bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinyl ester formation by lecithin:retinol acyltransferase (LRAT) is a key regulator of retinoid homeostasis in mouse embryogenesis
Authors: Wassef Lesley , Kim Youn‐Kyung , Hamberger Leora , Palczewski Krzysztof , Blaner William S , Quadro Loredana
Published: June 2021

The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids (vitamin A and its derivatives). The mechanisms through which mammalian developing tissues maintain adequate retinoid levels in the face of perturbations in maternal vitamin A dietary intake have not been established. We investigated the role of retinyl ester formation catalyzed by lecithin:retinol acyltransferase (LRAT) in regulating retinoid homeostasis during embryogenesis. Pregnant mice, lacking both LRAT and retinol‐binding protein (RBP), the serum specific carrier for retinol, were maintained on diets containing different amounts of vitamin A. Maternal dietary vitamin A deprivation during pregnancy generated a severe retinoid‐deficient phenotype of the embryo due to the retinoid deficient status of the double mutant dams rather than to the lack of embryonic LRAT. Moreover, LRAT acts together with Cyp26A1, one of the enzymes that catalyze the degradation of retinoic acid, and possibly with Stra6, the recently identified cell surface receptor for retinol‐RBP, in maintaining adequate levels of retinoids in embryonic and extraembryonic tissues.

Work supported by the National Research Initiative Grant no. 2006‐35200‐16580 from the USDA‐CSREES, Program 31.0.
Herpes oncolytic vectors effectively treat prostate carcinomas with neural invasion while preserving nerve function
Authors: Kelly Kaitlyn Jane , Brader Peter , Rein Avigail , Shah Jatin , Wong Richard , Fong Yuman , Gil Ziv
Published: June 2021

Prostate carcinoma has a high propensity for neural invasion (NI) and resection of these tumors often leads to erectile dysfunction. We hypothesized that oncolytic therapy using attenuated herpes simplex virus (HSV) will selectively target cancer cells invading nerves while preserving neural function.

Prostate cancer cell lines PC3, DU145, and LNCap were infected with HSVs (NV1023 and NV1066) in vitro and cytotoxicity was assessed over 7 days. HSV had significant oncolytic effect on all prostate cancer cell lines. An in vivo model of NI was established by implanting prostate carcinoma cells in the sciatic nerves of nude mice. Mice were treated with NV1023 or saline 7 days after establishment of tumors. Treatment with attenuated viruses had no toxic effect on nerves (n=30). Significant reduction in tumor size and inhibition of NI was found 6–8 weeks after treatment (P <0.005, n=30). The control animals developed complete paralysis <5 weeks post‐treatment, whereas most NV1023‐treated animals had preserved nerve function >12 weeks after treatment (P <0.0001, n=7–10).

Attenuated HSVs effectively treat prostate carcinomas with NI, while preserving nerve function. These findings may hold significant clinical implications for prostate cancer patients and for other neurotrophic tumors.

Research support was provided by FAMRI.
Disruption of androgen receptor activity by synthetic and dietary aryl hydrocarbon receptor ligands
Authors: Ralph Jody Love , Guns Emma T , Nelson Colleen C
Published: June 2021

There is a growing concern that many of the environmental and dietary chemicals that are ingested by humans but have not been thoroughly tested for endocrine activity may pose a significant health threat. These chemicals may have the potential to mimic or antagonize hormone action, possibly through binding to steroid receptors and interfering with transcriptional activity. The prostate is highly sensitive to androgens and requires precise hormonal control to regulate its growth and function.

Prostate cells express both androgen receptor and aryl hydrocarbon receptor (AhR) and cross‐talk between the two pathways may affect the function of the prostate cell. A number of methods were used to characterize the impact of dietary and environmental AhR ligands on androgen action in a prostate cancer‐derived cell line. Our in vitro cell culture‐based assay utilizes androgen‐responsive promoter constructs in conjunction with a luciferase reporter gene. These results combined with those from a binding assay indicate that several of the AhR ligands, including resveratrol, indole 3 carbinol, curcumin and hexachlorobenzene antagonize androgen‐initiated transcriptional activity without binding to the androgen receptor. Environmental and dietary compounds have been implicated in the rising incidence of prostate cancer and insight into the mechanisms of endocrine disruption will help to clarify their role.
ROLE OF PROSTAGLANDIN TRANSPORTER IN PROSTAGLANDIN E2 INDUCED COLONIC EPITHELIAL BARRIER ALTERATIONS
Authors: Virapin Manigandan Lejeune , Chadee Kris
Published: June 2021

