bluuebunny/crossref_metadata_2025 huggingface dataset:
FGF signaling in inner ear development
Authors: Mansour Suzanne L , Noyes C Albert , Li Chaoying , Wang Xiaofen , Hatch Ekaterina , Twigg Stephen R. F. , WIlkie Andrew O. M. , Urness Lisa
Published: June 2021

Inner ear development proceeds through phases of otic placode induction, otocyst formation and morphogenesis, and cell type specification. FGF signaling plays dosage sensitive roles in all of these processes. Fgfs3, 8 and 10 have essential roles in mouse otic placode induction. Microarray analysis identified targets of FGF signaling in otic ectoderm. We will discuss progress in determining their roles in otic development. The Fgf genes are also expressed during later phases of otic development, but later roles are obscured in global single and double KO mice by lethality and/or the sequelae to abnormal induction. To achieve temporal and spatial separation of the multiple functions of these signals throughout otic development, we studied embryos carrying various combinations of conditional Fgf alleles inactivated using different CRE drivers. We find that Fgfs3 and 8 provide redundant endodermal otic‐inducing signals. Initially, Fgf3 is required to maintain dorsal otic gene expression and initiate dorsal outgrowth and later, it functions redundantly with Fgf10 to control cochlear outgrowth. Increases in FGF signaling activity can also have adverse developmental consequences. We found that a mouse model of Muenke syndrome (FGFR3 P250R) has significant, predominantly low‐frequency hearing loss. This is accompanied by a cochlear supporting cell fate switch and excess outer hair cell development.
Enabling technology that allows the routine growth of cultured cells for applications in tissue engineering
Authors: Przyborski Stefan
Published: June 2021

Traditionally cells cultured in the laboratory grow on flat two‐dimensional (2‐D) plastic which is highly synthetic compared to the three‐dimensional (3‐D) environment in vivo. Consequently cells do not always function in a realistic fashion and there is demand to create models that enable cells to grow more like their native counterparts. Recreation of the complex interactions between neighboring cells is a step toward achieving this goal and producing engineered tissues in vitro. To address this issue, we have developed a polystyrene scaffold adapted for use in cell culture. The porosity of the scaffold is customized to within narrow tolerances optimal for 3‐D cell growth. Producing the scaffold in thin 200 micron membranes enables the entry of cells into its structure and it is of suitable thickness to allow cells sufficient exchange of gases and nutrients. Cell culture work has shown 3‐D cell growth and allows cells to form complex 3‐D interactions with their neighbours thus resembling real tissues. Cell viability is maintained at high levels and cells are not exposed to the un‐natural geometric stresses experienced when grown on flat surfaces. Cells also show enhanced ability to differentiate and respond to biochemical agents like their native counterparts in vivo. In summary, our technology can be used routine 3‐D cell culture and recreation of cell interactions similar to those found in tissues.
Molecular Cytogenetic Delineation of Chromosomal Structure and Complexity
Authors: Schwartz Stuart
Published: June 2021

There has been an explosion of technology in the entire field of genetics, but especially in the sub discipline of cytogenetics. Today we can not only visualize chromosomes microscopically, we can utilize FISH to look at any specific region of interest within a chromosome regardless of size, and to use array technology to look for subtle gains or losses of chromosome material. New syndromes and small changes leading to abnormal phenotypes have been identified utilizing this new technology. It has always been believed in cytogenetics that the most logical approach is to suspect the simplest route in the formation of abnormalities. However based on many studies using both FISH analysis and array studies, we have begun to learn that many abnormalities are more complex than originally envisioned. Utilizing an approach combining classical cytogenetics, FISH and array analysis, we have studied numerous abnormalities that now have been shown to be more complex than originally thought. These include: "balanced" translocations that have been shown to have deleted material; rearrangements thought to involve 2 or 3 breaks which actually have up to 12 different breakpoints; deletion which appear to be simple but have material within the deleted region present; ring chromosomes which are fragmented so that they contain discontinuous regions of the normal chromosomes; and inversions which can be shown to be insertions. These findings teach us several aspects concerning the formation of chromosome abnormalities. First, it shows that there are not always simple mechanisms leading to abnormalities and by studying these we may eventually better understand the underlying causes of structural abnormalities. Second, these complex rearrangements have an impact on the phenotype as genes initially thought to be involved may in fact not be.
Mesenchymal regulation of mineralization and bone mineral density in the jaw skeleton
Authors: Yu Jane , Jheon Andrew H. , Eames Brian F. , Schneider Richard A.
Published: June 2021

Proper bone mineral density (BMD) is crucial for skeletal function. Loss of BMD can lead to osteoporosis and fractures. From an evolutionary perspective, BMD helps couple morphology to the environment. For example, in aquatic mammals a higher BMD enables negative buoyancy during diving. BMD is governed by systemic and local factors throughout the life of an organism. On a cellular level, BMD is affected by osteoblasts, which secrete bone matrix that then mineralizes. During craniofacial development, osteoblasts are derived from neural crest mesenchyme (NCM). We use an avian chimeric system that exploits differences in morphology and maturation rates between quail and duck to investigate the role of NCM in establishing BMD. We unilaterally transplant quail NCM into duck, which maintains the host side as an internal control, and we analyze mineralization at the molecular and histological levels. We assess mineral deposition rates in quail, duck, and chimeric embryos by injecting fluorescent labels and by whole‐mount staining. We also measure BMD using DEXA. We find that duck have a slower rate and longer period of mineralization correlated with higher BMD, which is significant since duck use their bills for filter feeding and for diving. In chimeras, quail donor NCM alters the rate of mineral deposition. Thus, species‐specific differences in BMD have likely evolved through NCM‐mediated changes in mineralization.

