bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial signaling through Nod2/GSK3b protects the small intestine against ischemia‐reperfusion induced injury in mice
Authors: Jobin Christian , Kim Ji Won
Published: June 2021

Ischemia/reperfusion (I/R) injury represents an important mucosal injury and is one of the leading causes of death in critically ill patients.

Aim

To investigate the impact of bacteria/Nod2 signaling in I/R‐mediated injury.

Methods

The human colonic HCT‐116 (expressing endogenous Nod2) cells and rat IEC‐6 cells infected with Ad5Nod2 viral vector were used to study muramyl dipeptide (MDP; 50 ug/mL) signaling. GSK3β (Ser9), histone 3 (Ser10), β‐catenin phosphorylation levels were determined by Western blot or immunohistochemistry analysis. Germ‐free (GF), conventionalized (CNV), Nod2 knock‐out (Nod2−/−) and wild‐type (WT) mice (C57BL/6; 6–8 mice/group) were subjected to intestinal I/R by partial occlusion of the 2 distal branches of the superior mesenteric artery for 60 min, followed by 4 h reperfusion. Proliferation was determined using Ki67 IHC staining.

Results

GSK3β phosphorylation and nuclear accumulation of β‐catenin is induced within 60 min in MDP‐stimulated HCT‐116 cells and Ad5Nod2‐infected IEC‐6 cells. I/R‐induced ileal injury worsen in GF mice compared to CNV mice (1.5±0.22 vs 0.7±0.20, p<0.05) which associated with shorten crypt/villin length (11.9±0.19 μm vs 18.1±0.21 μm, p<0.01). Importantly, I/R‐induced ileal injury is more pronounced in Nod2−/− mice compared to WT mice (1.9±0.23 vs 0.9±0.29, p<0.05), with concomitant shorten crypt/villi length (11.8±0.17 μm vs 18.0±0.16 μm, p<0.05) and decreased IL‐6 mRNA accumulation (18.5±5.7 vs. 54.7±9.4). This increased injury response was accompanied by reduced Ki67 staining, GSK3β and H3 phosphorylation.

Conclusion

Bacteria/Nod2 signaling to GSK3β/β‐catenin pathway represents an important host response to the microbiota and may protect the small intestine against I/R‐induced injury.
Proximal tibia shape and locomotion
Authors: Sylvester Adam D
Published: June 2021

Since the size and shape of articular surfaces are correlated with habitual postures and movements, joint surfaces can provide important clues about animals' locomotor behaviors. Functional analyses of joint morphology are complicated, however, by the fact that variables other than locomotion influence joint morphology.

This presentation considers body size and alignment issues in the context of the primate knee, with particular attention to the morphology of the proximal tibia. Body size has been argued to directly affect joint morphology, with larger animals having flatter surfaces to accommodate their size. The nature of this proposed allometric relationship, however, is not clear. While some researchers have argued for a relationship between body size and articular curvature, I argue that joint morphology is primarily due to locomotor differences which are themselves highly correlated with body size. Thus any apparent allometric relationship is secondary to the effect body size has on locomotion.

Another critical issue for functional investigations is the alignment of specimens prior to measurement and analyses. Many analyses align specimens mathematically (e.g. Procrustes) or use functionally meaningless anatomical orientations. Because such alignments do not reflect equivalent in vivo orientations, they can not provide reliable bases for elucidating interspecific functional differences.
Role of the reprogramming factors in the induction of pluripotency
Authors: Sridharan Rupa , Tchieu Jason , Mason Mike , Kuoy Edward , Yachechko Robin , Plath Kathrin
Published: June 2021

Transcription factor induced reprogramming of somatic cells to induced pluripotent stem (iPS) cells, that are molecularly and functionally similar to ES cells, can be achieved by the expression of Oct4, Sox2, Klf4 and cMyc. We study the mechanism of the remarkable changes that accompany this process, including resetting of the somatic cell transcriptome, epigenome and, in female cells, the reactivation of the silent X‐chromosome. In a recent study, we have found that Oct4, Sox2, Klf4, and cMyc cooperate in binding to activate the embryonic housekeeping program, followed by the specific binding of Oct4, Sox2 and Klf4 to genes encoding pluripotency regulators leading to the self‐sustained, pluripotent state. Thus there are distinct temporal steps to the resetting of the transcription program. Currently, we are investigating the epigenetic roadblocks that impede the conversion of partially reprogrammed cells to complete pluripotency.

Grant Funding Source: CIRM
Epigenetic control of neural crest specifier genes by histone demethylases jmjd2A
Authors: Strobl‐Mazzulla Pablo Hernan , Bronner‐Fraser Marianne
Published: June 2021

The neural crest (NC) is a multipotent, migratory stem cell‐like population that is able to differentiate in many cell derivatives including the peripheral nervous system and facial skeleton of vertebrate embryos. Many studies have been focused on transcriptional regulation underlying NC formation, but less is known about epigenetic influences. Accordingly, we find unexpectedly dynamic epigenetic modifications are critical for proper temporal control of neural crest gene expression in vivo. The histone demethylase, JumonjiD2a (JmjD2A), is expressed in the forming neural folds and its loss of function using morpholinos causes a dramatic down‐regulation of neural crest specifier genes. Importantly, chromatin immunoprecipitation reveals stage‐specific interactions of JmjD2A with regulatory regions of neural crest genes as well as associated stage‐specific modifications in the methylation states of critical lysine residues. The results clearly show that epigenetic events are important for regulating the chromatin structure on neural crest genes, providing the first in vivo evidence for an epigenetic modification directly controlling a critical developmental event in the vertebrate embryo.