Prostaglandin E2 (PGE2) is an important pro‐inflammatory lipid mediator that is produced in excess during inflammatory bowel diseases (IBD). It exerts its action by signaling via four different EP receptor subtypes. In this study, we determine the role of EP4 receptor in the alteration of epithelial barrier functions. Screening for EP4 receptor expression in human colonic resection tissues (IBD patient vs. healthy control) revealed differential receptor expression/localization. EP4 receptors were normally localized on epithelial apical plasma membranes whereas; they were diffusely expressed throughout the cells from patients with IBD. To check how PGE2 is transported across colonic cells towards apical EP4 receptors, we probed for Prostaglandin Transporters (PGT) in human colonic epithelial cells. T84 monolayer modestly expressed PGT protein which increased 2‐fold following TNF‐a stimulation. A corresponding increase in the trans epithelial transport of 3H‐PGE2 was also observed. Moreover, EP4 receptor specific agonist caused a steep decrease in the Trans Epithelial Resistance (TER) of T84 monolayer indicating a loss of barrier function. Alteration in TER is associated with exclusive changes in the localization of a tight junction protein, Claudin4. Taken together, these studies unravel a complex mechanism whereby PGT play role in PGE2 induced alteration of colonic epithelial barrier function.
Role of Epac1, an exchange factor for Rap GTPase, in endothelial microtubule dynamics and barrier function
Authors: Sehrawat Seema , Cullere Xavier , Patel Sunita , Italiano Joseph , Mayadas Tanya
Published: June 2021

Vascular leakage leading to excessive tissue fluid accumulation often has severe pathological consequences and contributes to morbidity and mortality in disease. Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to co‐localize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8‐pCPT‐2′O‐Me‐cAMP (O‐Me‐cAMP) resulted in a net increase in the length of microtubules. This did not require cell‐cell interactions or Rap GTPase activation and was attributed to microtubule growth as assessed by time‐lapse microscopy of HUVEC expressing flourophore‐linked microtubule plus‐end marker end‐binding protein 3. An intact microtubule network was required for Epac mediated changes in cortical actin and barrier enhancement but was not required for Rap activation. Finally, Epac activation reversed microtubule dependent reductions in vascular permeability induced by TNFα and TGFβ. Thus Epac can directly promote microtubule growth in endothelial cells. This, together with Epac mediated Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability. Elucidation of the mechanism of Epac regulation of MT and actin may lead to potential therapies for edema in acute vascular syndromes.

Funding was provided by NIH HL (PPG), NIH HL036028 (TNM), HL68130 (JI). XC was supported by KO1AR054984, and SP by training grant HL066978 (L. Silberstein) and a Ruth L. Kirschstein fellowship HL082133‐01.
Endothelial Cells Become Phagocytic after CNS Injury and Inflammation
Authors: Lossinsky Albert S.
Published: June 2021

A major goal in understanding the vascular function of the blood‐brain barrier (BBB) is to characterize the nature of macromolecular and cellular transport of leukocytes and pathogens across the injured and inflamed BBB. Endothelial cells (ECs) can ingest particulate materials, pathogenic microorganisms, neoplastic and inflammatory cells since ECs and macrophages both originate from undifferentiated mesenchymal cells during embryologic development. Here we studied the phagocytic characteristics of BBB‐type ECs after exposure to HIV‐1 virus and Candida albicans yeast. We present ultrastructural and immunoultrastructural evidence that cultured human brain ECs can engulf viral particles and the yeast‐cells and filaments via EC caveolae defined previously as vesiculo‐canalicular/vesiculo‐vacuolar organelles (VCS/VVOs). These modified caveolae upregulate ICAM‐1that facilitates trafficking of the invading cells into and across the ECs. Elongated EC microvilli, fronds, and membrane ruffles embraced and engulfed the virus and yeast cells and transported them across the ECs via a transcellular pathway. Our studies demonstrate the importance of comparing both animal and cell culture models of the BBB to study the phagocytic properties of BBB‐type ECs. Supported by a grant from the NIAID: RO3 AI055636‐02.
Visualization of neuron‐astroglia interaction
Authors: Okabe Shigeo
Published: June 2021

Formation and maintenance of synapses are important in proper functions of the nervous system. Visualization of synapse structure and molecules localized in this micro‐compartment requires fluorescence imaging with high resolution and sensitivity. Two‐photon microscopy is a unique optical system suitable for the detection of submicron‐scale fluorescent structures deep inside the nervous tissue. By using selective labeling methods of neurons and glial cells in hippocampal slices in combination with two‐photon microscopy, dynamic interactions between postsynaptic spines and astrocytes were visualize. We labeled a small subset of astrocytes with GFP and introduced rhodamine‐dextran into pyramidal neurons in the close vicinity of GFP‐positive astrocytes. Two‐photon time‐lapse imaging of both dendritic spines filled with rhodamine‐dextran and GFP‐positive astrocytic processes revealed frequent physical interactions between two structural components. Dendritic spines with previous episodes of astrocytic contacts showed longer lifetime. Direct contacts with astrocytes also promoted maturation of dendritic protrusions into stable spines with prominent head structure. Stabilization of spines by astrocytic contacts requires intercellular signaling pathway dependent on ephrin/Eph interaction. These observations indicate importance of direct astrocytic contacts in stabilization and maturation of dendritic spines, where most excitatory synapses onto pyramidal neurons are formed.