Grant Funding Source

Departmental Startup
Evidence based medicine exercises in human gross anatomy
Authors: Rhodes Diana C. J.
Published: June 2021

This study evaluated the benefits of using evidenced based medicine (EBM) group exercises in a medical school gross anatomy course. Dissection groups formed, researched, and reported on "searchable clinical questions" centered on findings related to their cadaver. The quality of the research and the enthusiasm of the students for this optional project increased with time. A survey was administered to students in the pre‐EBM and EBM classes. Mann‐Whitney rank sum tests detected significant differences in responses between the two classes on 9 out of 17 items. Items with significant increases were ones that focused on the frequency with which students discussed the abnormalities found in cadavers and their agreement with statements attesting to their appreciation for the importance of anatomy in clinical medicine. Students with an overall more positive view of the gross anatomy dissection experience reported more time discussing the significance of the variations present in their cadavers. In conclusion, these optional EBM exercises were well received by students and seemed to positively affect their view of the importance of gross anatomy in clinical medicine. Supported by NIH‐NCCAM 1R25 AT003579‐01.
Very Small Embryonic Like (VSEL) Stem Cells: Characterization, Biological Significance, and Potential Applications
Authors: Ratajczak Mariusz
Published: June 2021

Bone marrow (BM) was for many years primarily envisioned as the "home organ" of hematopoietic stem cells (HSC). Augmenting evidence demonstrates, however that BM in addition to HSC also contains a heterogeneous population of non‐hematopoietic stem cells (non‐HSC). Recently our group identified in BM and other adult tissues including brain, kidney, pancreas, skeletal muscles, liver, spleen and lungs a population of very small embryonic like stem cells (VSELs) which express several markers characteristic for pluripotent stem cells (PSC) that are characteristic for epiblast/germ line‐ derived stem cells (Leukemia 2006;20:857‐869 & Leukemia 2007;21:297‐303). We hypothesize that VSELs are a population of epiblast‐derived cells that are deposited during early gastrulation in developing tissues/organs and play an important role in turnover of tissue specific/committed stem cells. In this context for example VSELs deposited in BM may give rise to long term repopulating HSC. We noticed that VSELs could be also mobilized into peripheral blood (PB) and their number of these cells circulating in PB increases during stress and tissue/organ injuries (e.g., G‐CSF administration, heart infarct and stroke). However, the number of these cells decreases with the age. Our recent data indicate that VSELs could be harnessed as a source of pluripotent stem cells for regenerative medicine. Finally, we envision that in pathological situations VSELs are involved in development of some malignancies (e.g., teratomas, germinal tumors, pediatric "small round blue cell" sarcomas).
Modulating the Modulator: Epigenetic Control Beyond the Histone Code
Authors: Zhou Ming‐Ming
Published: June 2021

Site‐specific post‐translational modifications of nucleosomal histones associated with chromatin structural change have been established to play an important role in epigenetic control of gene transcriptional activation or silencing. Growing evidence from recent studies argues that epigenetic gene transcription is governed not only by modifications of histones and DNA, but also by non‐coding RNAs in the cell nucleus. However, the molecular mechanistic understanding of the latter remains elusive. In this talk, I will present our structural and mechanistic analysis of the functional role of non‐coding RNA transcripts in histone H3 lysine 27 methylation mediated gene transcriptional silencing by Polycomb repressive complexes. I will discuss functional implications of the fundamental epigenetic interplay between RNA and histone lysine methylation in gene repression in human biology and disease.
Viruses, microRNAs and RNA Interference
Authors: Cullen Bryan R
Published: June 2021

In plants and invertebrates, RNA interference (RNAi) functions as a key innate immune response to viral infection. As a result, many plant viruses have evolved factors that can effectively inhibit RNAi and thereby facilitate virus replication. In contrast, efforts from several laboratories to identify an antiviral RNAi response in vertebrate cells infected by retro‐, flavi‐, or herpesviruses have so far failed to identify such a response, and artificial RNAi can be readily induced in virus infected human cells; i.e., viral infection of human cells does not result in a detectable inhibition of RNAi. In fact, rather than inhibiting RNAi, it is now clear that many DNA viruses encode microRNAs that are able to use the cellular RNAi machinery to inhibit the expression of cellular genes that have the potential to limit virus replication and/or that autoregulate viral gene expression. I will discuss our current understanding of the function of viral miRNAs and their potential contribution to viral pathogenesis, focusing on the identification of mRNA targets for the viral miRNAs encoded by the pathogenic human herpesviruses Herpes Simplex Virus 1 (HSV‐1) and Kaposi's Sarcoma‐associated Herpesvirus (KSHV).
Cellular quality control of protein aggregates
Authors: Bukau Bernd , Winkler Juliane , Tessarz Peter , Specht Sebastian , Mogk Axel
Published: June 2021