Grant Funding Source: USPHS grant HD037105
Diet, dietary adaptations and feeding biomechanics in Australopithecus africanus
Authors: Strait David Samuel
Published: June 2021

Australopithecus africanus is an early hominin (i.e., human relative) believed to exhibit stress‐reducing adaptations in its craniofacial skeleton that may be related to the consumption of resistant food items using its premolar teeth. Finite element analyses simulating molar and premolar biting tested the hypothesis that the cranium of A. africanus is structurally more rigid than that of Macaca fascicularis, an Old World monkey that lacks derived australopith facial features. Finite element models of crania of these species were subjected to isometrically scaled loads, permitting a direct comparison of strain magnitudes. Moreover, strain energy in the models was compared after results were scaled to account for differences in bone volume and muscle forces. Results indicate that strains in certain midfacial skeletal regions are higher in M. fascicularis than in A. africanus, and that the A. africanus cranium is, overall, more rigid than that of M. fascicularis during premolar biting. This is consistent with the hypothesis that this hominin may have periodically consumed large, hard food items. However, the strain energy data suggest that the A. africanus cranium is marginally less rigid than that of the macaque during molar biting, raising the possibility that results are being influenced by the allometric scaling of cranial cortical bone thickness. Supported by NSF BCS 0725126.
The Role of Mitochondrial Activity in the Differentiation of Human Embryonic Stem Cells (ESCs)
Authors: Marsboom Glenn , Paul Jonathan , Toth Peter T. , Zhang Yanmin , Malik Asrar B , Archer Stephen L. , Rehman Jalees
Published: June 2021

Introduction

Little is known about the role of metabolic pathways in regulating stem cell function and fate. We hypothesized that ESCs primarily use non‐mitochondrial glycolytic metabolism to avoid the production of ROS (reactive oxygen species) and promote their self‐renewal potential.

Methods

We studied metabolic gene expression and mitochondrial oxygen consumption in NIH‐approved WA01 human ESCs, adult human aortic smooth muscle cells and spontaneously differentiated ESCs. Mitochondrial membrane potential (ΨΔm) was assessed with the potentiometric dye JC1.

Results

The expression of the mitochondrial biogenesis transcription factor PGC1a was significantly higher in ESCs when compared to mature vascular cells. ESCs had 16.29±3.94 fold (P<0.05 vs AoSMCs) higher levels of PGC1a. However, oxygen consumption in ESCs was only 0.75nmol oxygen/min per million cells and showed minimal increase with addition of the mitochondrial uncoupler FCCP while AoSMC consumed 4.6nmol/min at baseline and increased to 14 nmol/min with FCCP.

Conclusion

ESCs express high levels of mitochondrial genes and contain substantial mitochondrial mass, but they show minimal evidence of mitochondrial oxygen consumption when compared to mature vascular cells. Understanding the metabolic mechanisms underlying ESC self‐renewal may permit optimization of ESC‐based regenerative therapies by modulating their metabolism.

Grant Funding Source: NIH and Heart Research Foundation
Reprogramming of Endothelial Progenitor Cells: Unipotency Towards Multipotency
Authors: Johnson Rajasingh , Yu Nanjia , Gomes Ignatius , Malik Asrar B
Published: June 2021

The development of autologous stem cells with enhanced lineage plasticity for subsequent testing in myocardial ischemia is an integral part of stem‐cell research. We tested the hypothesis that the treatment of EPC with chromatin modifying agents Trichostatin A (TSA‐ inhibitor for histone deacetylase) and 5Aza‐2‐deoxycytidine (5Aza‐ inhibitor for DNA methylation) influence histone acetylation and DNA methylation respectively thereby modify the chromatin structure and up‐regulates embryonic stem gene expression. In a series of preliminary experiments performed with EPCs that had been treated with 5Aza and TSA, we made the following observations: 1) 5Aza and TSA treatment induced expression of pluripotent genes (Oct4 and Nanog), cardiac stem cell marker Flk1 and down regulated expression of endothelial‐specific genes, 2) treated EPCs differentiated into ectodermal (neuronal) and mesodermal (cardiomyocyte) lineages, 3) aceH3K9 activity was higher and HDAC1 activity was lower in treated EPCs than in control cells, 4) functional and histological evidence from our preliminary studies in mouse AMI model also suggest that intracardiac transplantation of reprogrammed EPC improve left ventricular functions and differentiate into cardiomyocytes and endothelial cell phenotypes. Thus, the epigenetic modifying agents can de‐differentiate EPCs into cells with a more multipotent phenotype, and the de‐differentiated EPCs can subsequently differentiate into multiple cell lineages, including cardiomyocytes that could be used as potential source of cells for treating degenerative diseases.

