bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of PKC, RhoA and ERK signaling to serotonergic contractility of pulmonary arteries from chronic hypoxic fetal and adult sheep
Authors: Goyal Ravi , Mittal Ashwani , Nguyhn Dan , Loftin Mathew , Derrick Allison , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Serotonin (5‐HT) regulates pulmonary arterial (PA) contractility through Ca2+ and kinase dependent signaling pathways and many reports indicate these pathways are altered by chronic hypoxia (CH). Among the many kinase pathways activated by 5‐HT, protein kinase C (PKC), Rho kinase, and extracellular regulated kinase (ERK) are important to arterial reactivity. As we have found CH causes selective reductions in Ca2+‐dependent PA contractility in fetus and adult, we used wire‐myography and Western immunoblot techniques to test the hypothesis that CH upregulates kinase‐dependent contractility. PA rings were stimulated with 10 μM 5‐HT and comparative analyses of the kinase pathways were made in the absence and then presence of inhibitors of PKC (1 μM BIM I), RhoA (10 μM Y 27632), and ERK (10 μM U0126). CH did not affect the role for PKC in adult, which did not have an appreciable role in fetus. CH restricted the influence of Rho kinase in fetus and adult, and may cause ERK to become a negative regulator of contractility in adult. CH upregulated ROCK‐II in adult but did not influence the relative expression of PKCα, ERK‐I, ERK‐II, or ROCK‐I in either adult or fetus. In conclusion, maturation and CH cause complex changes in PKC, ERK and Rho kinase‐dependent contractility, which likely integrates with alterations in Ca2+‐signaling to regulate vascular reactivity. Support from NIH, UM, LLUMC, and Sigma Xi.
Peroxynitrite is a potent vasodilator agent in the pulmonary vascular bed of the rat
Authors: Murthy Subramanyam N , Uppu Rao M , Casey David B , Badejo Adeleke M , Dhaliwal Jasdeep S , Kadowitz Philip
Published: June 2021

It has been reported that the vasodilator effects of peroxynitrite (PN) are tachyphylaxic; however, responses have not been determined in the pulmonary vascular bed of the rat. In the present study iv injections of PN (1, 3 and 10 mmol) decreased pulmonary and systemic arterial pressure. Responses to PN could be repeated without the development of tachyphylaxis. The decreases in pulmonary arterial (PA) pressure in response to PN were modest under baseline conditions but were greatly enhanced when baseline PA pressure was increased with U‐46619. Also, the iv injections of PN decreased PA pressure when baseline tone was increased by ventilatory hypoxia or administration of the eNOS inhibitor L‐NAME. Sodium nitroprusside and PN produced similar vasodilator responses in the pulmonary and systemic vascular beds. The repeated administration of PN did not alter responses to both vasoconstrictor and vasodilator agents, and did not impair endothelium dependent responses. The results of the present study show that PN has potent pulmonary vasodilator activity in the rat and is consistent with the hypothesis that PN is converted into a substance that has NO‐donor like activity and that the beneficial effects of PN may be derived in part from the inactivation of superoxide anion and the activation of soluble guanylate cyclase.
Antagonism of orexin receptor 1 (OX1R) in the retrotrapezoid nucleus (RTN) inhibits the ventilatory response to hypercapnia in wakefulness.
Authors: Dias Mirela Barros , Li Aihua , Nattie Eugene E
Published: June 2021

Recent transgenic mice data suggest that OX plays an important role in CO2 sensitivity during wake periods. We hypothesized that OX1R in the RTN contribute to chemoreception. In unanesthetized rats, ventilation was recorded using a whole body plethysmograph, together with EEG and EMG. We dialyzed the vehicle and then SB‐334867 (OX1R antagonist) into the RTN to focally inhibit OX1R and studied the effects of both treatments on breathing in air and in 7%CO2. During wakefulness, but not during sleep, SB‐334867 caused a 26% reduction of the hyperventilation induced by 7%CO2 (135 ± 10 mL/100g/min) compared with vehicle (182 ± 10 mL/100g/min) (P< 0.01). This effect was due to both decreased Vt and f. Neither basal ventilation nor oxygen consumption was affected. The number and duration of apneas were similar between control and treatment periods. No effect was observed in a separate group of animals who had the microdialysis probe misplaced (peri‐RTN). Acute exposure to 7%CO2 tends to awaken or arouse rats and did so in the peri‐RTN group. Compared to the peri‐RTN group, treatment with SB‐334867 in the RTN decreased the awake period during 7%CO2 (P<0.05). We conclude that projections of orexin‐containing neurons to the RTN contribute via OX1R to the hypercapnic chemoreflex control during wakefulness, but not during the sleep. Further, RTN OX1R may be a part of the circuit that modulates the arousal response to hypercapnia. (HL 28066)
Hypercapnia engages parafacial respiratory group (pFRG) neuronal activity coincident with late expiratory motor outflow
Authors: Abdala Ana Paula Lima , Rybak Ilya A , Smith Jeffrey C , Paton Julian F.R.
Published: June 2021

