morin D E , Rodriguez‐Zas S L , Everts R E , Lewin H A
Published: June 2021

Objectives were to compare mammary tissue gene expression profiles between lactating cows subjected to dietary‐induced negative energy balance (NEB; n = 5/treatment) and cows fed ad libitum (AL) to maintain positive energy balance. NEB cows were feed restricted (FR) to 60% of calculated net energy for lactation requirements for 5 d. After 5 d of FR, both rear mammary quarters of all cows were biopsied for RNA extraction. A 13,257 oligonucleotide (70‐mers) array and qPCR were used for transcript profiling. Annotation was based on similarity searches using BLASTN against human, mouse and bovine RefSeq, human, mouse, and bovine UniGene, and bovine TIGR. Cy3‐ and Cy5‐labelled cDNA from mammary tissue and a reference standard were used for hybridizations. NEB resulted in 299 differentially expressed genes (P < 0.01). Among genes downregulated with NEB (156 genes total), Ingenuity Pathway Analysis® identified cell cycle (5), cell morphology (12), and cellular development (18) as some of the most enriched molecular functions. Genes upregulated by NEB (141 genes total) were associated with amino acid metabolism (11), small molecule biochemistry (18), gene expression (10) and lipid metabolism (8). Results indicate that NEB alters mammary gene expression. Project was funded by Section 1433 Animal Health and Disease, University of Illinois #ILLU‐538‐981, and NRI‐USDA #2007‐35204‐17758.
Proteomic analysis of tumor and adjacent non‐tumor intestinal tissue and tumor burden after iron supplementation in Apc Min/+ mice
Authors: Swain James , Alter Shawn , Chang Jinsook , Zao Hong , Chance Mark
Published: June 2021

To determine the effect of excess dietary iron on intestinal tumorigenesis, proteomic changes between tumors from intestines of ApcMin/+ mice fed adequate and excess iron (45 vs. 450 mg iron/kg AIN‐93M[M] diet) were analyzed using 2D‐DIGE and LTQ LCMS. In addition, tumor burden was measured and proteins differentially expressed in tumor and non‐tumor intestinal tissues were analyzed. There was a 2‐fold increase in tumor burden with excess iron. Of 980 analyzed protein spots, 78 spots were differentially expressed among normal and tumor tissues (adequate vs. excess iron (p<0.05; ANOVA, then Tukey's test)). Among these spots, 36 changed in tumor vs. normal tissue (12↓; 24↑) and 18 differed between tumors in mice fed adequate vs. excess iron (5↓; 13↑). Findings reveal that some of these proteins are involved in generation or reduction of reactive oxygen species (ROS), such as, by gene name, SOD1, SOD2, PRDX1, ITGB1 and ALB. Others are involved in protein degradation, such as ANPEP, DPP7, PSMA1, PSMA2, PSMA3, SERPINB1, HSPA5, HSPA9, PSMA4, and PEPD. Using Ingenuity Pathways Analysis (IPA), we found 4 highly significant functional networks in tumor as compared to normal tissue and 2 in tumors from mice fed adequate as compared to excess iron. Notably, the top network in both cases was associated with functions implicated in cancer and cell death. Supported in part by grants IRG‐91‐022 from ACS (to JS) and P30CA43703 from NIH.
Body composition and Bone Health of Obese Adolescents with vitamin D deficiency
Authors: Lenders Carine , Feldman Henry A , Merewood Anne , Sweeney Carol , Wilson Darrell M , Lee Phillip DK , Abrams Stephanie H , Gitelman Stephen E , Wertz Marcia S , Chen Tai C , Holick Michael F
Published: June 2021

Objectives:

To evaluate body composition and bone health of obese adolescents with and without vitamin D deficiency

Methods:

Data from 58 out of 92 obese adolescents aged 13–17.9 years evaluated for the Glaser Metformin Trial were obtained. Bone mineral content (BMC), lean body mass, and total body fat from DXA‐scan and visceral fat from CT‐scan were measured. Vitamin D status was determined using plasma 25(OH)D and iPTH. Vitamin D deficiency was defined as 25(OH)D<20 ng/mL. Osteocalcin was used as a biomarker for bone formation and CTX for bone resorption. Mean±SD and Student t test were used for analysis.