The aggregation of misfolded proteins in cells is controlled by chaperones including the small Hsp, Hsp70 and Hsp100. We have investigated the spatio‐temporal features of the aggregation of fluorescently labeled thermolabile proteins in heat‐treated bacteria and yeast cells. In bacteria, aggregates are almost exclusively deposited at the poles; upon cell division the old pole is the preferred site of aggregation. Membrane associated thermolabile proteins form aggregates along the longitudinal axis of the cell, and can serve as seed to relocalise aggregation‐prone proteins of the cytosol to the membrane. Upon heat treatment major components of the chaperone machinery of the cell almost completely relocalise to the aggregates. This occurs in a hierarchical fashion with distinct kinetics for each chaperone. Moreover, the relocalisation of some chaperones is prerequisite for the relocalisation of others. Mechanistic details of the disaggregation process by the ClpB/DnaK bi‐chaperone system will be provided. In yeast, the aggregation of thermolabile proteins initially occurs at multiple locations. During recovery typically 1‐2 prominent deposits are remaining, reminiscent of those identified recently by J. Frydman et al. We identified a role for some chaperones in the formation of these aggregates, indicating that chaperones are implicated in the cellular compartmentalisation of aggregates.
SIRT1, metabolism and calorie restriction
Authors: Bordone Laura
Published: June 2021

The Sirtuins NAD‐dependent family of deacetylases has been shown to mediate important diverse biological processes including extension of lifespan in multiple species including yeast, C. elegans, and Drosophila. Among the 7 mammalian sirtuins, SIRT1 regulates many important cellular processes, including cell survival, glucose homeostasis and fat metabolism. In particular, SIRT1 has been shown to mediate some of the effects of calorie restriction. In this talk I will discuss a mouse model that overexpresses SIRT1 that show phenotypes resembling calorie restriction. In addition, I will discuss the role that SIRT1 plays in metabolism, especially in the regulation of insulin secretion. These studies help to elucidate the major role that SIRT1 plays in metabolism and calorie restriction.
Clathrin‐mediated endocytosis: Dynamics and Dynamin
Authors: Schmid Sandra L. , Danuser Gaudenz , Loerke Dinah , Mettlen Marcel , Pucadyil Thomas , Yarar Defne
Published: June 2021

Dynamin is essential for clathrin‐mediated endocytosis (CME), but its exact function and mechanism of action remain unknown. Using TIR‐FM together with new particle tracking software and statistical analyses we identified 3 kinetically‐distinct subpopulations of eGFP‐labeled clathrin coated pits (CCPs): 2 short‐lived abortive species and 1 longer‐lived productive species. siRNA‐mediated knock‐down of dynamin‐2 and reintroduction of WT or mutant dynamin‐1 showed that it plays an early regulatory role and controls the rate of CCV formation. We have also developed fluid supported bilayers with excess membrane reservoir, (SUPER) templates, to assay vesicle formation and membrane fission. Under physiological conditions in the constant presence of GTP, dynamin cooperatively organizes into self‐limited assemblies at the necks of emergent vesicles and is sufficient to mediate membrane fission leading to continuous vesicle release. Together, these in vivo and in vitro approaches provide evidence that dynamin plays a dual role in CME, functioning at early stages to regulate CCV formation and at later stages to directly mediate membrane fission. Supported by NIH GM42455, GM73165, MH61345.
Coupling of initiation of DNA replication and establishment of sister chromatid cohesion in Xenopus egg extracts
Authors: Takisawa Haruhiko , Tanaka Hiroshi
Published: June 2021

Sister chromatid cohesion, which is mediated by a multi‐protein complex cohesin, is established during S‐phase. Recent studies have clarified that a cohesin ring concatenates sister DNA molecules, and that the cohesion is established during S‐phase through acetylation of cohesin subunit. However, little is known about how DNA replication is coupled to the establishment reaction. Since cohesin is loaded onto chromatin before the initiation of replication, the cohesion establishment reaction should involve the passage of replication fork through a cohesin ring. Accumulating evidences suggest that a fairly large complex called replisome progression complex (RPC) is formed at the replication fork. Yeast RPC contains essential components of DNA helicase (CMG complex) and highly conserved but non‐essential components such as Ctf4, Tof1 and Csm3 that are implicated in the establishment process. With Xenopus egg extracts, we found that Xenopus homolog of yeast Ctf4 (AND‐1) and Tof1‐Csm3 (Tim1‐Tipin) are assembled into Xenopus RPC depending on CMG complex formation, and they are required for proper establishment of the cohesion. Double depletion of AND‐1 and Tim1 lead to aberrant sister chromatid cohesion which is similarly induced by depleting cohesin from the extracts. These results demonstrate that AND‐1 and Tim1‐Tipin are key factors for connecting DNA replication and the establishment of cohesion.
How proteins find their genomic targets in vivo? Implications for epigenetic inheritance
Authors: Struhl Kevin
Published: June 2021

DNA‐binding proteins have a specificity of ~104 to 105, which corresponds to approximately 7‐8 bp of DNA sequence information. As a consequence, sequence motifs for DNA‐binding proteins should occur many times in genomes simply by chance expectation. This is true for E. coli (4 million bp), even more so for yeast (12 million bp), and dramatically so for human (3 billion bp). How do DNA‐binding proteins select their "correct" genomic targets in vivo while avoiding "incorrect" motifs that occur by chance? It turns out that organisms have different strategies. In E. coli, all motifs are bound, and selectivity is attained by DNA‐binding proteins with high specificity and evolutionary selection against motifs in non‐biological locations. In yeast, promoter regions are have DNA sequences that intrinsically disfavor nucleosome formation, thereby favoring binding (by a factor of 5‐30) to sites in promoter regions as compared to coding regions. In mammalian cells, the vast majority of motifs are unbound in vivo, and many bound sites lack motifs. This indicates that site selection in human cells requires cooperative binding, and a model for such cooperative binding in the absence of direct protein‐protein interactions is proposed. The issue of how proteins find their genomic targets underlies the issue of epigenetic inheritance, and I will discuss a model for position‐effect variegation and more generally how patterns of gene regulation are epigenetically inherited. In this model, epigenetic inheritance is mediated primarily through transcriptional regulatory proteins and transcription itself, with the role of chromatin being to reinforce the specific decisions made by these proteins. In particular, epigenetic inheritance is ultimately reduced to a question of binding affinities for different genomic regions, and the combined contributions of the various protein‐DNA and protein‐protein interactions.
Mechanisms of protein translocation across membranes
Authors: Rapoport Tom A
Published: June 2021