Recently, we found that suppression of the parafacial respiratory group (pFRG) by bilateral microinjection of isoguvacine (GABAA agonist, 60 nL, 20 mM) abolished late expiratory (late‐E) activity recorded from the abdominal nerve (AbN) during hypercapnia. Thus, we tested the hypothesis that hypercapnia would activate pFRG neurons with late‐E discharge. We recorded phrenic (PN), central vagus (cVN) and AbN outflow (L1) during 7‐10% CO2 while recording from pFRG in the arterially perfused in situ preparation. During normocapnia, AbN exhibited a low amplitude post‐inspiratory discharge only, but a high amplitude late‐E discharge was evoked with 7‐10% CO2. Using a ventral approach to the brainstem, we recorded both population and single unit activities from the caudal half of the pFRG. Two types of neurons were found: (1) tonically firing units that were sensitive to hypercapnia and responded to peripheral chemoreceptor stimulation (n=5) and, (2) rhythmically active units that fired coincidently with AbN late‐E discharge in hypercapnia but were quiescent in the absence of late‐E discharge in AbN during normocapnia (n=5). We conclude that pFRG contains a heterogeneous population of hypercapnia sensitive neurons including a subpopulation of late‐E neurons, which may be the source of the late‐E AbN discharge that emerges during hypercapnia.

Research funded by the NIH (RO1 NS057815)
NHE1, NHE3, and NHE5 mRNA expression in brainstem and cortex of Sprague‐Dawley rat
Authors: Perkins Matthew Huntoon , Pritz Maria Magdalena , Cordovez Juan M , Solomon Irene C
Published: June 2021

In brainstem CO2 chemosensitive neurons, an increase in CO2 (hypercapnia) leads to a maintained reduction in intracellular pH (pHi) while in non‐chemosensitive neurons, pHi recovery is observed. Although the precise mechanisms for this difference in pHi regulation remain to be determined, it has been suggested that different isoforms of the Na+/H+‐exchanger (NHE) may be responsible. To begin to explore this possibility, real‐time RT‐PCR was used to examine mRNA expression for three NHE isoforms ‐ NHE1 (Slc9a1), NHE3 (Slc9a3), and NHE5 (Slc9a5) ‐ in brainstem and cortex (non‐chemosensitive control) of 21 day‐old SD rat. Preliminary data indicate that each of the NHE isoforms is present in both brainstem and cortex. To further investigate the distribution within the medulla, mRNA expression levels for the NHE3 and NHE5 in dorsal (dM) versus ventral (vM) medulla were compared. These analyses revealed that NHE5 mRNA levels are ~4‐fold higher in the vM than in the dM (when normalized to Actb), but NHE3 mRNA levels are similar between the two regions. Additional studies are needed to further evaluate the relative abundance of different NHE mRNAs in chemosensitive areas such as the NTS/DMV, VLM, the caudal medullary raphe, and the LC. To date, however, our data suggest that differential expression of NHE isoforms may participate in pHi regulation in chemosensitive and/or non‐chemosensitive neurons. Supported by NS045321.
Ventilatory responses to chronic intracerebroventricular leptin injections in obese leptin‐deficient mice
Authors: Bassi Mirian , Glass Mogens L.
Published: June 2021

Previous studies suggest that leptin may play an important role in the regulation of respiratory function. Obese leptin deficient mice (ob/ob) exhibit ventilatory depression when subjected to hipercarbic condition (high CO2). However, how leptin regulates pulmonary function is still unclear. We hypothesized that leptin modulates pulmonary function by acting on chemoreceptors in the brainstem. We evaluated pulmonary ventilation (VE), tidal volume (VT) and ventilatory rate (fR) of ob/ob mice under hipercarbic conditions before and after a 4‐day leptin or vehicle treatment regimen (4th ventricle injections at 10 ?g/2 ?l, n=7/group). We also determined the presence of leptin receptor protein (OB‐R) in brainstem nuclei by the imunohistochemistry. Our results demonstrate that VE (ml.kg−1.min−1) and VT (ml.kg−1) increased significantly after leptin treatment (from 3,372 ± 364 to 6,176 ± 347 and from 9.7 ± 0.9 to 16.7 ± 0.8, respectively). Leptin treatment did not alter fR. These variables were not affected by vehicle treatment. OB‐R staining was observed in various brainstem nuclei associated with the control of respiratory function, including: nucleus of the tractus solitarii, locus coeruleus, raphe nucleus and facial nucleus. These results suggest that leptin may be a powerful stimulator of the ventilatory responses by acting directly in brainstem sites responsible for the control of respiratory function.
Effect of Hydrogen Sulfide on [Ca2+]i Regulation in Airway Smooth Muscle
Authors: Ryu Alexander , Thompson Michael , Venkatachalem Sathish , Pabelick Christina , Prakash Y S
Published: June 2021