Results:

Adolescents were 15±1.4 y. old, had a BMI of 36±5 kg/m2, 55 were Tanner Stage>2, 38 were female, and 8 were black. Their level of 25(OH)D was 25±10 ng/ml, iPTH 16±10 pg/ml, osteocalcin 45±22 ng/ml, CTX 1.5±0.9 ng/ml, and BMC 2273±402 g. A total of 17/58 were vitamin D deficient but none had high iPTH (max 48 pg/ml). The group with vs. without vitamin D deficiency had similar lean body mass (57±3 vs. 56±1 kg, p=0.77), differed in total body fat (43±1 vs. 39±1%, p=0.002), but not visceral fat (11±1 vs. 13±1%, p=0.17). They had lower osteocalcin (35±3 vs. 49±4 ng/ml, p=0.04) but similar CTX (1.2±0.2 vs.1.6±0.1 ng/ml, p=0.10) and BMC (2345±93 vs. 2243±65 g, p=0.39).

Conclusion:

Vitamin D deficiency was associated with fat mass and lower osteocalcin, but was not a predictor of BMC in obese adolescents.
Plasminogen activator inhibitor‐1 is negatively associated with fasting plasma monounsaturated fatty acids but not influenced by postprandial polyunsaturated fatty acid composition in men with high fasting triacylglycerol
Authors: Dekker Mark J , Mazurak Vera C , Wright Amanda J , Graham Terry E , Robinson Lindsay E
Published: June 2021

Increased consumption of dietary monounsaturated fatty acids (MUFA) is associated with a favorable risk factor profile associated with the metabolic syndrome (MetS), including lowered plasminogen activator inhibitor (PAI)‐1. In addition, the postprandial response to dietary fat has emerged as a dynamic metabolic period associated with MetS. Plasma fatty acid composition was measured in men with high fasting triacylglycerol (n=8, HTAG>1.69 mmol/L) and low fasting TAG (n=8, LTAG<1.7 mmol/L). Each subject underwent three postprandial oral fat tolerance tests (OFTT) of 1g/kg body weight emulsified lipids consisting of varied PUFA/saturated fatty acid (P/S) ratios of 0.2, 1.0 and 2.0, with blood collected through 8h. Fasting MUFA was negatively correlated with total PAI‐1 antigen (P<0.001, r=−0.98) and PAI‐1 activity (P<0.02, r=−0.8) in HTAG, but not LTAG. Fasting total PUFA concentration was positively correlated with total PAI‐1 antigen (P=0.03, r=0.75) in HTAG, but not LTAG. There was no difference in postprandial PAI‐1 total or activity in response to three OFTTs with similar MUFA but varied PUFA TAG composition. These data suggest that elevated MUFA concentrations may have beneficial effects on PAI‐1 in HTAG subjects and that postprandial PUFA concentrations do not influence postprandial PAI‐1 in this population.

Supported by the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) and NSERC
Opposing roles of omega‐3 and trans polyunsaturated fatty acids in sudden cardiac death and vascular remodeling
Authors: Siddiqui Rafat Ali , Ruzmetov Nargiz , Harvey Kevin , Antalis Caryl , Miller Steven , Zaloga Gary
Published: June 2021