Many proteins in bacteria are transported during their biosynthesis across or are integrated into the plasma membrane, a process that is similar to protein translocation across endoplasmic reticulum (ER) membrane in eukaryotes. Transport occurs through a protein‐conducting channel that is formed from a conserved heterotrimeric membrane protein complex (SecY or Sec61 complex). The channel associates with different partners in different translocation pathways. In bacteria, the SecY channel can associate with the translating ribosome (co‐translational translocation) or with the cytoplasmic ATPase SecA (post‐translational translocation).

The crystal structure of an archaeal SecY complex shows the architecture of the closed channel. The structure suggests that a single SecY copy forms the translocation pore, a prediction that was confirmed for SecA‐mediated translocation by disulfide bridge crosslinking experiments. However, translocation appears to be mediated by oligomers of the SecY complex; nucleotide‐binding fold 1 (NBF1) of SecA interacts with a non‐translocating SecY copy, while other domains of SecA "push" the polypeptide chain through a neighboring SecY copy.

A recently determined crystal structure shows one copy of SecA in its transitions state of ATP hydrolysis bound to one copy of SecY complex. The latter corresponds to the translocating SecY copy. Both SecA and SecY undergo major conformational changes upon interaction. The structure suggests mechanisms for how SecA moves polypeptides through the SecY channel. In addition, the data reveal that SecA binding opens the lateral gate of SecY for signal sequence intercalation, and induces plug movement. These results indicate that the channel is opened for translocation in two distinct steps, induced by SecA and signal sequence binding.
Chronic food intake patterns among Filipino women: Trends and associations with aging‐related disabilities
Authors: BORJA JUDITH B. , DUAZO PAULITA L. , LEE NANETTE R. , ADAIR LINDA S.
Published: June 2021

Using 24‐hour food recall data, we examined trends in food intake patterns of 1548 women (aged 38‐71) for over 2 decades (7 surveys between 1983‐2005). We identified those who tracked or maintained the same food group intake rankings over time, and assessed how these patterns influence disabilities occurring with age i.e., difficulty in performing basic functions, physical limitations, and cognitive impairment. Evident of the nutrition transition occurring in the Philippines, we observed a decrease in vegetable and high‐fiber grain consumption, and an increase in meat and sugar consumption over time. We also noted market‐driven changes such as an increase in noodle consumption starting in the mid 80s reflective of the proliferation of cheap instant noodles, and a downward trend in corn as basic staple. Multivariate models were run to assess the diet‐aging relationship, controlling for education, morbidity status, calorie intake, physical activity, and other maternal and household characteristics. Our findings show that tracking of certain food groups influence disabilities. For example, predominantly being in the highest vegetable intake quartile (relative to each other)protect women against having physical limitations at later ages (Odds Ratio: 0.60 [95% CI: 0.42, 0.86]). This merits attention given the secular downward trend in vegetable consumption for this women.

NIH Grant No. 1R03TW008133‐01
Linkages between hepatic vitamin A storage and metabolism and hepatic steatosi
Authors: Wongsiriroj Nuttaporn , Jiang Hongfeng , Piantedosi Roseann , Blaner William S.
Published: June 2021

Mice lacking lecithin:retinol acyltransferase (LRAT) and cellular retinol‐binding protein, type I (CRBPI) (Lrat−/−/CrbpI−/− mice) fed a chow diet or the AIN‐93M diet accumulate no hepatic retinyl ester stores and have low hepatic retinol levels compared to matched wild type, CrbpI−/− and Lrat−/− mice. Further study showed that CrbpI−/− and Lrat−/−/CrbpI−/− mice spontaneously accumulate significantly more (> 2‐fold) hepatic fat than wild type mice. This suggested a link between low hepatic vitamin A levels and development of fatty liver. Consequently, we assessed gene expression levels by qRT‐PCR for a number of vitamin A responsive genes and a number of genes involved in mediating hepatic fat metabolism. Cyp26A1 and RARβ, which are responsive to retinoic acid, were upregulated in livers of CrbpI−/− and Lrat−/−/CrbpI−/− mice. Genes involved in hepatic fat metabolism, including PPARγ and PPARβ/δ, were also altered. We suggest that the impaired ability of CrbpI−/− and Lrat−/−/CrbpI−/− mice to store vitamin A results in increased retinoic acid synthesis, which elevates retinoic acid responsive gene expression and dysregulates hepatic fat metabolism and accumulation. (Supported by R01 DK079221)
Zinc‐regulated ZnT1 (SLC30A1) and Glucocorticoid‐regulated ZnT2 (SLC30A2) Influence Zinc Efflux from Pancreatic Acinar Cells
Authors: Guo Liang , Cousins Robert J.
Published: June 2021