There is increasing interest in the role of hydrogen sulfide (H2S) as a gasotransmitter similar to nitric oxide and carbon monoxide. In this study, we examined the role of H2S in airway smooth muscle (ASM) inflammation. H2S is produced by the enzymes cystathionine beta‐synthase (CBS) and cystathionine gamma‐lysase (CTH). Both of these enzymes were detected in human ASM by Western blot analysis. In tissues exposed to the pro‐inflammatory cytokine tumor necrosis factor‐a (TNFα) both CTH and CBS expression were upregulated. In ASM strips contracted with 10 µM ACh, only exposure to 1 mM of the H2S donor NaHS induced relaxation. Human ASM cells loaded with fura‐2 demonstrated a dose‐dependent decrease in intracellular calcium concentration ([Ca2+]i) to NaHS (50‐250 μM), in the presence of either 1 μM ACh or 10 μM histamine. However, sodium sulfide (Na2S, 100 µM up to 1 mM) showed no effect on [Ca2+]i. Exposure to 60 mM KCl in the presence of either NaHS or Na2S (50 μM‐3 mM) did not affect [Ca2+]i. Relaxant effects of NaHS or Na2S on ACh‐induced [Ca2+]i elevation were absent when experiments were conducted in zero extracellular Ca2+. These results suggest that H2S relaxes ASM and reduces [Ca2+]i by affecting Ca2+ influx, potentially via voltage‐gated channels, but does not affect sarcoplasmic reticulum Ca2+ release. The relevance of endogenous H2S generation in ASM, and its role in infalmmation needs to be explored further.

Supported by the Flight Attendants Medical Research Institute (FAMRI), NIH grant HL74309, and the Department of Anesthesiology, Mayo Clinic.
Forward transport of the receptor P63 is necessary for secretagogue‐enhanced surfactant protein‐A (SP‐A) binding to type II pneumocytes
Authors: Bates Sandra R. , Kazi Altaf S. , Tao Jian‐Qin , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

SP‐A plays an important role in surfactant lipid turnover in the lung. P63 (CKAP4) located on both the plasma membrane (PM) and ER of type II pneumocytes has been identified as an SP‐A receptor. Interference with specific calcium (Ca++)‐dependent binding of SP‐A to type II cells occurs in competition studies using anti‐P63 pAb and upon treatment with siRNA to reduce P63 protein levels. A 2.5‐fold enhancement of 125I‐SP‐A Ca++‐dependent binding to type II cells plated on microporous membranes was achieved by exposure of the cells to the secretagogue cAMP (0.1mM), without effect on non‐specific (Ca++‐independent) SP‐A cell association. Anti‐P63 Ab blocked the cAMP‐augmented specific binding of SP‐A, evidence for the importance of P63 in this process. Inhibitors of receptor recycling were utilized to evaluate whether the forward transport of the ER‐resident P63 to the PM was necessary for receptor function. Monensin, a carboxylic ionophore, increases the pH of intracellular vesicles and brefeldin A (BFA) blocks formation of transport vesicles. In the presence of monensin (2uM) or BFA (10ug/ml), cAMP‐augmentation of the specific binding of SP‐A was abolished. Thus, secretagogue stimulation of SP‐A binding required the movement of P63 protein from the ER to the PM. Our results provide further evidence for the role of P63 as a SP‐A receptor protein on lung type II cells. [HL 19737]
Activation of Glucose‐6‐Phosphate Dehydrogenase by Depolarization of Membrane Potential Mediates Vascular Smooth Muscle Contraction
Authors: GUPTE Rakhee S , Abe Madoka , Rawat Dhwajbahadur K , Ochi Rikuo , Gupte Sachin A
Published: June 2021

We have previously demonstrated that inhibition of glucose‐6‐phosphate dehydrogenase (G6PD) evokes relaxation of vascular smooth muscle (VSMC). So, to elucidate the mechanism of relaxation, we examinedwhether G6PD activation affects contraction of VSMC. We found,using ultracentrifugation, two isoforms (62 & 58 KD) of G6PD were present in bovine coronary artery (BCA); the longer (62 KD)isoform was localized in caveolae fraction and the shorter(58 KD) isoform was detected in cytoplasmic fraction. Both isoforms were functionally active; depolarization of the membrane potential to ‐9mV by KCl (30 mM; n=5) increased G6PD activity in the membrane fraction by ~2‐fold. Mechanistic studies revealed that PKCδ mediated activation of G6PD evoked by depolarization in a time‐dependent manner.Silencing of PKCδ in VSMC and HEK293 decreased (P<0.05; n=5) basal activity (0.25 nmol/min/mg protein) of endogenous G6PD and ectopically expressed G6PD in HEK293 (PKCδ−/G6PD+) cells by 50% as compared to cells transfected with non‐targeting siRNA, and in these PKCδ−cells 30mMKClwas unable to activate G6PD. Interestingly, inhibition of G6PD expression in BCA by siRNAand in aorta of G6PD mutated mice, decreased KCl‐induced contractions. In conclusion, activation of G6PD by depolarization of membrane potential plays a pivotal role in mediatingVSMC contraction.