Highly correlated structural changes of synapses and glial cell processes illustrate importance of two‐photon time‐lapse imaging of living nervous tissue in understanding molecular mechanisms of neural circuit development and its maintenance in the brain.
From 3‐D Cell Culture to Reparative Medicine: Matrix Materials that Meet the Translational Challenge
Authors: Prestwich Glenn Downes
Published: June 2021

Two‐dimensional (2‐D) cell culture on coated plastic is being replaced by culture on matrices with more appropriate composition and stiffness, or by encapsulation of cells in three‐dimensions (3‐D). The acceptance of this new 3‐D paradigm has been constrained by the lack of a commercially available, biocompatible material that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. The standard for 3‐D cell culture should be a bio‐inspired, biomimetic material that can be used “as is” in drug discovery, toxicology, cell banking, and in the clinic. Such a biomaterial should be highly reproducible, manufacturable, approvable, and affordable. These criteria are met by a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3‐D culture of cells in vitro and enables tissue formation in vivo. These semi‐synthetic hyaluronan‐derived sECMs materials were developed for in vivo tissue repair, and have been reverse‐engineered for in vitro applications in research. Specific applications for different sECM compositions include stem cell expansion with control of differentiation, scar‐free wound healing, growth factor delivery, cell delivery for osteochondral defect and liver repair, and development of vascularized tumor xenografts for personalized chemotherapy.
Capillary reconstruction in skin and blood vessels by tissue engineering
Authors: Germain Lucie
Published: June 2021

An important aspect of engineered tissues is the addition of the microvasculature. Preexisting capillaries within the construct could enhance engraftment and allow the survival of thicker tissues. We first reconstructed in vitro a human capillary‐like network in a sponge using dermal fibroblasts, endothelial cells and keratinocytes. The spontaneous formation of capillary‐like structures in an extracellular matrix (ecm) was promoted. The vascularization of these skin constructs was accelerated after transplantation. We have shown that the production of completely biologic endothelialized tissue is feasible. The self‐assembly approach we developed allows the production of a tissue from cells only (no exogenous ecm or biomaterials). The self‐assembly approach consists of culturing mesenchymal cells (fibroblasts or smooth muscle cells) in the presence of ascorbic acid in order that cells produce and organize a dense ecm. The sheets are then superposed to reconstruct a dermis or rolled over a mandrel to produce a tubular structure such as a tissue‐engineered blood vessel (TEVB, 5mm i.d.). Capillary‐like structures may be produced by adding endothelial cells. In this manner, a tissue‐engineered capillary network forms in the TE‐dermis or in the adventitial layer of the TEVB. In conclusion, our tissue‐engineered models allowed us to progress towards the reconstruction of microvasculature in vitro.
Diffusion of innovations: wireless and wearable anatomy learning resources
Authors: Trelease Robert
Published: June 2021

Since 1999, the author has explored and reported on the use of handheld computers, PDAs and personal media players, for anatomy and health sciences education. Attention has been consistently directed to the anticipated integration of handheld computer functions into ubiquitous cellular telephones. However, modest distribution of first‐generation “PDA‐phones” (e.g., Palm Treo and Blackberry) has provided few potential users for basic health sciences learning resources. In contrast, with the widespread popular adoption of the iPod media player, “podcasting” has become a successful portable medium for distributing lectures, images, and video‐based content to students. With the recent introduction of the widescreen Apple iPhone and the iPod touch wireless handhelds, scalable World Wide Web access has been added to the iPod's basic media player capabilities. The author's successfully distributed iNatomy limb muscle “flashcard” resource was converted from iPod Notes Reader format to (X)HTML, with cascading style sheets optimized for the iPhone/touch Safari Web browser. This new application was tested and openly distributed on public Web servers. Existing PDF documents and image resources were also successfully tested, including video lectures, clinical and 3D imaging arrays. The iPhone and iPod touch provide readable high‐resolution displays (320 × 480 pixels at 160 dots/inch) that can readily display, zoom, and pan images with simple finger‐touch gestures. They represent just the first of a new generation of touch‐interfaced, “wearable” computing devices that provide practical, scalable wireless Web access, also suited for audience response systems. With widespread self‐adoption of such new personal technology by incoming students, educators can look forward to increasing portability of well‐designed, multiplatform “learn anywhere” resources.
“An asymmetric response to fibroblast growth factor during heart development in the primitive chordate, Ciona intestinalis”
Authors: Davidson Brad
Published: June 2021

Fibroblast growth factors (FGFs) impact many aspects of cell behavior, playing critical roles in normal development and cancer biology. However, FGF signaling has not been linked to cell polarity. Modifications of cell polarity often direct asymmetric cell division resulting in differential specification of embryonic cell lineages. Here we employ the simple chordate, Ciona intestinalis, to investigate the role of fibroblast growth factor (FGF) in an asymmetric cell division closely tied to differential cardiac specification. Midway through embryogenesis, FGF signaling causes four founder cells to undergo an asymmetric division, generating four small heart precursor cells and four large tail muscle precursors. Targeted expression of a dominant negative FGF receptor blocks both asymmetric division and heart specification. However, application of the MAP kinase pathway inhibitor U0126 blocks heart specification, but does not appear to block asymmetric division. We are currently investigating this apparent uncoupling of specification and asymmetry downstream of FGF. Additionally, we have employed comparative microarray analysis of gene expression from sorted heart cells to identify the primary transcriptional changes mediated by FGF signaling. We are currently investigating the potential role of these target genes in signal modulation, cell polarity and heart specification.
Overview of the Lymphatic System/Cell and Tissue Biology of the Lymph Node
Authors: Bolender David , Paulsen Douglas
Published: June 2021