The exocrine pancreas plays an important role in the maintenance of the whole body zinc homeostasis by regulating zinc excretion into the intestine. We identified two zinc transporters, ZnT1 and ZnT2, as potentially being involved in zinc efflux from the exocrine pancreas. Dietary zinc restriction in mouse decreased the zinc concentration in pancreatic cytoplasm and zymogen granules and was correlated to decreased expression of ZnT1 and ZnT2. In contrast, in oral zinc gavage studies increases in pancreatic zinc contents and up‐regulated ZnT1 were observed. Regulation of these two zinc transporters were also examined in vitro with AR42J acinar cells. ZnT2 was found to be stimulated by the glucocorticoid analog dexamethasone during AR42J differentiation. We also identified that the dexamethasone‐induced ZnT2 expression in mice was accompanied by a reduction of pancreatic zinc content. Knocking‐down ZnT2 resulted in increased cytoplasmic zinc and decreased zymogen granule zinc further establishing ZnT2 as the transporter mediating the sequestration of zinc into zymogen granules. In pancreatic acinar cells, ZnT1 controls zinc efflux through the apical membrane in a zinc‐dependent manner, while ZnT2 controls zinc sequestration into secretory granules to influence entero‐pancreatic zinc circulation. Supported by NIH Grant DK 31127.

Grant Funding Source

NIH
Early consumption of blueberry diet protects against sex steroid deficiency‐induced bone loss in adult female rats
Authors: Ronis Martin J , Chen Jin‐Ran , Lazarenko Oxana P , Badger Thomas M
Published: June 2021

We studied the effects of blueberry consumption in early development on bone loss in ovariectomized (OVX) adult female rats. Weanling female rats were fed AIN‐93G diets supplemented with 10% whole blueberry powder from PND 21 to PND34 (short‐term group), or PND21 to PND81 (chronic group). The short term group was fed AIN‐93G after PND34. Animals were sacrificed at 1 week or 3 weeks following OVX on PND60. In both blueberry groups, tibial total and trabecular bone mineral density (BMD) were higher following 3 weeks of OVX (p < 0.05), and total bone mineral content (BMC) were higher (p < 0.05) 1 week post‐ OVX compared to control casein AIN‐93G fed animals. In the chronic group, but not the short term group, tibial total and trabecular BMD were also higher after 1 week of OVX. Interestingly, cortical BMD, cortical thickness and total BMC were all higher in the short term blueberry group after 1 week of OVX (p < 0.05), but there are no differences in cortical thickness and total BMC after 3 weeks of OVX as compared to control casein groups. Higher BMD in both blueberry groups was tightly associated with elevation of the serum bone formation marker osteocalcin (p < 0.05). These results indicate preventive effects against sex steroid deficiency‐induced bone loss in adults associated with blueberry consumption in early life may result from long term increases in bone formation. Supported in part by ARS CRIS #6251‐51000‐005‐03S.
Effects of parathyroid hormone on porcine mesenchymal stem cell differentiation
Authors: Mahajan Avanika , Stahl Chad H.
Published: June 2021

Parathyroid hormone (PTH) effects on mesenchymal stem cell (MSC) differentiation are dependent on the timing of treatment and culture conditions. In this study, we have characterized the effects of either continuous (CONT) or intermittent (2h/day, INT) PTH treatment on porcine MSC (pMSC) differentiation in media designed to promote proliferation. After 6d of culture, proliferation and gene expression were analyzed and alkaline phosphatase (ALP) activity was determined. The expression of Runx2, ALP, PPARG, LPL and PTHR were increased (P < 0.05) with either PTH treatment. Either PTH treatment also significantly increased (P < 0.05) the percentage of ALP+ cells (10% and 25% for CONT and INT compared to the control, respectively), and decreased (P < 0.05) cell proliferation (20 and 25% compared to the control for CONT and INT, respectively). We have previously shown that 1,25(OH)2D3 stimulates adipocytic differentiation of pMSC and wanted to examine the interaction of PTH with 1,25(OH)2D3 treatment. The ability of PTH to stimulate osteoblastic differentiation was completely inhibited by co‐treatment with 1,25(OH)2D3 as seen by decreased (P < 0.05) ALP+ cells and reduced (P < 0.05) expression of Runx2, ALP and PTHR by greater than 2 fold. These data suggest that PTH stimulates differentiation of pMSC. Also, 1,25(OH)2D3 inhibits the ability of PTH to stimulate MSC toward osteoblast differentiation.
Dietary protein holds positive and negative effects on bone health in rats
Authors: Thorpe Matthew P , Valentine Rudy J , Moulton Christopher J , Wagoner Johnson Amy J , Evans Ellen M , Layman Donald K
Published: June 2021

The effect of high protein intakes on bone health is controversial, with both positive and negative effects reported. Human studies are clouded by correlations of protein, calcium and other factors. To control these, 48 Sprague‐Dawley rats aged 52d were randomized to 10 wk diets: PRO: 35% energy as casein, 40% as carbohydrate; CHO: 15% casein and 60% carbohydrate. All other nutrients were matched. Measures included serum IGF‐1 by RIA, mechanical strength of the femur midshaft by 3‐point bend test, bone density by dual x‐ray absorptiometry and, in a random subsample (n=16), trabecular microarchitecture by micro‐computed tomography. Energy intakes did not differ. Respective PRO and CHO M±SD were: trabecular spacing, 232±6, 224±4 um; trabecular number, 2.97±0.16, 3.09±0.09 per mm (p=0.08); trabecular thickness, 120±4, 124±5 um (p=0.08); IGF‐1, 266±61, 204±62. No difference in mechanical strength was observed; however after covarying for IGF‐1 protein had both a negative effect (ß=‐0.37) on work to failure, and an opposing positive effect (ß=0.43) mediated by IGF‐1. Opposing effects were roughly equal in magnitude, producing no net effect on bone strength; however the PRO diet was detrimental to microarchitecture, increasing the average space between trabecular scaffolds. Results are consistent with the hypothesis that protein has both positive effects, mediated by IGF‐1, and negative effects on bone health.