Omega‐3 polyunsaturated fatty acids (n‐3 PUFAs) have well‐recognized cardio‐beneficial effects. In contrast, premature coronary deaths are associated with consuming high levels of trans polyunsaturated fatty acids (TFAs). We studied effects of n‐3 PUFAs and TFAs on cardiac health using sudden cardiac death and compensatory vascular remodeling models. Male rats were subjected to coronary ligation and were randomly assigned to diets high in n‐3 PUFAs or TFAs. Feeding an n‐3 PUFA‐enriched diet caused an increase in plasma n‐3 PUFAs by 7 fold compared to that of TFA fed group whereas TFA‐enriched diet caused an increase in plasma TFAs by 2.5 fold compared to n‐3 PUFA group. A diet high in TFAs was associated with a lower survival rate (6‐month post MI) due to sudden cardiac death. Animals on a TFA diet also exhibited variable degrees of aortic atherosclerotic lesions. For compensatory vascular remodeling, male mice were subjected to femoral artery ligation. After 3 months on the diets, animals on TFA diet did not exhibit any collateral vascular growth whereas animals on n‐3 PUFA diet exhibited compensatory vascular growth around ligated regions. In conclusion our data suggest that TFAs decrease whereas n‐3 PUFAs improve animal survival after Myocardial Infarction. TFAs induce whereas n‐3 PUFAs prevent development of atherosclerotic lesions and TFAs prevent whereas n‐3 PUFAs stimulate compensatory vascular remodeling.
Dietary DHA and ARA alter the expression of 1700 hepatic genes including the tumor suppressor TOB1 in baboon neonates
Authors: Brenna J. Thomas , Anthony Joshua C. , Pan Bruce S. , Hsieh Andrea T. , Nathanielsz Peter W. , Kothapalli Kumar S.D.
Published: June 2021

Background

. We have recently shown that 1100 cerebral genes are differentially expressed in 12 week old baboon neonates consuming medium and high levels of DHA, with ARA. Here we report on differential hepatic gene expression induced by these diets.

Methods

. Baboon neonates were randomized at birth to three formulas: C, 0/0 DHA/ARA; L, 0.33/0.67%w/w; L3, 1.0/0.67. At 12 weeks, liver was collected and Affymetrix Gene chips assessed gene expression. Significance was determined by t‐tests, and Ingenuity® pathway analysis detected gene relationships.

Results

. More than 1700 of 54,000 probe sets (ps) were differentially expressed. Most significantly upregulated was TOB1, hepatic tumor suppressor associated with ERBB2. SCD (9‐desaturase) and FADS1 (5‐desaturase) were downregulated. ELOVL6, a PUFA elongase and BHMT (one carbon metabolism) were upregulated with increasing DHA. Ingenuity pathway analysis identified network TOB1 and SCD with many genes interacting with CTNNB1 and ERBB2. A number of (n = 52) transcripts with predicted peroxisome proliferator response elements were differentially expressed, including TOB1.

Conclusions

. Dietary DHA/ARA within the normal range found in breastmilk had widespread influence on gene expression, including TOB1 and many genes associated with PUFA metabolism. Funding: Mead Johnson Nutritionals, Evansville, IN.
Vitamin E forms inhibited IL‐13‐induced eotaxin secretion by blocking stat6 activation in human lung epithelial A549 cells
Authors: Jiang Qing , Moreland Michelle , Ames Bruce , Illek Beate , Wagner Jim , Peden David B
Published: June 2021