The lymphatic vascular system is a network of vessels (lymphatics) responsible for collecting tissue fluid from intercellular spaces and returning it to the venous system. Only the central nervous system lacks lymphatic vessels. Tissue fluid, originating as an ultrafiltrate of blood in capillary beds, enters the system through lymphatic capillaries and flows though a series of progressively larger vessels, the largest of which is the thoracic duct. Flow through the system is driven by compression of the surrounding tissues and directed by valves. At specific locations in the body, lymph vessels connect with encapsulated accumulations of lymphatic tissue known as lymph nodes. Lymph entering the nodes through afferent vessels percolates through sinuses allowing contact with macrophages and other antigen‐presenting cells. Foreign matter and cellular debris are filtered from the lymph and collect in the node. As reservoirs for tissue debris, metastatic cells, and infectious agents, lymph nodes improve the efficiency of interactions between reactive lymphocytes and these heralds of disease. Lymphoid nodules in the nodes' cortex contain abundant B‐lymphocytes that when activated form antibody‐secreting plasma cells. The nearby paracortical zone is a key point of contact between the blood and lymphatic vascular systems. Its high‐endothelial venules provide an entry point for T‐lymphocytes circulating in the blood and thus access to the reservoir of antigenic material in the node. Lymph nodes therefore play a critical role in lymphocyte activation for both humoral and cell‐mediated antigen disposal mechanisms. Filtrate exiting the node through the efferent vessels contains less antigenic material, but additional antibodies and activated lymphocytes.
Regenerative medicine: enabling technologies for clinical translation
Authors: Yoo James J.
Published: June 2021

Tissue engineering and regenerative medicine has emerged as an innovative scientific field that focuses on development of new approaches to repairing cells, tissues and organs for clinical applications. Recent advances in this field have offered new therapeutic opportunities that facilitate the restoration and maintenance of normal tissue function. Consistent with the goals of tissue engineering and regenerative medicine, we have followed a strategy that has shown to be effective clinically. This approach involves the use of biocompatible matrices either with or without cells. The matrices are either used as cell delivery vehicles or as scaffolds to promote and enhance tissue regeneration. When cells are used, donor tissue is dissociated into individual cells, which are expanded in culture, attached to a support matrix, and reimplanted into recipient for recovery of tissue function.

Over the past years, research investigations have been actively pursued in various tissue systems using tissue engineering and regenerative medicine concepts and technologies. Although these tissues have shown their potential applicability experimentally, only a limited number of tissues have been successfully engineered for clinical use to date. This is due to various obstacles encountered in the tissue building process. The progress toward clinical translation, current challenges hampering organ development and potential solutions to overcome technical hurdles will be discussed.
An Investigation into the Cellular and Molecular Basis for Newt Spinal Cord Regeneration
Authors: Odelberg Shannon J , Kent David , Zukor Katherine , Atkinson Donald L , Stevenson Tamara J , Hoang William
Published: June 2021

An adult newt has the remarkable ability to regenerate its spinal cord and regain function after suffering a paralysis‐inducing injury. Function is usually regained in about 6–9 weeks following a complete transection of the thoracic spinal cord. Following tail amputation, the spinal cord regenerates in synchrony with the regrowth of the tail. Spinal cord regeneration is dependent upon several factors: 1) a robust and organized ependymal cell response; 2) the ability of the newt axons to regenerate; and 3) the absence of an axon growth‐inhibiting scar at the lesion site. We have characterized the timing of the ependymal cell response following both thoracic spinal cord transections and tail amputations and have determined that DNA synthesis is initiated by the seventh day in both types of injuries. Using differential display, microarray analysis, and real‐time RT‐PCR, we have identified several members of the matrix metalloproteinase (MMP) gene family as well as a few novel cytokines that are excellent candidates for regulating newt spinal cord regeneration. We are currently assessing the role of these genes in the regenerative process.

Supported by The Craig H. Neilsen Foundation
Imaging the steps of lymphatic metastasis
Authors: Padera Timothy P.
Published: June 2021

The mechanisms used by cancer cells to form lymph node metastasis are starting to be understood, with the hope of identifying treatment strategies to lower mortality due to disseminated cancer. In contrast to hematogenous metastasis in which the primary tumor has functional blood vessels for cancer cells to invade, the story for lymphatic metastasis is likely more complicated. Solid tumors seem to lack functional intratumor lymphatic vessels which means tumor cells that enter intratumor lymphatic vessels will not be carried by lymph flow to lymph nodes. Functional lymphatic vessels in the margin of tumors, however, seem sufficient for lymphatic metastasis to occur. Using intravital microscopy, we have investigated the individual steps of lymphatic metastasis. We will discuss at which step molecules affecting lymphatic metastasis act and how this will affect the design of anti‐metastatic therapeutics.
Differential Muscle Cell Recruitments and Functions in Mouse Lymphatic Tissue Beds
Authors: Muthuchamy Mariappan , Foskett Andrea M , Gashev Anatoliy , Greiner Steve , Zawieja David C
Published: June 2021