Grant Funding Source

University of Illinois Division of Nutritional Science Margin of Excellence Award
Human bioavailability of lunasin after consumption of soy protein products
Authors: Gonzalez de Mejia Elvira , Dia Vermont P , Torres Sofia , de Lumen Ben O. , Erdman John W.
Published: June 2021

Lunasin is a bioactive peptide with anticancer properties originally isolated from soy. The objective of this study was to assess the presence of lunasin in blood of men fed soy protein products. Five healthy male subjects age 18‐25 were fed with 50 g of soy protein for 5 days, blood was taken at the beginning of the study and at 30 min and 1 h after soy protein ingestion. Lunasin was isolated from plasma by ion exchange chromatography using magnetic beads and quantified by ELISA. At day 5, lunasin in plasma ranged from 50.2 to 110.6 ng/ml (average, 66.0 ± 25.4 ng/ml) after 30 min and 33.5 to 122.7 ng/ml (average, 71.0 ± 32.8 ng/ml) after 1 h. An average 4.5% lunasin absorption (range 2.2‐7.8%) was estimated from the total lunasin ingested. MALDI‐TOF profiling showed a 5 kDa peptide similar to synthetic lunasin in plasma samples of subjects fed soy protein while it was absent in the baseline plasma samples from the same individuals. LC/MS‐MS analysis also showed the presence of amino acids from lunasin in plasma samples after soy intake but not in plasma samples after a wash out period and before soy consumption. The results of the present study suggest that lunasin is bioavailable in humans; an important requirement for its anticancer potential.

Supported by ACES Global Connect, University of Illinois and CONACYT Mexico
Net protein anabolic response is not altered during hyperaminoacidemic (hyperAA) clamp in men with Type 2 Diabetes (T2D)
Authors: Bassil Maya , Marliss Errol B. , Morais José A. , Gougeon Réjeanne
Published: June 2021

Reduced 24h net protein balance in hyperglycemic T2D was explained by less protein accretion during hyperinsulinemic euglycemic, isoaminoacidemic (isoAA) clamps. We tested whether protein anabolism would worsen with glycemia at 8.0 mM and AA at iso (fasting) or hyper (postprandial). Eight T2D men (57±2 y; BMI: 34±2; FPG: 8.4±0.6mM) underwent hyperinsulinemic (600 pM), hyperglycemic clamps with glucose and leucine kinetics measured at 2 steady‐states: isoAA and hyperAA. Results are compared to 10 T2D and 10 lean nondiabetic men studied euglycemic, isoAA and 9 studied hyperglycemic, hyperAA.

In T2D 1) Leucine flux was higher (4.3±0.1 vs. 3.1±0.2 micromol/FFM.min) but net protein balance not different (0±.03 micromol/FFM.min) with isoAA when glycemia was at 8.0 vs. 5.5; net balance was less than in lean (0.25±0.02); however response to hyperAA was as in lean (0.9±.09) 2) endogenous glucose production was less suppressed (1.7±0.4 vs. 0.7±0.2mg/FFM.min) and glucose uptake higher (4.8±.6 vs. 3.6±0.3 mg/FFM.min) with glycemia at 8 vs. 5.5 and with hyperAA. 3) hyperAA did not lower glucose metabolic clearance rate (3.3±0.3 vs. 3.6±0.4 ml/FFM.min), as in lean (9.3±0.7 vs. 5.7±0.4). Thus, protein anabolic response to postprandial AA in T2D is not altered despite indication of insulin resistance of protein. (CIHR)
Beverage Consumption Patterns in the U.S. Population
Authors: Storey Maureen L , Anderson Patricia A
Published: June 2021

Beverages play an important role in hydration and beverage choices have changed over the last few years. Therefore, it is important to understand beverage consumption patterns. Using data from the 2003‐2004 and 2005‐2006 National Health and Nutrition Examination Survey (NHANES), we examined beverage consumption for males and females aged 6‐11, 12‐19, 20‐39, 40‐59, and 60+ years and compared the results with those we published previously using NHANES 1999‐2002 data. Appropriate survey weights were used to calculate average consumption of fruit juices, coffee, tea, milk products, regular fruit drinks/ades, diet fruit drinks/ades, regular carbonated soft drinks (RCSD), and diet carbonated soft drinks (DCSD). Preliminary results show that beverage consumption patterns have changed. On average, RCSD, milk products, and fruit juice consumption have decreased, while consumption of diet fruit drinks/ades has increased across most age groups. Many of the consumption pattern differences between race/ethnicities found using the 1999‐2002 data persist.