Grant Funding Source: NHLBI
The sensitive man: an interactive computer program to teach cutaneous nerves, dermatomes, and sensory testing
Authors: Downs Mary Beth
Published: June 2021

To understand the rationale for sensory testing, a common step in the diagnosis of neurological deficits, students must know the pathways from the skin to the sensory cortex and understand how a pattern of abnormal sensation correlates with a central or peripheral lesion. Students often have difficulty in learning dermatomes, cutaneous nerve maps and neuronal pathways and in applying that knowledge to clinical situations. The Sensitive Man is an interactive computer program which addresses these learning goals. The program has four modules: a teaching module with the image of a man that when clicked with a cotton swab or pin cursor displays the cutaneous nerve field and pathway to the sensory cortex, a teaching module with detailed information on the CNS pathways showing animated central neurons, a knowledge testing module which displays a dermatome or cutaneous nerve for identification, and a clinical testing module designed to facilitate the students' problem‐solving skills through testing of a virtual patient. The program was designed using Poser Pro (SmithMicro) and Director (Adobe) software. Students who have beta‐tested modules of the program indicated in a questionnaire that its interactive nature and the visualization of the nerve pathways helped them remember the material. A controlled trial of the program is planned in order to determine its impact on student learning outcomes. This work was funded by the Alabama State Department of University Physical Therapy.
Wnt/beta‐catenin signaling in patterning and differentiation of the vertebrate endoderm
Authors: Murtaugh Lewis Charles
Published: June 2021

The endoderm gives rise to organs as different in anatomy and function as the lungs, colon and liver, and we want to understand the signaling processes controlling this diversification. Our primary interest is in the pancreas, and our work indicates multiple roles for the Wnt/beta (β)‐catenin signaling pathway in its development. Early, Wnt signaling inhibits pancreas specification within the foregut; later, it promotes expansion of multipotent progenitor cells and their differentiation into exocrine acinar tissue. Until recently, our studies have focused on response to Wnt ligands, mediated by the non‐redundant functions of β‐catenin. To better understand the role of Wnt ligand production, while bypassing redundancy between the ligand‐encoding genes, we have targeted the mouse Porcn gene, homologous to Drosophila porcupine. Porcupine is required for wingless secretion and bioactivity, and we find that mouse cells lacking Porcn cannot produce any of several Wnt ligands tested, but retain normal responsiveness to exogenous Wnts. The Porcn mutant defect thus closely resembles that of fly porcupine, indicating a conserved and non‐redundant role for this gene in Wnt production. Wnt production by Porcn mutant cells is rescued by wildtype human PORCN, but not a PORCN mutant recently identified in a pleiotropic congenital syndrome, Focal Dermal Hypoplasia. Mice carrying a conditional Porcn allele are viable, and will provide a novel genetic tool to understand Wnt function in endoderm development and beyond.

Supported by the NIH (R01‐DK075072) and Searle Scholars Program (06‐B‐116).
Forefoot varus malalignment: anatomical origin and association with signs of patellofemoral joint osteoarthritis in cadavers
Authors: Lufler Rebecca , Stefanik Joshua , Niu Jingbo , Hoagland Todd , Foster Timothy , Gross Doug
Published: June 2021

Forefoot varus (FFV) is a non‐weightbearing foot alignment deformity. FFV can cause foot pronation, which may increase stress in the patellofemoral joint (PFJ). The etiology of FFV may be insufficient valgus torsion of the talar head and is a potential risk factor for PFJ osteoarthritis (OA). The purpose of this study was to determine the relationship of: 1) talar torsion to foot alignment and 2) foot alignment to signs of PFJ OA. Alignment and talar torsion were measured in 50 cadaver feet and cartilage damage was scored 0–4 on patellar and trochlear facets of 45 knees. We dichotomized cartilage damage into presence (≥3) or absence of pathology. Pearson's correlation and logistic regression were used to determine the association of foot alignment to talar torsion and PFJ cartilage damage respectively. Regression analyses were adjusted for gender and age; GEE was used to account for correlation between measurements of the same subject. There was no linear association between foot alignment and talar torsion, r = 0.2, p = 0.2. Those in the highest tertile of foot alignment had 3.9 (95% CI 1.0, 15.3) times the odds of medial PFJ cartilage damage and 2.3 (95% CI 0.7, 7.7) times the odds of lateral PFJ cartilage damage as feet in the lowest tertile. The increased odds of medial PFJ cartilage damage suggest that FFV may present a risk to the PFJ. These findings may have important treatment implications, as FFV is modifiable using orthoses. This research was supported by the American College of Rheumatology Research and Education Foundation.

Grant Funding Source: None
Distribution and dynamics of nonmuscle myosins IIs in cardiac and in skeletal muscle cells
Authors: Sanger Joseph W. , Wang Jushuo , White Jennifer , Sanger Jean M.
Published: June 2021

We have proposed a three‐step model for myofibrillogenesis: premyofibrils to nascent myofibrils to mature myofibrils that involves nonmuscle myosin II presence only in the first two steps. However, it has been reported (Takeda et al., 2000) that antibodies directed against nonmuscle myosin IIs stained the Z‐bands of mature myofibrils and intercalated discs of striated muscle cells. We have used similar nonmuscle myosin II antibodies against intact hearts and cultured cardiac and skeletal muscle cells and did not obtained these results. We have transfected cultures of embryonic avian cardiac and skeletal muscle cells with GFP plasmids encoding the three different isoforms of nonmuscle myosin II (A, B or C). The cells were cotransfected with CeruleanFP plasmids encoding sarcomeric alpha‐actinin, a marker for z‐bodies and Z‐bands. The three different GFP‐nonmuscle myosin II proteins localized to the premyofibrils and nascent myofibrils alternating with z‐bodies (as identified by CeFP‐alpha‐actinin localizations). The nonmuscle myosins never localized to the Z‐bands (identified by CeFP‐alpha‐actinin) or any other region of the mature myofibrils in the muscle cells. Our results support the idea that nonmuscle myosin IIs participate in aligning the z‐bodies and their attached actin filaments in premyofibrils and nascent myofibrils, preceding the formation of the mature myofibril.