The molecular mechanisms that regulate the lymphatic vessel maturation, particularly the recruitments of lymphatic muscle cells are completely unknown. We propose that muscle cell recruitment to different lymphatic beds is dependent upon its functional requirements. While mesenteric lymphatic vessels consistently stain strongly positive for LYVE1 and podoplanin, and weakly positive for CD31 in all ages of mice, muscle staining for neither vascular nor enteric actin is observed even in 4 month old mice. Iliac and thoracic duct lymphatics show actin positive cells on the walls of the vessels. In situ measurements of contractile activity of mesenteric lymphatics show phasic contractions are either absent or very limited. Thoracic duct exhibits comparatively strong basal tone and shows contractile responses to substance P. Iliac lymphatics exhibit some phasic contractions. The diastolic diameters for the iliac preparations were 245±6 μm; the systolic diameter was 225±5 μm (n=5). The contractile frequency was 19±3 per minute. Acetylcholine at 10 μM completely blocked the phasic contraction; the diastolic diameter increased to 280±6 μm. Our data suggest that the mouse mesenteric lymphatics lack muscle cells and therefore may serve merely as passive conduits for lymph flow without active influencing it, whereas, iliac and thoracic duct lymphatics might actively control lymph flow.
Fashion News: Using clothing to demonstrate the formation of the inguinal canal
Authors: Lyons Virginia Taylor
Published: June 2021

The formation of the inguinal canal and the descent of the testis are challenging concepts that students typically have trouble grasping. Particularly confusing is the fact that transversus abdominis does not contribute to the layers of the spermatic cord. Additionally, the anatomy of the superficial inguinal ring is often misunderstood, as students don't realize that the external spermatic fascia is continuous with the external oblique aponeurosis at the margins of the superficial ring. Understanding the anatomical organization of the inguinal region is important, due to its clinical relevance in hernia repair. One way to explain the anatomy in this region will be demonstrated using various articles of clothing.
Drawing the heart and great vessels: What Grant can teach us about surface anatomy and cardiac imaging
Authors: DePhilip Robert
Published: June 2021

In study of the thorax, the renowned anatomist, J. C. B. Grant, believed that the ability to plot the sternocostal projection of the heart and great vessels on the anterior chest wall was of such obvious importance that he made it the “key to the region” (Grant, 1948). His recommendation is as valid today as then. This presentation will show how Grant's original drawing exercise can be adapted to a large audience setting and expanded to correlate cardiac anatomy with the vertebral levels at which specific structures are found on computed tomography and magnetic resonance images.
Perpetuating peritoneal reflections in the pelvis
Authors: Granger Noelle A
Published: June 2021

The organs of the pelvis project so far into the true pelvic cavity that understanding the organization of their peritoneal coverings, especially the broad ligament and its relationship to the uterus, the uterine tubes and the ovaries, can be a difficult. In addition, the pelvic peritoneal reflections are important to a number of clinical concepts. This arrangement of these reflections can be explained and visually reinforced with the use of a bed sheet in a simple demonstration.
Of extracellular matrix, the genome and the microenvironment in breast cancer: No cell is an island
Authors: Bissell Mina J
Published: June 2021

Development of robust and versatile 3‐D culture models for studying functional differentiation of normal mammary gland and steps in breast cancer has allowed us to conclude that tissue structure and epithelial polarity are overarching suppressor mechanisms that play crucial roles in maintenance of the differentiated state and how the loss of these has significant ramifications in cancer and in response to therapeutic agents. These assays and the concepts derived from the experiments are now pursued by many other laboratories. To move these concepts to the next stage, we have embarked on developing complementary assays:

1) Can we apply the knowledge gained so far to finding more effective therapeutics? 2) how to make other versatile models to understand the process of branching morphogenesis of the mammary gland, in particular, how the epithelial cells invade the fat pad in organized branching patterns without breaching the basement membrane. This knowledge is crucial for understanding where the regulation goes awry in breast cancer cells when they invade and metastasize; and 3) how to improve the monocellular 3D models to reflect the complexity of the mammary gland. I will discuss the progress the laboratory has made in these and other areas.
Mechanistic studies of ribosome function and potential implications for translational control
Authors: Green Rachel
Published: June 2021

Translation of the genetic code is mediated by the adaptor tRNA substrates and protein factors interacting with two distinct active sites in the RNA‐rich functional centers of the ribosome. We are using mutant analysis coupled with pre‐steady state kinetic approaches to decipher the molecular details of ribosome function in these active sites. Recent studies have focused on the protein class I release factors (RF1 and RF2) and (1) how they interact with the large subunit active site to trigger the hydrolytic reaction of peptide release and (2) how their interactions with the decoding center of the small subunit are critical to this function. In related studies, we have focused on the process of decoding and how the tRNA itself and conserved nucleotides in the small subunit of the ribosome conspire in this rapid and high fidelity process. These studies by us (and by others) are converging on a common theme that the ribosome samples discrete on and off states during each step of the translation cycle. We are intrigued by the idea that these same states are directly manipulated by extra‐ribosomal factors that regulate translation and are currently extending our studies in bacterial and eukaryotic systems to characterize such potential effects. Through these approaches we hope to provide some mechanistic insights into broad aspects of translation regulation.
Structures of the E. coli ribosome with ribosome recycling factor and antibiotics
Authors: Cate Jamie HD , Borovinskaya Maria A , Pai Raj D , Zhang Wen , Schuwirth Barbara S , Holton James M , Hirokawa Go , Kaji Hideko , Kaji Akira , Shoji Shinichiro , Fredrick Kurt
Published: June 2021

Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. Aminoglycosides are widely used antibiotics that, among their effects, inhibit ribosome recycling. I will present x‐ray crystal structures of the E. coli 70S ribosome with RRF and/or aminoglycosides bound that provide a structural explanation for aminoglycoside inhibition of ribosome recycling. I will also present structures of the ribosome with the antibiotic spectinomycin, which inhibits mRNA and tRNA translocation on the ribosome.
Automated, continuous analysis of embryonic gene expression with cellular resolution in C. elegans
Authors: Waterston Robert H. , Murphy John Isaac , Bao Zhirong , Blick Daniel , Boeck Max E. , Boyle Thomas J. , Mericle Barbara , Preston Elicia , Sandel Matt J. , Zhao Zhongying
Published: June 2021

We have developed methods that automatically analyze the kinetics of gene expression in the C. elegans with cellular resolution continuously during embryogenesis. From 3D movies of developing embryos expressing histone‐GFP fusion proteins in all nuclei, we use computational methods to detect cell divisions and determine the lineage. We simultaneously image a histone‐RFP reporter driven by a promoter of interest. By interrogating the nuclear positions determined in the lineage tracing we obtain quantitative measurements of expression for each cell at each time point. Because the cell lineage is invariant, we can directly map each independent expression pattern onto the reference lineage to obtain composite pictures of gene activity. We are applying the method systematically to reveal the expression of each transcription factor in embryonic development, and have already generated expression patterns for key developmental regulators, including pha‐4 (forkhead), cnd‐1 (NeuroD), hlh‐1 (MyoD) and the GATA factors end‐3, elt‐2 and elt‐7. Comparing the expression of multiple genes expressed within a single cell and its ancestors reveals the subtleties of expression and regulatory pathways. We have begun testing some of these predictions by examining the impact of RNAi knockdown of the regulator on expression levels of predicted targets.

The automated lineage methods also allow new phenotypic analyses.
How Proteins Fold Deciphered by Hydrogen Exchange
Authors: Englander Walter
Published: June 2021

Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates that live for milliseconds cannot be isolated and solved. It has been possible to determine the structure and properties of intermediates during kinetic folding by hydrogen exchange (HX) methods. Having reached the native state proteins repeatedly unfold and refold. The normally invisible intermediates can be characterized by HX. Three concepts emerge. Proteins act like accretions of cooperative unfolding/refolding units called foldons. They account for the unit steps in folding pathways (and other functions). During folding the stepwise formation of foldons is guided by pre‐existing structure in a sequential stabilization process. These two factors generate predetermined stepwise pathways that progressively assemble the foldon units of the target protein. Experimental results that have been interpreted differently, in terms of multiple independent pathways, are due to chance misfolding errors that cause different population fractions to fold differently and so appear to represent different pathways. The integration of these three concepts ‐ foldon units, sequential stabilization, and optional errors ‐ explains much of the vast literature on the folding behavior of protein molecules.
The Mre11/Rad50 complex and its roles in processing of DNA double‐strand breaks
Authors: Paull Tanya T. , Nicolette Matthew , Lengsefld Bettina
Published: June 2021

The repair of DNA double‐strand breaks by homologous recombination pathways requires that the ends of the DNA be processed into 3′ single‐strands. In E. coli this processing is catalyzed by the RecBCD enzyme, but functional orthologs of this complex do not appear to be present in eukaryotes. The Mre11/Rad50/Xrs2 (MRX) complex has been implicated in DNA processing during meiosis in budding yeast. In addition, the MRX complex has been shown to affect DNA processing events in vegetative cells, and recent evidence suggests that the human Mre11/Rad50/Nbs1 (MRN) complex also contributes to end processing in human cells as well.

It is clear from the work of many laboratories that Mre11/Rad50 does not act alone in processing DNA in vivo, but functions in concert with other conserved factors. The Sae2 protein in S. cerevisiae has been identified as a factor required for efficient DNA end processing in both mitotic and meiotic yeast cells. We have investigated the biochemical properties of the Sae2 protein in vitro and found unexpectedly that Sae2 possesses endonuclease activity on single‐stranded DNA and at single‐strand/double‐strand junctions. This is consistent with the proposed role of Sae2 in processing 5′ ends of DNA at double‐strand breaks in vivo. These and other experiments investigating the roles of both Sae2 and Mre11/Rad50/Xrs2 in DNA end processing will be discussed.
Following the synthesis, transport and translation of mRNA in living cells
Authors: Singer Robert H. , Shav‐Tal Yaron , Xavier Darzacq , de Turris Valeria , Gruenwald David , Shenoy Shailesh
Published: June 2021