Airway epithelial cells release eotaxins (CCLll, CCL24, CCL26) upon being stimulated by IL‐13, a cytokine generated from Th2 cells. Eotaxins are potent chemotactic agents for eosinophils, which play an important role in the pathogenesis of allergic airway diseases. The goal in the current study is to investigate the potential effect of vitamin E forms on eotaxin secretion by IL‐13 stimulated human lung epithelial A549 cells. Vitamin E forms differentially inhibited IL‐13 induced CCL26 formation at a relative potency of gamma‐tocotrienol (IC50 at 15uM) > delta‐tocopherol, gamma‐tocopherol (IC50 at 25–50uM)> alpha‐tocopherol. Cell viability was not significantly affected by the treatment of vitamin E forms. Studies using inhibitors of p38 mitogen‐activated protein kinase, NF‐kB, and JAK‐STAT suggest that JAK‐STAT and p38 may be important to eotaxin formation. Western blot showed that gamma‐tocotrienol inhibited IL‐13 induced STAT6 phosphorylation in a dose‐dependent manner. On the other hand, no apparent activation of or effect of vitamin E on p38 was evident. These results suggest that vitamin E forms may attenuate allergic airway inflammation by inhibition of eotaxin secretion, which, in turn, may result in a reduction of eosinophil infiltration to the airway. (Supported by NIH P01AT002620)
Effects of diets restricted in fat and carbohydrate with and without resistance training on body composition and cardiovascular risk
Authors: Ballard Kevin David , Quann Erin E , Puglisi Michael J , Forsythe Cassandra E , Volk Brittanie M , Labonte Cherise C , Fernandez Maria‐Luz L , Kraemer William J , Scheett Timothy P , Volek Jeff S
Published: June 2021

To examine the effects of carbohydrate restricted (CRD) and low fat diets (LFD) alone and in combination with resistance training (RT) on body composition and cardiovascular risk, 47 overweight (BMI 31.26±3.78) men (age 33.7±1.5y) were assigned to LFD (25% of energy) or a CRD (15% of energy). Two additional groups performed a whole body, nonlinear periodized RT program 3 d/wk while consuming either the LFD or the CRD diet. Body composition (via DXA), fasting blood lipids and exercise performance were determined before and after the 12 wk intervention. RESULTS: Reductions in body mass, fat mass, percent body fat and abdominal fat were greater in the CRD than the LFD (p<0.001). The addition of RT to the CRD resulted in greater improvements in percent body fat than diet alone (−4.8 vs. −5.5% for CRD and CRD‐RT, respectively). Gains in lean body mass were demonstrated in the RT groups only (p=0.004). CRD resulted in greater reductions in plasma triglycerides (TG) (p=0.023) and TG/HDL‐C ratio (p=0.035), while increasing LDL peak particle size (p<0.001). Maximum bench press and back squat strength were greater (p=0.002) in the RT groups, with no diet effect detected. CONCLUSION: Resistance exercise is an effective strategy to combat diet induced losses of lean body mass. The combination of CRD and RT resulted in the greatest improvements in body composition and cardiovascular health. Source of research funding: Nature's Bounty.
High dose lycopene supplementation increases hepatic CYP2E1 protein and TNF‐α expression in alcohol fed rats
Authors: Veeramachaneni Sudipta , Wang Xiang‐Dong
Published: June 2021

Lycopene (LYC) has attracted considerable attention due to its antioxidant and anti‐inflammatory effects. However, in vivo knowledge of possible interactions between escalating doses of LYC and chronic alcohol ingestion are lacking. F‐344 rats (6 groups, n = 10) were fed either a liquid ethanol Lieber‐DeCarli diet or a control diet with or without LYC for 11 weeks. Two doses of LYC were tested, 1.1 and 3.3 mg/kg BW/day, which are equivalent to 15 mg and 45 mg LYC/day in humans, respectively. Hepatic and plasma concentrations of LYC and its isomers (HPLC), hepatic cytochrome P450 2E1 (CYP2E1) protein (Western Blotting) and tumor necrosis factor‐α (TNF‐α) expression (qPCR) were assessed. A dose‐dependent increase in plasma and hepatic LYC concentrations were observed in both control and alcohol fed rats. Hepatic LYC levels increased 74–157% and plasma LYC levels increased 48–127% (low and high dose, respectively) in alcohol fed rats vs. control rats supplemented with identical doses of LYC. Additionally, much lower concentrations of LYC cis isomers were observed in alcohol fed rats vs. controls. High dose LYC also significantly increased hepatic CYP2E1 and TNF‐α in alcohol fed rats. These data indicate an interaction between chronic alcohol consumption and high dose LYC, suggesting a need for caution among individuals consuming high amounts of alcohol and LYC.