Research support: NIH.
Impact of aging on dermal wound healing
Authors: Brubaker Aleah L , Mahbub Shegufta , Deburghgrave Cory , Kovacs Elizabeth J
Published: June 2021

Age‐related changes in wound healing have been characterized by alterations in the early inflammatory, proliferative and late remodeling phases. Aging has also been associated with decline of the innate and adaptive immune response, contributing to morbidity and mortality following infection. This study will characterize the effect of advanced age on the inflammatory response to full‐thickness dermal injury in a murine model. In response to injury, aged mice exhibited decreased neutrophil infiltration into the wound as compared to young mice. Further, macrophage inflammatory protein‐2 (MIP‐2) levels remained elevated in aged mice 72 hours post injury, whereas the levels of this chemokine decrease in young mice after 48 hours. Additionally, the dendritic cell population was decreased by 65% in uninjured aged skin. Interestingly, after injury, the dendritic cell population decreased in young mice and increased in aged mice. These data suggest an age‐related differential response to dermal wound healing that may impact the innate immune response to infectious challenge.

Grant Funding Source: National Institute of Health R01 AG506080 (EJK) and the Ralph and Marian C Falk Medical Research Trust
Design of Smart Nanofilms for Regenerative Medicine
Authors: Thangaraj Mridhula , Mills David
Published: June 2021

Wound healing can be classified into several categories based their severity. There are many modalities to achieve wound healing but, the significant variety in wound types fosters a need to develop more advanced modalities that accelerate the wound healing process. This project developed a smart nanofilm using biocompatible polyelectrolyte nanofilm multilayers (PNMs) with embedded growth factors. PNMs were achieved using nebulization, an expedient tool for nanoassembly. Nebulization significantly reduces deposition time and does note compromise nanofilm characteristics. Oppositely charged PNMs, Chondroitin Sulfate (CS), Protamine Sulfate (PS), Poly‐L‐lysine (PLL) and Poly‐Glutamic acid (PGA), served as effective nanoarchitectures. PNMs (with/without platelet derived growth factor (PDGF) were formed on organtypic keratinocyte cultures (with/without wound creation). PNMs were assembled with PDGF as an active component in deep, double layered, or superficial ten‐layer architectures. AFM analysis demonstrated that heightened surface roughness and thickness is achieved with the nebulization technique when compared to the nanoassembly. Nanofilms further enhanced cell adhesion, proliferation and protein synthesis. The study suggests that bioactive, PNMs, acting as a local drug delivery system and dressing can enhance the initial stages of cutaneous wound healing.

Grant Funding Source: National Science Foundation
The extracellular matrix plays an active role in muscle regeneration
Authors: Calve Sarah , Simon Hans‐Georg
Published: June 2021

In urodele amphibians like the newt, skeletal muscle provides a reservoir for uncommitted stem‐like blastema cells that participate in the regeneration of the lost structure. Using 3D immunohistochemical analyses we found dynamic expression of a transitional extracellular matrix (ECM) rich in hyaluronic acid (HA), tenascin‐C (TN) and fibronectin (FN) around skeletal muscle. To test the hypothesis that the ECM plays key instructive roles early in blastema formation, we investigated the behavior of muscle cells under defined culture conditions using time‐lapse microscopy. Our results revealed that distinct components of the early transitional matrix supported myoblast migration and inhibited fusion (HA and TN) whereas ECM expressed around differentiating muscle induced both proliferation and fusion (FN and laminin). In addition, we developed silicone elastomer substrates with a tunable stiffness that can mimic the transverse stiffness of newt skeletal muscle and blastema (17 kPa and 14 kPa, respectively). By combining defined ECM components with a substrate of appropriate native stiffness, we aim to control the behavior of muscle‐derived cells with high precision. The establishment of a physiologically relevant environment and its controlled manipulation in vitro will provide new insights into the control of the differentiated state of the cell. Funded by NIH, Searle Foundation.

Grant Funding Source: NIH, Searle Foundation
A histochemical and immunohistochemical investigation of epimorphic regeneration in the representative lizard, Eublepharis macularius
Authors: McLean Katherine , Vickaryous Matthew
Published: June 2021

Epimorphic regeneration involves cell proliferation and the formation of a regeneration blastema to replace lost or damaged tissues. Although well described for teleosts and urodeles, this type of regeneration is also characteristic of many lizards. The objective of this study was to investigate the events that characterize epimorphic tail regeneration in the leopard gecko, Eublepharis macularius using both histochemistry and immunohistochemistry. Changes in tissue structure and composition of the regenerating tail were examined at various times post‐tail loss. A phase of wound healing follows immediately after tail loss during which time basal cells of the epidermis proliferate and migrate across the wound site. Once the wound is capped proliferation begins deep to the wound epithelium, giving rise to a localized mass of undifferentiated cells, the regeneration blastema. Blastema formation coincides with the onset of angiogenesis and redevelopment of axons and the ependymal tube of the spinal cord. Skeletal tissues differentiate somewhat later during tail regeneration, matching with the expression of Sox9. We also observe Sox9 expression in both mesenchymal and ependymal cells. Our findings demonstrate the utility of lizard tails as a model system for ongoing studies of epimorphic regeneration.