This study was supported by the American Beverage Association.
Netrin‐1 Over Expression Protects Kidney From Ischemia Reperfusion Injury By Suppressing Apoptosis
Authors: Ramesh Ganesan , Pays Laurent , Mehlen Patrick , Reeves Brian William , Wang Weiwei
Published: June 2021

Netrin‐1, a diffusible laminin‐related protein, is highly expressed in the kidney. The pathophysiological roles of netrin‐1 in kidney are unknown. To address directly, we used transgenic mice which over express chicken netrin‐1 in the kidney. Over expression was confirmed by real time RT‐PCR and western blot analysis. 8 weeks old WT and transgenic mice were then subjected to 26 minutes of renal ischemia followed perfusion for 72hrs. WT mice developed severe renal dysfunction by 24hrs whereas netrin‐1 transgenic mice were showed better renal function at 24hr (BUN: 56±3 vs. 151±10 and creatinine: 0.58±0.1 vs. 1.2±0.1, p<0.005). Functional improvement was associated with better preservation of morphology and peritubular capillaries, reduced inflammation, and reduced oxidative stress in transgenic mice kidney as compared to WT mice. In addition, both basal and reperfusion induced cell proliferation was dramatically increased in transgenic as seen by Ki67 staining. Interestingly, ischemia reperfusion induced a large increase of apoptosis in WT mice but not in netrin‐1 transgenic mice (319±16 vs. 1±0.3, p<0.0001). These results suggest that netrin‐1 protects renal tubular epithelial cells against ischemia reperfusion induced injury by increasing proliferation and suppressing apoptosis.
Paradoxical roles of beta‐catenin in Fas and TNF‐alpha‐mediated hepatocyte death
Authors: Nejak‐Bowen Kari D , Tan Xinping , Monga Satdarshan S
Published: June 2021

Suppression of β‐catenin signaling in developing and regenerating hepatocytes causes a basal increase in apoptosis. However, the mechanism by which this cell death occurs is currently unknown. Here, we demonstrate that loss of β‐catenin exacerbates Fas‐mediated apoptosis, but protects against TNF‐α mediated apoptosis. Mice injected with either Jo‐2 antibody (Fas pathway) or galactosamine (GalN) and lipopolysaccharide (LPS) (TNF‐α pathway) were monitored for morbidity and examined morphologically and histologically upon sacrifice. β‐catenin conditional knockout (KO) mice show a dramatic increase in liver injury and a decreased survival upon Jo‐2 administration as compared to their wild‐type (WT) counterparts, with extensive loss of architecture and hepatocyte apoptosis. Conversely, the KO mice are refractory to GalN/LPS treatment: the livers are protected from damage and apoptosis, and the mice show decreased morbidity. Analysis of liver enzymes confirms the presence of massive injury in WT but not in KO mice. Finally, levels of the anti‐apoptotic transcription factors GSK‐3β and NF‐κB are elevated in KO mice as compared to WT both before and after insult, suggesting a mechanism for this protection. In conclusion, our results demonstrate an essential role for β‐catenin in hepatocyte survival.
Treatment of Muscle Stem Cells with N‐acetylcysteine Increases Cardiac Repair
Authors: Drowley Lauren , Okada Masaho , Keller Bradley , Tobita Kimimasa , Huard Johnny
Published: June 2021

We have previously shown that muscle‐derived stem cells (MDSCs) significantly reduce negative cardiac remodeling compared to myoblasts after myocardial infarction (MI). One major issue with cellular transplantation for cardiac repair has been the poor survival of the transplanted cells. Cells that have increased survival under stressful conditions could improve functional repair, and MDSCs survive oxidative stress at significantly higher levels than myoblasts. We hypothesized that by altering the level of antioxidants in the cells, we could influence cardiac repair. To examine this, we reduced levels of the antioxidant glutathione with diethyl maleate (DEM). We also treated cells with N‐acetylcysteine (NAC), which is a direct antioxidant and increases glutathione. We examined in vitro characteristics as well as cardiac repair after a MI. Treatment with NAC significantly improved cell survival after inflammatory and oxidative stress compared to DEM and untreated cells. Cardiac function showed a trend that NAC groups improved contraction above control, and this was significant when compared to DEM and saline. The NAC group had higher angiogenesis and decreased scar tissue formation compared to the other groups. Treatment with NAC is simple, inexpensive, and does not cause a permanent transformation of the cells, making this option extremely attractive as a potential clinical treatment for cell therapy.
CD44v6 mediates neutrophil clearance from the apical surface of the intestinal epithelium
Authors: Louis Nancy A. , Lee Winston Y. , Lockard Catherine M. , Parkos Charles A.
Published: June 2021

Neutrophil (PMN) infiltration and accumulation are pathognomonic of multiple inflammatory processes of the bowel, including ulcerative colitis, however, the signals governing PMN clearance remain to be defined. Our goal was to identify inflammation responsive epithelial ligands regulating PMN transepithelial migration and clearance. A panel of antibodies was generated against interferon‐gamma (IFNγ)‐stimulated T84 intestinal epithelial cell membranes. Once such antibody, GM35, recognizes an IFNγ‐responsive antigen and reduces PMN transepithelial migration in a time and dose dependent manner. Immunohistochemical staining and selective surface biotinylation localize the GM35 antigen to the apical surface of T84 cells, and further work indicates that the GM35 epitope is cleaved from T84 cells during PMN transepithelial migration. Antigen immunoprecipitation and confocal microscopic analysis of immunofluorescently labeled T84 cells indicate that GM35 binds a member of the CD44 family, while microsequencing more specifically identifies the GM35 ligand as the CD44 variant CD44v6. These studies identify CD44v6 as a candidate mediator of intestinal epithelial PMN clearance and a potential target for therapeutic intervention. This work is supported by R01 funding (Parkos), an NIH Career Development Award (Louis), and an Emory University Research Council seed grant.
Novel Stem/Progenitor Populations: Implications for Cell Therapy
Authors: Keating Armand
Published: June 2021