In mammalian cells we developed a system using fluorescent proteins to analyze gene expression in real‐time, and to follow individual mRNPs. An array of genes coding for a functional mRNA that contains 24 repeats of the MS2 coat protein binding motif combined with the MS2 coat protein fused to GFP or YFP allowed us to analyze the kinetics of transcription in real time and to detect single molecules of RNA in live cells. In these studies we used photo‐bleaching of GFP‐labeled mRNAs and of a YFP‐polII fusion protein and photoactivation of paGFP labeled mRNA. Analysis of the complex process of transcription using fluorescent polymerase as well as fluorescent MS2 proteins provided an opportunity to model the kinetic steps of RNA synthesis. These results yield rate constants for each of the steps of promoter assembly, initiation and elongation. They demonstrate that transcription is inefficient and that polymerases can elongate each faster than thought, but can pause stochastically. When RNAs leave the transcription site, they can be followed as single molecules in the nucleus. The analysis demonstrated that subsequent RNA mobility was not directed, but governed by rules of simple diffusion. The transport through nuclear pores can be determined as well. Once in the cytoplasm, mRNAs diffuse but also can be directed to their destinations by virtue of a zipcode motif in the RNA. A zipcode binding protein (ZBP1) is essential for this localization and inhibition of translation. Beta‐actin mRNA can be localized to sites of active F‐actin polymerization in migrating fibroblasts or growing neurons. The movement of the RNA follows rules of diffusion, alternating with probabilistic motor‐driven events on microtubles as actin filaments.
Design Implications for an Ensemble‐based View of Proteins
Authors: Hilser Vincent J. , Whitten Steven T.
Published: June 2021

It has become clear, especially over the past decade, that conformational fluctuations in proteins can be dramatic, and that in their aggregate probability can account for a significant fraction of the total probability under native conditions. This poses potential challenges to design efforts. Namely, a designed ligand must either be compatible with one low energy conformations of the protein, or multiple higher energy conformations in order to result in a high binding affinity. Furthermore, because recent experimental results indicate that protein use the intrinsic fluctuations to facilitate their functional role, design processes that can manipulate the native state ensemble to favor or disfavor different conformations, can in principle facilitate the development of inhibitors or diagnostics. These possibilities can be explored using our ensemble‐based computational approach. I will discuss the latest results of our efforts to design peptide therapeutics based on these ideas.
Perceptions and Beliefs of the Role of Nutrition and Diet on Brain Health
Authors: Sharkey Joseph R , Sahyoun Nadine , Liu Rui , Laditka James N , Laditka Sarah
Published: June 2021

Evidence suggests that good nutrition may help to maintain brain health. Little is known about attitudes about nutrition and brain health among older adults. Fifty focus groups with 436 older participants were conducted in 9 states. Core questions from a standard guide were used in all locations. Participants also completed a 9‐item survey. Using grounded theory and constant comparison methods, transcripts were independently coded and analyzed for themes, issues, and beliefs. Participants reported limiting fat intake, and eating at least as many fruits and vegetables as other people they know. Participants reported hearing or seeing media messages about diet, especially dietary supplements. However, when asked, “How can we keep our brains healthy?” participants rarely mentioned a good diet. Participants emphasized that environmental barriers to good nutrition were common. Older Americans are interested in brain health. They are uncertain about their ability to follow recommended health behaviors. Public health messages should address the fact most participants did not associate nutrition with healthy aging.
Body mass index (BMI) and serum parathyroid hormone (sPTH) influence cortical bone in premenopausal women
Authors: Shapses Sue a , Sukumar Deeptha , Brolin Robert , Riedt Claudia , Ambia‐Sobhan Hasina , Gordon Christopher , Stahl Ted
Published: June 2021

Continuous endogenous sPTH excess has been shown to have catabolic effects on trabecular and cortical bone in normal‐weight women. In this study we examined the relationship between sPTH levels and BMI on cortical and trabecular volumetric bone mineral density (vBMD) and geometry in 87 normocalcemic obese and non‐obese women. Measurements were made by peripheral quantitative computed tomography at 4% and 38% from the distal tibia. In 43 premenopausal women, we used the median to categorize sPTH (pg/mL) as higher (70 ± 24) and lower (29 ± 4), and categorized BMI (kg/m2) into obese (39 ± 9) and non‐obese (26.5 ± 1.8). A similar analysis was performed for a postmenopausal population. Not surprisingly, we found that obesity increased cortical vBMD and content (BMC), geometric properties (area, thickness, circumference), polar moment of inertia and the stress:strain index in all women (P < 0.05), and also increased trabecular vBMD and BMC (P < 0.02) in the premenopausal women. We found an interaction (2‐way ANOVA) between BMI and sPTH for cortical BMC and geometric variables (p < 0.05) in the premenopausal women. Hence, the positive influence of BMI on cortical bone geometric properties is dependent on higher sPTH in the younger women. These findings suggest that the altered hormonal milieu associated with obesity is an important determinant in modulating the effects of excess weight on the skeleton.
Aged Garlic Extract Improves Vascular Function in Asymptomatic Individuals with Subclinical Atherosclerosis
Authors: Budoff Matthew Jay , Ahmadi Naser , Gul Khawar , Hajsadeghi Fereshteh , Usman Nudrat , Nuguri Vivek , Flores Ferdinand , Nasir Khurram , Naghavi Morteza
Published: June 2021

This study evaluates changes associated with Aged Garlic Extract (AGE) use in vascular reactivity during cuff‐occlusive reactive hyperemia by Digital Thermal monitoring (DTM) and progression of coronary artery calcium scores (CACS).

Methods: A placebo‐controlled, double‐blind, randomized trial, 65 (mean age: 60±9 years, 78% males) intermediate risk patients were enrolled and 60 completed the study protocol in which they either received four capsules of AGE (250 mg), Vitamin B12 (100mcg), Folic Acid (300 mcg), Vitamin B6 (12.5mg) and L‐Arginine (100mg) or placebo daily 1 year. Changes in vascular endothelial function, TMP AUC (Post‐cuff deflation Area Under the temperature Curve), index of thermal vascular reactivity, and CAC were assessed over 1 year follow up in both groups.