Grant Funding Source: NSERC
Systems approaches to biology and disease: integrating discovery and hypothesis‐driven paradigms
Authors: Hood Leroy
Published: June 2021

The challenge for biology and medicine in the 21st century is the need to deal with its incredible complexity. One powerful way to think of biology is to view it as an informational science requiring systems approaches. This view leads to the conclusion that biological information is captured, mined, integrated by biological networks and finally passed off to molecular machines for execution. Systems approaches are holistic rather than atomistic—and employ both hypothesis‐driven as well as discovery‐driven approaches. Hence the challenge in understanding biological complexity is that of using systems approaches to deciphering the operation of dynamic biological networks across three time scales of life—development, physiological and disease responses. I will focus on our efforts at a systems approach to disease—looking at prion disease in mice. We have just published a study that has taken more than 5 years—that lays out the principles of a systems approach to disease including dealing with the striking signal to noise problems of high throughput biological measurements and biology itself (e.g. polymorphisms). I will also discuss the emerging technologies (measurement and visualization) that will transform medicine over the next 10 years—including next generation DNA sequencing, microfluidic protein chips and single‐cell analyses. It appears that systems medicine, together with pioneering changes such as next‐generation DNA sequencing and blood protein measurements (nanotechnology) and as well as the development of powerful new computational and mathematical tools will transform medicine over the next 5–20 years from its currently reactive state to a mode that is predictive, personalized, preventive and participatory (P4).
Translational control of breast cancer
Authors: Schneider Robert J.
Published: June 2021

The role of translational control and changes in the protein synthetic machinery will be described that are associated with development of the most common form of advanced breast cancer, known as locally advanced breast cancer (advanced stage, large tumor size >5 cm, but non‐metastatic). These alterations have been compared to the changes identified in the translation machinery of metastatic breast cancers and inflammatory breast cancers, the most lethal form of breast cancer that typically initiates as a non‐solid tumor and rapidly metastasizes in a period of weeks. The alterations found in patient tumor specimens have been used to develop animal models that also recapitulate locally advanced and metastatic breast cancers, demonstrating the central role of translational control and alterations in the translation machinery in development and progression of human breast cancer. Studies have identified the overexpression of components of the Akt/mTOR/4E‐BP1 pathway and overexpression of several translation initiation factors specific to LABC or IBC and their changes with metastatic progression of disease. These studies will also show the importance of specific translational control changes in human breast cancers that enable tumor angiogenesis (vascularization) and their ability to respond to hypoxia (oxygen deprivation), when tested in animal models.
Pharmacological suppression of nonsense mutations to treat genetic diseases
Authors: Bedwell David Michael , Du Ming , Buckley Jessica , Wang Dan , Keeling Kim Marie , Welch Ellen , Peltz Stuart W
Published: June 2021

We are exploring the use of pharmacological agents to suppress nonsense mutations that cause genetic diseases. This approach could theoretically restore the synthesis of full length, functional proteins and relieve disease phenotypes. Much of our work focuses on cystic fibrosis (CF), a life‐threatening genetic disease that compromises function of the lungs and pancreas. CF is caused by mutations in the CFTR gene, which encodes the CFTR protein, a cAMP‐activated chloride channel. In cultured cells, we have shown that readthrough agents such as gentamicin and ataluren (formerly PTC124®) suppress CFTR nonsense mutations, resulting in a partial restoration of CFTR protein and function. Using a transgenic mouse model expressing the human CFTR gene with the G542X nonsense mutation found in CF patients, we have shown that these compounds can restore human CFTR expression and function in vivo. Recent data from a phase II clinical study support these conclusions, and show that ataluren can reduce the electrophysiological defects associated with CFTR nonsense mutations in CF patients. This therapeutic approach is also currently being applied to Duchenne Muscular Dystrophy and the lysosomal storage disease Hurler syndrome (MPS I‐H). Together, these results suggest that this approach could benefit a genotypic subset of patients with a wide range of genetic diseases caused by nonsense mutations. Supported by NIH RO1 NS057412 and P30 DK072482.
Dynamic Domains in Lipid Membranes near a Miscibility Critical Point
Authors: Keller Sarah L. , Honerkamp‐Smith Aurelia R.
Published: June 2021

Micron‐scale liquid domains appear in membranes containing at least three lipid types (lipids with high melting temperature, lipids with low melting temperature, and cholesterol or a similar sterol) as long as the membrane is at a temperature below a miscibility transition. When the transition occurs at a critical point, distinctive phenomena occur. Just below the critical temperature, the edges of domains fluctuate. Just above the critical point, domains are replaced by submicron fluctuations. We find that the size of the largest fluctuations (the correlation length) and their lipid composition (the order parameter) scale in a way that is consistent with predictions from a reductionist physical model (the two‐dimensional Ising model) (Honerkamp‐Smith et al. BJ 2008). Other researchers have found that complex mixtures of lipids and proteins derived from cell membranes in GPMVs (giant plasma membrane vesicles) exhibit the same critical behavior (Veatch et al. ACS Chem. Biol. 2008). Recently, we became interested in how long composition fluctuations persist in lipid membranes. We measured the effective dynamic critical exponent relating the decay time of membrane composition fluctuations to the wavenumber (an inverse length). We find that at temperatures far from the critical point, the exponent is 2, as expected from diffusion. As the temperature approaches the critical point, the exponent increases
High Resolution Field Cycling for Analysis of Lipid Dynamics in Membranes
Authors: Roberts Mary Fedarko , Pu Mingming , Redfield Alfred G.
Published: June 2021