This study is supported by AHA and NIH grant# 0435070N and R01HL85352
Myo‐Inositol monophosphatase, inositol production and osmoregulation in the euryhaline European eel, Anguilla anguilla
Authors: Kalujnaia Svetlana , Cramb Gordon
Published: June 2021

The European eel (Anguilla anguilla) can survive in both freshwater (FW) and seawater (SW) environments with only minor changes in the osmolality of body fluids. A microarray technique used to screen for differential gene expression following transfer from FW to SW highlighted a group of genes related to the synthesis and transport of solutes known to act as osmolytes in mammalian cells. One gene associated with the production of one such osmolyte, inositol, is myo‐inositol monophosphatase (IMPA1). Northern blot, qPCR, Western blotting and immunohistological analyses were used to compare the expression of IMPA1 in the gill and kidney as fish move between FW and SW environments. After 2 days acclimation to SW, 1.9‐ and 2.5‐fold increases in IMPA1 mRNA expression were found in kidney and gill respectively and these levels increased further to 4.9‐ and 3.4‐fold respectively, 5 months after SW transfer. IMPA1 protein expression was up‐regulated fold in the gill following SW transfer, however, no significant changes in protein abundance were detected in the kidney. Preliminary immunohistological studies have revealed that IMPA1 is expressed in most cells within the gill and especially within chondrocytes found within the branchial arch and primary filaments. Our studies have indicated a potential role for IMPA1, and the osmolyte inositol, in salinity adaptation in the European eel.
Expression of the let‐7 family of microRNAs is elevated in older human skeletal muscle
Authors: McCarthy John Joseph , Drummond Micah J , Volpi Elena , Rasmussen Blake B. , Esser Karyn A.
Published: June 2021

A defining characteristic of aging in humans is sarcopenia, the progressive loss of skeletal muscle mass and strength. Although the etiology of sarcopenia remains ill defined, a recent study suggested altered microRNAs expression may be a contributing factor (Drummond et al., 2008). We hypothesized that there would be a significant difference in microRNA expression of skeletal muscle from young and old males. To test this hypothesis, a comprehensive microarray analysis was performed to determine the expression level of 837 human microRNAs (miRBase 11.0) in skeletal muscle biopsies (vastus lateralis) from young (n=12; 30±2yr) and older (n=12; 72±2yr) male humans. The most notable finding was that expression of all members of the let‐7 family were significantly elevated in the older subjects as compared to the young (P<0.05). In addition, expression of the muscle‐specific microRNAs miR‐133a and miR‐206 were significantly decreased and increased, respectively, in the older versus younger subjects (P<0.05). The let‐7 family, as well as the muscle‐specific microRNAs, have numerous predicted target genes which are known to be involved in muscle cell growth and development. Future experiments will focus on the validation of predicted let‐7 target genes and their potential role in the age associated loss of muscle mass in older humans.
Histology Challenge: an interactive program for 1st year students
Authors: Dallemagne Catherine , Richardson Neil , Weyand Peter
Published: June 2021

Our students come from diverse backgrounds. They need flexibility in their learning, and opportunities to review aspects of curriculum they are less confident with. An online teaching and learning programme called the Histology Challenge has been developed to supplement learning experiences offered in several first year anatomy and anatomy&physiology units at QUT. The programme is designed to be integrated with the existing Blackboard sites.

The Histology Challenge emphasises the foundation concept that a complex system, such as the human body, can be better understood by examining its simpler components. The tutorial allows students to examine the cells and tissues which ultimately determine structural and functional properties of body organs. The program is interactive, asking students to make decisions and choices, demonstrating an integrated understanding of systemic and cellular aspects. It provides users with the ability to progress at their own pace and to test their understanding and knowledge. For the developer the learning activity can be easily controlled and modified via the use of text files.

There are several key elements of this programme, designed to promote specific aspects of student learning. Minimum text is used, instead there is a strong emphasis on instructive artwork and original, high quality histology images presented within a framework that reinforces learning and promotes problem solving skills.
Development and Assessment of Creative Writing Activities within an Interdisciplinary Biotechnology Program
Authors: Clase Kari
Published: June 2021

Science and technology are changing rapidly resulting in a society that needs graduates who can identify problems and creatively design working solutions. An enmeshing of disciplines within the biological sciences and engineering is creating an interdisciplinary environment in the workplace and academia, which has created a need for interdisciplinary educational environments that enhance creative thinking and innovative problem solving. But how do you teach students creativity and innovation? How do you teach students to work effectively and solve interdisciplinary, ill‐defined problems? Curriculum has been developed and integrated into a multidisciplinary biotechnology program where students are engaged and contribute "hands on" to the pursuit of authentic bioengineering research. The learning goals of the inquiry‐based biotechnology curriculum are to produce students who acquire basic research skills and are able to:

Function in interdisciplinary teams to solve ill‐defined problems effectively and creatively

Transfer knowledge across disciplinary contexts.