Results: At baseline there was no difference between both groups in their CAC scores (AGE: 291±50 vs. Placebo: 347±67, p=0.3) as well in TMP AUC (AGE: 291±50 vs. Placebo: 95±11, p=0.7). As shown in figure, Over 1 year follow‐up there was significantly higher improvement in TMP AUC and less progression of CACS with AGE compared to placebo independent of age, gender and risk factors. (TMP AUC: 2.9, 95%CI 1.5–4.7, p0.02) and (CAC: 0.71, 95% CI 0.53=0.9, p0.02).

Conclusion: This study suggests that AGE can improve thermal vascular function and reduce CAC progression in asymptomatic adults with intermediate risk.
Erythrocyte (RBC) copper chaperone for Cu,Zn Superoxide Dismutase (CCS) levels increase in copper‐deficient cattle
Authors: Hepburn Joeva Jade , Arthington John , Spears Jerry , Knutson Mitchell D.
Published: June 2021

Copper (Cu) deficiency can lead to hypochromic anemia and immune dysfunction. Bovine Cu deficiency is often undiagnosed due to limitations in current status indicators. We examined CCS as a potential Cu‐status indicator in the bovine, as it has been shown to increase in liver and RBCs of Cu‐deficient rats and mice. Weanling calves were randomized to one of 3 diets: Cu adequate (Cu+; n = 6), Cu deficient (Cu−; n = 8), and Cu deficient + manganese, Mn (Cu−+Mn; n = 7). After 190 days, liver Cu concentrations were 208 ± 39, 6.9 ± 1.5, and 5.2 ± 1.5 for the Cu+, Cu−, and Cu−+Mn groups, respectively. Western blot analyses indicate that RBC CCS levels were 63% higher (P < 0.05) in the Cu− calves compared to the Cu+ calves. To examine if RBC CCS levels respond to inflammation, 11 yearling heifers were given a Mannheimia haemolytica vaccination, and blood samples were taken for the next 4 days. Inflammation was confirmed by increased plasma ceruloplasmin concentrations, but RBC CCS levels did not change. We conclude that RBC CCS protein levels increase in Cu‐deficient calves and do not exhibit acute‐phase properties.
Alternative splicing of the Menkes Copper Atpase (Atp7a) gene; regulation during iron‐deficiency
Authors: Collins James F. , Hua Ping , Ranganathan P. N.
Published: June 2021

Atp7a is strongly induced during iron‐deficiency in rat intestine. The Atp7a gene is also induced in iron‐deprived IEC‐6 cells; actinomycin D treatment abolished this induction indicating transcriptional regulation. We thus sought to characterize the rat Atp7a gene promoter. We first identified the 5’ end of the Atp7a transcript. Results revealed 3 splice variants present at the 5’ end of the gene. All clones began with a sequence that mapped ~60 kb upstream of the rat Atp7a gene, which was homologous to exon 1 in mouse and human. We found that exon 1 was spliced to exon 1A (a novel exon), to exon 2 and to exon 3. The first 2 splice variants would encode a full length Atp7a protein, but the ex1/ex3 splice variant would encode a protein that was truncated by 69 amino acids (missing metal binding domain 1). Preliminary studies also suggest that these splice variants exist in mouse and human. A qRT‐PCR strategy was devised that allowed quantification of all 3 variants; results revealed ~7‐fold induction of all splice variants in the intestine of iron‐deficient rats. We further developed specific antibodies against the extreme N‐terminus of the rat Atp7a protein and showed strong induction of protein expression in iron‐deficient rats and unique immunolocalization in IEC‐6 cells. Our studies have thus revealed a novel variant of Atp7a that may display altered trafficking and function that could be important during iron‐deficiency.
Storage of retinyl esters in hepatic stellate cells: The role of LRAT in stellate cell lipid droplet formation
Authors: O'Byrne Sheila M , Libien Jenny , Kluwe Johannes , Schwabe Robert , Plczewski Krzysztof , Blaner William S
Published: June 2021

Hepatic stellate cells represent 5–8% of the total number of cells in a healthy liver and are responsible for the majority of the liver's retinoid stores. Retinyl esters are stored within the characteristic lipid droplets of these cells and comprises 40% of the droplets total lipid content along with triglycerides, phospholipids, cholesterol esters, cholesterol and free fatty acids. The amount of stored retinyl ester decreases progressively in liver injury when stellate cells are activated. We investigated the role of the enzyme Lecithin:Retinol Acyl Transferase (LRAT) and show that it is the most important enzyme for catalyzing the formation of retinyl esters in the majority of tissues in the body. LRAT knockout (KO) mice have undetectable levels of retinyl esters in their livers and depend on regular intake of retinoids to maintain their health. Livers examined by electron microscopy showed no lipid droplets in their hepatic stellate cells. Yet these mice showed no signs of fibrosis. showed earlier signs of hepatic fibrosis compared to wild type controls. Our studies indicate a novel role for LRAT in However, LRAT KO mice challenged with CCl4 the formation of lipid droplets in the hepatic stellate cell, and possibly in other cells, and this can make the mice more susceptible to experimentally induced liver fibrosis.