Phospholipid dynamics often play a role in binding of peripheral membrane proteins. Whether there are discrete phospholipid binding sites on the proteins is often difficult to assess unless binding is quite tight. Yet for many proteins that bind reversibly to membranes, weaker interactions with specific phospholipids can have important functional consequences. For example, phosphatidylinositol‐specific phospholipase C (PI‐PLC) enzymes can be activated by nonsubstrate phospholipids with the extent of activation tuned by the lipid composition. Activity of the Bacillus thuringiensis PI‐PLC is significantly enhanced by a small amount of phosphatidylcholine (PC) in the target membrane. Whether (and where) there is a discrete site for an activator PC molecule or whether lipid mixtures alter bilayer properties is hard to distinguish. High resolution 31P and 13C NMR relaxation studies from 0.005 to 11.7 T can be used to explore interactions of such a peripheral membrane protein with mixed phospholipid vesicles as well as with small molecules. If spin‐labeled proteins are used, estimates of site‐off rates and phospholipid binding site location can be made. With this novel approach for PI‐PLC, we see that (i) discrete complexes for each type of phospholipid on the enzyme with lifetimes around a microsecond must exist; (ii) the discrete site for a substrate analog is spatially different from that for activator PC; and (iii) modeling coupled with the NMR results suggests a PC site, near a series of tyrosine residues, that would aid in anchoring the protein to membranes. The high resolution field cycling method illustrated here may be extended to a wide range of proteins that reversibly bind to membranes to explore differential interactions with phospholipid components as well as other intermolecular interactions.
Regulation of Ion Channel Trafficking
Authors: Pearce David
Published: June 2021

The epithelial sodium channel (ENaC) is central to the control of blood pressure in all mammals. It is regulated by both hormonal and non‐hormonal factors, which modulate primarily its cell surface expression. The serum‐ and glucocorticoid‐induced kinase 1 (SGK1) plays a central role in controlling ENaC cell surface expression, and SGK1, in turn, is regulated at the level of gene transcription, subcellular localization and posttranslational modification. SGK1 activity is controlled by two phosphorylations, one of which occurs in a carboxy‐terminal domain termed the hydrophobic motif (HM). Little is know about the mechanistic basis of this phosphorylation, and in particular the identity of the HM kinase has been unclear. We have found that that the highly conserved serine‐threonine kinase mTOR, is essential for SGK1 HM phosphorylation and activation of ENaC‐dependent Na+ transport. mTOR is organized into two distinct complexes, mTORC1 and mTORC2. Our data show that mTORC2—but not mTORC1—is the essential SGK1 HM kinase. In concert with mTORC2 regulation of HM phosphorylation, the small chaperone, GILZ regulates SGK1 subcellular localization. Together, these factors allow selective stimulation by SGK1 of ENaC‐dependent Na+ transport. Dysregulation of this signaling network can lead to hypertension or sodium wasting states.
The B.S. in Molecular Biology and Business Administration at UT Dallas
Authors: Zhang Li
Published: June 2021

The rapid advance of biological sciences and technologies in the past decade has revolutionized the ways by which diseases are diagnosed and treated, and the ways by which drugs are designed and discovered. The concepts and principles of biochemistry and molecular biology not only impact on research and applications in medical and health sciences, but they have also penetrated many professional fields ranging from business administration and stock analysis to law enforcement. At the University of Texas at Dallas, the molecular biology and biology programs currently train over 1000 undergraduate students to prepare for careers in medical and health professions, as well as in business administration and law enforcement. Particularly, our Biology degree offers a streamlined double major with Business Administration or Crime and Justice Studies. The specifics of such opportunities will be discussed.
SIRTUINS, AGING AND DISEASE
Authors: Guarente Leonard
Published: June 2021

SIR2 and related genes are NAD‐dependent deacetylases that slow aging in yeast, C. elegans, and Drosophila. In yeast and flies, SIR2 genes are also involved in the longevity conferred by dietary or calorie restriction (CR). The mammalian SIR2 homologs termed SIRT genes, or sirtuins, are involved in changes in stress resistance and metabolism and are known to be associated with CR. Critically, the CR diet in rodents not only extends life span, but also protects against many diseases of aging. In this talk, I will describe recent findings in the lab regarding SIRT1 function in specific mammalian tissues in relation to murine disease models. Our findings indicate that SIRT1 can influence many of the major diseases of aging, includingmetabolic diseases like diabetes, neurodegenerative diseases (Alzheimer's and Huntington's), cancer and osteoporosis. Therefore small molecules that alter the activity of SIRT1 (i.e. CR mimetic drugs) offer a new approach to prevent and possibly treat the major diseases of aging.

Supported by NIA, Glenn Foundation, Hi‐Q, HDF, Estee Lauder. Guarente consults for Sirtris/GSK

Guarente, L. (2007). Sirtuins in aging and disease. CSH Symp. Quant. Biol. 72, 483–488.