We are exploring the effectiveness of the writing activities to enhance student creativity and increase their knowledge of the nature of science. This poster will describe the writing activities that have been developed to integrate with the hands‐on laboratory activities and the use of calibrated peer review for assessment.
Use of the APS Medical Physiology Objectives as a foundation for solo teaching of medical physiology
Authors: Wright Bruce E.
Published: June 2021

At many small medical schools, one instructor teaches medical physiology alone, including preparation and delivery of up to ten hours of lecture per week. For such day‐to‐day lecture preparation within a systems approach to physiology, the materials provided by the APS Medical Physiology Curriculum Objectives Project (http://www.the‐aps.org/education/MedPhysObj/medcor.htm) are invaluable starting point for direct student use but lacking in organization. For his course, the author compressed and collated many overlapping objectives, both within and between systems, to provide a more general understanding of systems topics. For example, renal objectives R20, R26, and R29 all involve Starling forces at the glomerulus. These were combined and associated with related cell and cardiovascular objectives CE45, CE47, and CV71. Four other objectives, e.g. R22‐23‐24 (CV94‐95), were also integrated within the topic of glomerular filtration. Other examples of this integration will be provided. The author's approach to application of these objectives over an entire course might be of use to others faced with similar challenges.
Physiology Understanding Week: Developing a national outreach program
Authors: Limson Mel , Lakes Matyas Marsha
Published: June 2021

Physiology Understanding (PhUn) Week is the annual APS member‐based outreach program that fosters partnerships between physiologists and K‐12 teachers. PhUn Week seeks to increase student awareness of physiology in their lives and to introduce them to physiology as a possible career. The successful development of the program models how formative evaluation is instrumental. Pilot tests in 2005 concentrated on select APS members and teachers committed to the new initiative. The program was moderately expanded in 2006 by doubling the targeted numbers of participants. Formative assessment in this phase helped design a program website (www.PhUnWeek.org) with downloadable resources, including event planner forms, instructional materials, and career presentations. Planning partners and their teams adapt the models of outreach and develop innovative activities. After pilot testing for feasibility, the program was nationally launched in 2007. More than 3,000 students, 72 teachers, and 100 APS members participated in 32 events across 24 states and Puerto Rico. The program website has facilitated the processes for coordinating a PhUn Week event. The early years of the program have seeded a base of volunteers that continues to build momentum for the participation of other physiologists and teachers. The APS sponsors and funds the outreach program since conception with additional support from ADInstruments.
The Interactive Textbook
Authors: Goldberg Harry
Published: June 2021

The Interactive Textbook is a virtual learning environment that has been designed and developed to serve as a next‐generation textbook. This software integrates multiple media: video, full chapter‐length animation, illustration, simulation, and text into an interface that addresses the needs of students with a variety of learning preferences. One of the major attributes of this system is that it enables faculty to spend less class time relaying facts and more time actively engaging students in discussion of scientific theory and application.

The theoretical underpinnings of this system have been tested and shown to be effective in delivering content. The Interactive Textbook is currently being assessed in a variety of university environments. The potential use of this technology in the curriculum will be discussed.
Mechanisms underlying corticosterone mediated inhibition of angiogenesis
Authors: Shikatani Eric A , Haas Tara L
Published: June 2021

Angiogenesis involves cell proliferation, invasion and migration of endothelial cells. Corticosterone (CORT), elevated in diabetes and metabolic syndrome, is known to be angiostatic, but it's mechanisms of action are unknown. Previously, we found that CORT inhibits matrix metalloproteinase (MMP)‐2 production and activity. We treated rat microvascular endothelial cells with 600 nM CORT for 48 hrs and found a significant decrease in full length MMP‐2 promoter activity using a luciferase reporter assay. This effect was localized to a region between ‐1560 and ‐1386 bp. However, pretreatment of cells with 10 μM RU 486 (glucocorticoid receptor (GR) antagonist) for 2 hrs did not block the CORT mediated inhibition of MMP‐2 production or activation. To observe the effect of CORT on endothelial cell sprouting, we cultured cell spheroids in type‐1 collagen and treated them with CORT for 48 hrs. At 24 hrs CORT treated cells displayed numerous sprouts, similar to controls, but at 48 hrs, sprouts had destabilized and regressed in response to CORT. Our data suggest that the mechanism of CORT‐dependent inhibition of MMP‐2 production may be due to reduced transcription, but that this may not be GR‐mediated. In future studies, we will identify intracellular signals affected by CORT treatment that may contribute to sprout repression. Funding by NSERC & HSFO.

Grant Funding Source

NSERC
Role of the sodium‐dependent phosphate co‐transporter PiT‐1 in mouse development and vascular calcification
Authors: Festing Maria H , Giachelli Cecilia M
Published: June 2021