Mesenchymal stromal cells (MSCs) are increasingly used in human cell therapy protocols. Two major issues, however, have yet to be addressed with this intriguing cell population: the likelihood of true transdifferentiation and isolation of MSC stem cells. To date, neither has been convincingly demonstrated. We have attempted to address both issues here. The demonstration of self‐renewal and differentiation ‐ key criteria for stem cell characteristics ‐ have yet to be accomplished. To address this issue, we rigorously generated true clones from the highly proliferative population of MSCs derived from non‐transformed human umbilical cord perivascular cells (HUCPVCs) that share the properties and immunophenotype of bone marrow‐derived MSCs. We demonstrated a hierarchy of differentiation potential along adipocytic, osteogenic, chondrogenic, myogenic and fibroblastic lineages. Future studies will be aimed at characterizing clones for the regeneration of specific tissues.

To address the issue of transdifferentiation, we employed a novel in vitro system in which embryonic rat cardiomyocytes are co‐cultured with adult murine MSCs derived from transgenic animals carrying the cardiac‐specific myosin heavy chain promoter driving green fluorescence protein (GFP). We found that some cells expressed GFP and, additionally, had acquired several cardiac‐specific markers but nevertheless had retained all previously assayed MSC determinants, suggesting the generation of a unique cell population that we also showed had not arisen from cell fusion. Furthermore, electrophysiological studies revealed that these unique GFP+ cells do not generate action potentials or ionic currents typical of cardiomyocytes, indicating that the cells are functionally, stromal and not cardiomyocytes. The presence of this population in vivo was confirmed in studies with a murine experimental acute myocardial infarction model. The implications of these findings on the nature of tissue regeneration with exogenous MSCs will be discussed in detail.
Advanced multimedia applications for teaching anatomy: a comparison of software used to generate 3D anatomical models
Authors: Midgley Michael , Wilson Timothy D
Published: June 2021

Reform in medical education has resulted in decreasing anatomy instructional hours and increasing emphasis on medical imaging and clinical application. Recent trends necessitate exploration of alternative teaching methods that augment traditional lecture and cadaveric dissection experiences. Currently, there are a variety of advanced multimedia applications emerging as potential teaching tools. The aim of this study is to compare and contrast the development of 3D anatomical models using two different segmentation and rendering software programs: Amira and OsiriX. Models of the upper limb will be assessed on ease of creation and applications within learning environments. With Amira (v.4.1), accurate reconstructions of anatomical structures are generated from cryosection images obtained from the Visible Human Project. Both manual and semi‐automatic techniques are used to highlight and segment structures of interest. Through stereoscopic projection, the highly detailed model may enhance understanding of complex spatial relationships in lecture or laboratory settings. In contrast, OsiriX is a free program that provides an intuitive interface for managing and viewing cross‐sectional CT and MRI data. It is capable of quickly constructing 3D models through an automatic process. Subsequently, models can be integrated into presentation software.

Grant Funding Source

Internal
Development of a 4‐dimensional model of the human heart using cardiac CT data
Authors: Yim Harold , Kiaii Bob
Published: June 2021

A new and interactive model for presenting human anatomy may allow students, instructors, and clinicians to better understand and integrate knowledge of the body. The aim is to create an anatomically correct three‐dimensional (3‐D) model of a human heart that will be able to rhythmically contract with time, resulting in a four‐dimensional (4‐D) heart model. ECG‐gated cardiac computed tomography (CCT) scans taken at ten time phases (5%‐95%, at 10% intervals) were processed by 3‐D visualization and modeling software (AMIRA). Using manual and automatic segmentation techniques, voxels with gray scale values representing different parts of the heart anatomy were selected and compiled to create the model. A model was constructed for each heartbeat phase, and all phase models were consolidated into one dynamic moving model. The resulting stereoscopic 4‐D model will retain the structure of the original anatomy. An effort will be made to detect any pathological anomalies in the patient using the model. It will feature interactive and explorative capability, eg. zooming in/out, addition and removal of different areas of the heart anatomy, and alteration of model opacity for better viewing of inner compartments.

Grant Funding Source

Internal
Modelling the nuclei and pathways of the limbic system using the BrainTower functional neuroanatomy system
Authors: Greene John
Published: June 2021

Students studying the limbic system need their knowledge to progress from recognizing the morphological features to understanding how particular nuclei and cortical regions are interconnected. This knowledge is difficult to obtain by looking at traditional anatomical models or sections of preserved brains so an interactive aid called the BrainTower® has been developed. It is built from plates representing sections of the nervous system (relating to axial MRI images). These plates can be drawn on, printed or etched to provide further detail and are perforated to show the positions of particular pathways. Some plates are interconnected by wires that run in the positions of functional columns and divisions of the reticular formation. Components, representing nuclei or cortical regions, are connected to the plates and wires and interconnected by monofilaments depicting particular pathways. BrainTower® is easy to clean ‐ so it can be used alongside preserved specimens. Construction uses a few simple processes so students can quickly acquire the skills to build their own models. Work is proceeding to evaluate the role of the model in undergraduate and postgraduate teaching.