Guarente, L. (2008). Mitochondria – a nexus for aging, calorie restriction and sirtuins?. Cell 132, 171–175.
General Methods to Conditionally Regulate Protein Function
Authors: Wandless Thomas J
Published: June 2021

Advances in our understanding of complex, sometimes interconnected, signaling networks will likely require the implementation of tunable perturbation techniques that enable sensitivity analyses of these circuits. We have developed a general experimental technique in which the stability of a specific protein is regulated by a synthetic small molecule. Mutants of the E. coli dihydrofolate reductase (ecDHFR) protein were engineered to be degraded when expressed in mammalian cells. Trimethoprim (TMP) is a high‐affinity ligand for ecDHFR that stabilizes fusion proteins in a predictable, dose‐dependent manner, allowing investigators to tunably regulate genetically engineered alleles of any protein of interest. DD‐regulated proteins are degraded very rapidly upon withdrawal of TMP, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood‐brain barrier enables the tunable regulation of proteins expressed in cells found within the mammalian central nervous system.
Skills in demand
Authors: Ainsworth Susan Jones
Published: June 2021

This talk will provide an overview of the skills that corporate employers seek in current job candidates. Although most students of biochemistry or molecular biology graduate with strong technical and problem‐solving skills that allow them to be successful in academic research, they don't always have the skills to allow them to soar within a corporate environment. Today, companies are looking for strong scientists who have good communication skills and positive leadership characteristics. They also want to find those who have the ability to work collaboratively on teams made up of professionals from a range of disciplines and diverse cultural backgrounds. In a less‐than‐ideal job market, these skills allow scientists to gain an edge in competing for the best jobs. They can also open doors to an array of careers away from the bench.
VP Regulatoy, Quality Assurance, Medical & Clinical Affairs
Authors: Barber Cynthia M
Published: June 2021

The attractiveness of having a science degree is that there is an array of diverse and exciting jobs outside of the laboratory. Currently there is a shortage of regulatory and quality assurance (QA) personnel. And firms are seeking scientists for these position.

The need arises because it is crucial that all pharmaceutical, medical device, veterinary, infant nutritional and cosmetic products are registered before they can be marketed for sale. Working in regulatory affairs involves the preparation of scientific and technical information to support product approval globally, keeping abreast of changes in legislation and informing colleagues according. Job roles include quality control and assurance, clinical trial design and management, process development chemist, product formulation scientist, medical writing for regulatory dossiers and acting as a company representative to regulatory agencies.

Many companies seek PhD scientist for these positions. Most regulatory agencies employee scientist and there is a need to counter these skills within the company. The expectation is that you bring your scientific research experience and thought process to the company.

Learn on the job and after a couple of years experience you will be considered a seasoned regulatory affair and quality assurance professional.
The Dietary Intervention of Tomato and Broccoli for the Prevention of PhIP Induced Prostate Carcinogenesis in Rats
Authors: Canene‐Adams Kirstie , De Marzo Angelo M.
Published: June 2021

The heterocyclic amine, 2‐ amino‐1‐methyl‐6‐phenylimidazo[4,5‐B]pyridine (PhIP), is found in well‐cooked meats and has been implicated by epidemiology and rodent carcinogenesis studies for causing breast, prostate, and colon cancers. The combination of tomato and broccoli powders has been shown to reduce cancer burden in other models of prostate cancer, but have not been utilized to study PhIP induced prostate carcinogenesis previously. Our objective was to determine if a diet of tomato and broccoli could reduce PhIP induced prostate carcinogenesis. Fifty male Fisher 344 rats were put into 1 of 3 treatment groups, control, PhIP (Nard Institute, Ltd., Japan, 200 ppm in the diet and fed for 20 wks), or tomato + broccoli + PhIP. Tomato and broccoli powders, Future*Ceuticals, Illinois, were mixed into AIN93G diets at a level of 10% each and fed with PhIP for 20 wks, and then without PhIP for an additional 32 wks. Rats were euthanized as needed based on health status and tumor burden and survival rates for each group were calculated. After the 52 wks, all remaining rats were euthanized and various tissues collected for histological analysis.

Research support: DOD PCRP and the Maryland Cancer Restitution Fund
COX‐1 mediates the proliferative effects of Western‐type dietary fat in normal colonic mucosa
Authors: Neilson Andrew Peter , Reuven Shony L. , Djuric Zora , Ren Jianwei , Hong Yu H. , Smith William L. , Brenner Dean E.
Published: June 2021

Male C57BL/6 mice [wild type (WT), COX‐1 null, or COX‐2 null, from Dr. R. Langenbach, n=15/group] were fed a modified AIN−93 diet containing 10% Western fat blend and either 10% olive oil (Western‐type, WD) or 10% fish oil (FO) (n=5/diet) ad libitum for 11 weeks. Colonic mucosa was stained for Ki67 marker of cellular proliferation. The epithelial proliferative zone (as fraction of crypt height) was significantly lower in COX‐1 null (0.53 ± 0.06) vs. COX‐2 null (0.67 ± 0.04) and WT (0.68 ± 0.03) mice fed WD. Similar trends were observed for Ki67‐labeling index (LI) between groups. These data suggest that the influence of fatty acids on epithelial proliferation in normal colonic mucosa is modulated by COX‐1, which is constitutively expressed. The effect of COX‐2 knockout was likely not significant due to low expression in normal mucosa. FO‐fed mice had slightly decreased proliferation that was not affected by COX status. This could be due to distinct metabolic products from n‐3 FAs in FO vs. n‐6 FAs present in the WD. Preliminary analysis of eicosanoids in the colonic mucosa indicated that COX‐1 null mice had greatly decreased PGE2 levels while levels in COX‐2 null mice were similar to that of control mice. Although FO decreased PGE2, COX‐1 knock‐out had a much stronger effect. These data have implication for prevention targets in normal mucosa. Supported by the Michigan Institute for Clinical & Health Research and NIH grant GM 48864.
Consumption of high‐fat diet enhanced the malignant progression of human colon cancer in xenograft mouse model
Authors: Tang Feng‐Yao , Chiang En‐Pei Isabel
Published: June 2021