Vascular calcification is correlated with morbidity and mortality in coronary artery disease, diabetes and chronic kidney disease. In vitro, elevated phosphate levels induced smooth muscle cell calcification in a PiT‐dependent manner. To determine the in vivo role of PiT‐1, a mouse conditional allele was generated in which loxP sites flanked exons 3 and 4 of the PiT‐1 gene. Recombination occurred in the presence of Cre driven by the ubiquitous Sox‐2 promoter, and the resulting PiT‐1Δ allele would produce a nonfunctional protein with a shift in the reading frame and production of a stop codon at amino acid 121. Wild‐type and PiT‐1Δ/+ mice were produced at expected Mendelian ratios, but no PiT‐1Δ/Δ mice were born. Examination of early development revealed that the PiT‐1Δ/Δ embryos were arresting at embryonic stage 10.5 and most were resorbed by E13.5. Affected embryos displayed cardiac defects and an under‐vascularized yolk sac as compared to siblings. Semi‐quantitative RT‐PCR analysis confirmed that PiT‐1Δ/Δ embryos expressed a truncated form of mRNA, but only embryos harboring at least one wild‐type allele expressed wild‐type mRNA. Thus, we have successfully generated conditional and null PiT‐1 alleles and confirmed that PiT‐1 is required for normal mouse embryonic development.
Effect of Fluid Shear Stress on Embryonic Stem Cells
Authors: Ahsan Taby , Doyle Adele M , Nerem Robert
Published: June 2021

Physical forces, such as compression, tension, and shear, have long been applied to differentiated cells. The effects of applied mechanical cues on stem cell self‐renewal and differentiation, however, have yet to be well characterized. In these studies, we focus on the effects of applied fluid shear stress on mouse embryonic stem cells. Previously, we had shown that the application of 15 dynes/cm/cm of shear stress to mouse embryonic stem cells significantly (p<0.05) increased protein expression for endothelial markers (e.g. Flk1, VECAD, PECAM) and promoted endothelial functionality (structure formation on Matrigel(tm) in vitro). Recent mRNA expression studies have found that the application of shear over a range of magnitudes (5‐15 dynes/cm/cm) resulted in similar changes on embryonic stem cell differentiation in Flk1, FLT, VECAD, and PECAM. towards an endothelial phenotype. Changes in magnitude, however, differentially regulate (p<0.05) genes associated with atherosclerosis, such as PPARG and VCAM1. The authors would like to acknowledge the Parker H. Petit Institute for Bioengineering & Bioscience and the NSF ERC "Georgia Tech/Emory Center for Living Tissues" for funding.
Monocyte CD11c/CD18 expression is upregulated postprandially and mediates firm arrest on VCAM‐1
Authors: Gower R Michael , Wu Huaizhu , Knowlton Anne A , Ballantyne Christie M , Simon Scott I
Published: June 2021

Atherosclerosis is an inflammatory disease initiated by hyperlipidemia and monocyte recruitment. Low shear stress and cytokine activation increases VCAM‐1 expression, the principle endothelial receptor facilitating monocyte arrest. We reported that monocyte integrin CD11c/CD18 binds VCAM‐1 and cooperates with VLA‐4 to arrest monocytes on inflamed endothelium. In this study, we determined if CD11c expression on monocytes from 60 human donors increased in a hyperlipidemic state. Blood was drawn from fasting donors and again at 3.5hrs after a 45% fat meal. Resting and MCP‐1 stimulated levels of CD11c on monocytes were increased after the meal. Highest CD11c expression correlated with high blood levels of total cholesterol (>200mg/dL) and low HDL (<35mg/dL). To assess the role of CD11c in recruitment, monocytes were isolated from ApoE−/−/CD11c−/− mice and perfused through a microfluidic chamber derivatized with VCAM‐1 and E‐selectin. When CD11c was absent there was a 45% decrease in firm arrest compared to CD11c+/+ cells. Antibody blocking of VLA‐4 inhibited monocyte arrest to a similar extent for both genotypes. When firmly arrested monocytes were exposed to a 5‐fold increase in shear stress, 40% more CD11c+/+ cells remained firmly arrested. We conclude hyperlipidemia associated with a high fat meal can upregulate monocyte CD11c, and its binding to VCAM‐1 is a critical step in high avidity monocyte adhesion. Research supported by HL082689 to S.I.S. and T32 HL086350‐01A1 to R.M.G.
Dynamic Analysis of Vertebrate Cardiogenesis
Authors: Yang Jennifer , Lansford Rusty , Fraser Scott
Published: June 2021

Vertebrate heart morphogenesis is a complex process that integrates different structures and cell types driven to interact by genetic and epigenetic factors. We have taken an interdisciplinary approach to studying heart formation by combining the power of molecular genetics with advanced optical imaging within living embryos. We have developed a Tie1‐H2B.eYFP transgenic quail and novel imaging methods that permit dynamic observation and manipulation of vascular development. Avian embryos are ideal for imaging because they are accessible at all stages of development outside of their vertebrate mother, unlike mammals, yet they develop a heart that has four chambers. Our over‐arching goal is to analyze heart morphogenesis and assess the relative roles of epigenetic and genetic mechanisms. Our experimental plan directly tests the hypothesis that flow is a major determinant of cardiac development and offers experimental tools that can be applied in a variety of experimental settings.

Grant Funding Source

NIH Cardio
Exploring the Chondrocytic Potential of Cranial Neural Crest Cells
Authors: Ainsworth Sophie Jane , Evans Darrell
Published: June 2021

Osteoarthritis is a degenerative joint disease where the joint cartilage becomes damaged. Despite the ability of cranial neural crest cells (CNCC) to form cartilage in the embryo, this population of cells has been overlooked when designing cell‐based therapies for osteoarthritis and associated diseases We have begun to explore the potential of CNCC in developing tissues. A LacZ‐encoded replication‐deficient retrovial vector was used to tag explants of CNCC excised from stage 10 chick embryos before injecting into the joint regions of developing limbs of stage 18‐28 embryos. Tagged chondrocytes were subsequently identified in the developing cartilage, particularly in the region of the developing joints. The results appear to demonstrate that cranial neural crest derived cells (CNCDC) retain their ability to give rise to cartilage when implanted into the developing limb and are restricted to particular regions of the limb. The chondrocytic potential of CNCC is now being refined to investigate the integrative properties of these cells in developing and regenerating cartilage. Funded by Brighton & Sussex Medical School.
Quantifying relative shape variation in modern human femora and humeri
Authors: Sylvester Adam David , Mahfouz Mohamed R.
Published: June 2021

Lower limb bones are thought to be more developmentally stable (more symmetric) than upper limb bones, presumably because of the functional importance of locomotion. However, the relative degree of canalization (limits on between‐individual variation) in the limbs is less clear. Here we assess the 3D shape variation between individuals in a sample of modern human femora and humeri.

Femora and humeri from 47 human males were CT scanned and 7002 landmarks were distributed across the surface of the bone using a statistical shape atlas (Merkl and Mahfouz, 2007). Each bone was scaled by its first principal component score to remove size and size‐correlated shape, and then by maximum length so that the femora and humeri were the same size. Bone shape variation was assessed as the average root‐sum‐squared distances of bone vertices from their average positions within each element. Bootstrap samples (1000) were used to create distributions of shape variation for each bone type, and distributions were compared using an ANOVA.

The analysis reveals that after accounting for differences in size and size‐correlated shape, the humerus is more variable in 3D shape than the femur (p< 0.0001), indicating that the 3D shape of the femur is more canalized than the humerus.

Grant Funding Source

None
Elastic properties and microstucture of external cortical bone in the craniofacial skeleton of the baboon
Authors: Wang Qian , Lin Angela S.P. , Guldberg Robert E.
Published: June 2021

Bone diseases have imposed tremendous clinical needs in bone health maintenance and reconstructive surgery. It has been realized that successful bone regeneration must go beyond reproducing shape and structure to restore biological and mechanical function as well as long‐term integration with surrounding native tissues. Recent studies have demonstrated that bone material properties and microstructure vary throughout the skulls. However, how the bone microstructure is related to the mechanical properties of bone at the tissue level needs systematic investigation. Samples from the outer cortical table in the neurocranium, supraorbital torus, zygomatic arch, and face of a baboon skull were assessed using ultrasonic and micro‐CT analyses. Results demonstrated that there were significant variations in bone elasticity and morphology of osteonal canals in different areas, and the principal orientation of osteonal canals roughly agreed with the axis of maximum stiffness and further with the long axis of the bone at some sites. These findings and further studies on the microstructural basis of bone mechanical quality at tissue level and skeletal function at the organ level will greatly enhance our ability to address questions derived from both evolutional and craniofacial medicine fields about the development and biomechanics of skulls. Supported by MEDCEN and Mercer Seed.
Interplay between Np95 and Eme1 in the DNA damage response
Authors: Mistry Helena Sumantrai , Gibson Lianne , Yun Ji Weon , Sarras Haya , Tamblyn Laura , McPherson John Peter
Published: June 2021

Methyl methansulfonate UV sensitive clone 81 (Mus81) and essential meiotic endonuclease 1 (Eme1) form a heterodimeric endonuclease that preserves genomic integrity through the repair of DNA replication‐associated damage. Despite its role as genome guardian and tumour suppressor, our understanding of how this endonuclease is coordinated during the DNA damage response is poor. In order to identify other proteins that interact with Mus81‐Eme1, our laboratory conducted a protein interaction screen to identify and characterize proteins that interact with Eme1. One candidate identified in this screen was found to encode the carboxy‐terminal domain of Np95, a chromatin‐associated ubiquitin ligase. Np95 and Eme1 co‐localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co‐localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. These findings for the first time, link the tumour‐suppressor Mus81‐Eme1 with the replication‐associated chromatin modifier functions of Np95 in the DNA damage response. Supported by grants from the Canadian Cancer Society through the National Cancer Institute of Canada and the Cancer Research Society.
Saccharomyces cerevisiae G2 checkpoint and double‐strand break (DSB) repair genes synergistically suppress the formation of homology‐directed translocations
Authors: Fasullo Michael Thomas
Published: June 2021