Obesity and diet‐induced overweight have been demonstrated as risk factors of colon cancer. Several studies suggested that loss of E‐cadherin adherent molecule has been associated with epithelial– mesenchymal transition (EMT) and the malignant progression of human colon cancer cells. However, the effects of high fat (HF) diet intake on the expression of E‐cadherin and in vivo malignant progression of human colon cancer have not been demonstrated well yet. Our results indicated that consumption of HF diet (45% of total energy intake) for 4 weeks would induce the tumor growth in human colon cancer HT‐29 cell bearing mice. Bioluminescence imaging analysis also confirmed that consumption of HF diet would stimulate the proliferation of human colon cancer. HF diet modulated the expression of E‐cadherin and β‐catenin in xenograft mouse model. Furthermore, consumption of HF diet enhanced the cellular proliferation through the modulation of proliferating cellular nuclear antigen (PCNA) and p21cip proteins. The possible molecular mechanisms of actions were, in part, through the activation of the MAPK/ERK signaling pathway and the suppression of E‐cadherin adhesion molecule. Take together; these results suggested that consumption of HF diet could modulate the growth and the malignant progression of human colon cancer cells in vivo.
Ascorbic acid does not offset the inhibitory effect of bioactive dietary polyphenolic compounds on heme iron absorption in human intestinal Caco‐2 cells
Authors: Ma Qianyi , Kim Eun‐Young , Han Okhee
Published: June 2021

Heme iron is an important source of dietary iron but its absorption mechanism has not been completely elucidated. Recently, bioactive dietary polyphenols have been attracting more and more attention due to their reported health benefits. We previously reported that (−)‐epigallocatechin‐3‐gallate (EGCG) and grape seed extract (GSE) inhibit heme iron absorption by reducing basolateral iron export rather than by decreasing apical heme iron uptake in intestinal cells. In current studies, we determined the effect of three dietary polyphenols, green tea extract (GTE), EGCG and GSE, on heme iron absorption in human intestinal cells and tested whether ascorbic acid counteracts the inhibitory effects of polyphenols on heme iron absorption. The results demonstrated that GTE, EGCG and GSE all strongly reduced basolateral iron transport. Addition of ascorbic acid to polyphenol treatment stimulated apical heme iron uptake but did not reverse the decreased basolateral iron transport. These results suggest that the inhibitory effects of bioactive polyphenolic compounds on heme iron absorption cannot be offset by ascorbic acid.
Ground Beef Fatty Acid Composition Alters HDL Functionality
Authors: Wu Xiuzhi
Published: June 2021

High dense lipoprotein (HDL), also referred to as the “good cholesterol” has various atheroprotective functions. In this study, the functionality change of HDL induced by beef consumption, with a focus on the capability to promote reverse cholesterol transport was assessed. Human serum and plasma samples used for the assays were collected from 28 individuals that successfully completed the beef feeding trial, in which hamburger patties containing low and high monounsaturated fatty acid (MUFA): saturated fatty acid (SFA) ratios were consumed. Each subject completed a three 5‐week diet in a random order with a 4‐week wash period between the diets. The activities of lecithin:cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) were measured by commercially available kits from Roar Biomedical, Inc. Lipoprotein Profiling was achieved by ultracentrifugal density gradient separation in CsBiEDTA. Sphingosine 1phosphate, an important component of HDL, was extracted by a two step liquid‐liquid extraction and quantified by HPLC. The concentrations of serum and HDL3 apoproteins including Apo A‐I, A‐II, B, E were measured by fluorescent microsphere immunoassay. The results of the lipid profiling indicated that the consumption of high MUFA:SFA and low MUFA:SFA diets significantly increased the blood level of both HDL and HDL3. This work is supported by the National Cattlemen's Beef Association.
The effect of activated‐mitogen activated protein kinase (MAPK) activity on 1,25 dihydroxyvitamin D (1,25D)‐mediated gene transcription in colon cancer cells
Authors: DeSmet Marsha , Fleet James C.
Published: June 2021

The active form of vitamin D (VD), 1,25D, induces anti‐cancer effects in many cell lines by activating the VD receptor (VDR). In cancer, MAPK pathways (ERK, JNK, p38) are activated and previous research shows that activated MAPK impairs 1,25D action in skin and prostate cell lines. To determine whether activating MAPK influences 1,25D action in colonocytes, Caco‐2 cells were pretreated with an agent that induces multiple MAPK pathways, As2O3, prior to 1,25D treatment. As2O3 decreased 1,25D‐mediated induction of CYP24 mRNA (−55%) but also decreased VDR mRNA levels by 50%. In contrast, a specific JNK activator, anisomycin, did not alter 1,25D‐mediated CYP24 mRNA induction indicating the effect of As2O3 was not due to JNK activation. KRAS activating mutations are common in colon cancer. Caco‐2 cells transfected with either a constitutively active (ca) KRAS plasmid or a caMEK1 plasmid have reduced 1,25 D‐induction of both 3XVDRE (−30%) and CYP24 promoter (−60%) reporter genes. Conversely, in HCT116 colon cancer cells with a RAS‐activating mutation 1,25D‐induced CYP24 mRNA was enhanced 285% by the MEK inhibition. These data demonstrate that a common mutation in colon cancer, i.e. KRAS activation, suppresses 1,25D action in colon cancer cells and may limit the chemopreventive effects of high vitamin D status.

Supported by NIDDK Award DK54111 to JCF