Checkpoint and DNA repair mutants exhibit more genetic instability compared to wild‐type yeast. We previously observed that G2 checkpoint and rad51 mutants exhibit elevated rates of spontaneous translocations. We measured rates of spontaneous translocations in diploid mutants defective in checkpoint and/or DSB repair genes. Directed translocation events were measured by selecting for His+ recombinants that result from mitotic recombination between two truncated his3 genes. The rate of spontaneous translocations in wild type is 4.4 x 10−8. Whereas rad51 and rad9 mutants exhibited fourfold and sevenfold higher rates of spontaneous translocations, rad51 rad9 double mutants exhibited 39‐fold higher rate. Compared to rad51 rad9, we observed similar rates in rad55 rad9 and rad57 rad9 mutants. While xrs2, rad50, and mre11 mutants exhibited 10‐20 fold higher rates, compared to wild type, xrs2 rad9, rad50 rad9, and mre11 rad9 exhibited similar rates compared to rad51 rad9. Interestingly, rate of spontaneous translocations in mec1‐21, a S phase checkpoint mutant that retains some G2 checkpoint function, was 23‐fold higher than wild type, while rates in mec1‐21 rad51 double mutants were 100‐fold higher. Pulse field gel electrophoresis confirmed that recombinants contain translocations. The results illustrate that both DSB repair pathways and G2 checkpoint pathways suppress translocation frequencies.

Grant support: CA70105 from the National Cancer Institute
The transcription factor Lc‐Maf participates in Col27a1 regulation during chrondrocyte maturation
Authors: Holden Devin Neal , Mayo Jamie L. , Barrow Jeffery R. , Bridgewater Laura C.
Published: June 2021

The transcription factor Lc‐Maf has previously been shown to play an important role in endochondral ossification. In this study, enhancer/reporter assays were used to determine whether Lc‐Maf could regulate cartilage‐specific enhancers from the Col27a1 and Col11a2 genes, which are both expressed in chondrocytes during endochondral ossification. The Col27a1 enhancer was upregulated over 4‐fold, while the Col11a2 enhancer was affected very little by Lc‐Maf. To confirm these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with an Lc‐Maf expression plasmid, and quantitative RT‐PCR was performed to measure expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was found to be upregulated approximately 5‐fold by Lc‐Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in embryonic mouse limbs undergoing endochondral ossification. Results demonstrated that Lc‐Maf and Col27a1 mRNAs were most prominent in prehypertrophic and proliferating chondrocytes, and that type XXVII collagen protein was similarly most abundant in prehypertrophic and proliferating chondrocytes in these samples. This work suggests that Lc‐Maf may affect endochondral ossification by participating in the regulation of Col27a1 expression. Supported by NIH grant AR4883 and by the Fulton Family Foundation.
Enhancing polyadenylation in vivo with bioinformatically identified auxiliary cis‐elements
Authors: Darmon Sarah K. , Tian Bin , Lutz Carol S.
Published: June 2021

Polyadenylation is a processing event eukaryotic cells use to make mature translatable mRNA. Cis‐elements in the 3' untranslated region (UTR) of mRNA aid in efficiency of polyadenylation. Upstream of the polyadenylation site (PAS) is a hexamer that is most often AAUAAA. Downstream is a U/GU‐rich sequence. Both elements bind core polyadenylation factors. Additional auxiliary cis‐elements further upstream or downstream of the PAS were previously identified in a bioinformatic survey. We used this predicted information to direct our in vivo validation studies. Auxiliary elements were placed in their respective sites in the cyclooxygenase‐2 (COX‐2) 3'UTR. An in vivo polyadenylation assay determined the strength of the polyadenylation signal. Upstream elements (USEs) enhanced usage of the COX‐2 PAS three to four fold. Downstream elements (DSEs) enhanced usage two fold. A luciferase assay showed increased protein levels with the insertion of the auxiliary elements. USEs had greater enhancement in protein levels compared to DSEs. Overall, some auxiliary elements had more enhancing effects than others, with some elements giving little effect as compared to wild type. We conclude that auxiliary elements have a role in enhancing polyadenylation and may aid in polyadenylation choice during alternative polyadenylation. Research supported by NIH and the Foundation of UMDNJ grants.
The switch between unextendable and extendable telomere states is mediated by the Hsp82 molecular chaperone
Authors: DeZwaan Diane C. , Toogun Tunji A , Freeman Brian C.
Published: June 2021

Telomeres are heterologous protein assemblies localized at linear chromosome ends that serve to both protect and maintain the DNA. Remarkably, the telomere system functions effectively in vivo despite a common DNA binding specificity by multiple telomere‐proteins. Why the factors do not interfere with each other and how transitions between the different telomere structures are mediated is not understood. We sought to test directly whether the Hsp82 molecular chaperone might function with a telomere capping structure, by establishing and in vitro telomere capping complex, demonstrating whether Hsp82 affects the cap, and determining how a switch occurs between different functional telomere states. We have established a novel two‐state in vitro telomere system in which the telomeric DNA forms either an unextendable protein structure or arranges into a telomerase‐extendable complex. Notably, we find that Hsp82 triggers the switch between the telomere capping and extending structures by modulating the DNA binding activity of the central telomere‐protein Cdc13. We suggest that Hsp82 regulates the DNA‐bound state for heterologous proteins, thus enabling a dynamic protein environment along the genome.
Characterization of substrate binding to the Group II Archael Chaperonin from Methanococcus maripaludis (Mm‐Cpn)
Authors: Goulet Daniel , Knee Kelly M , Jameel Shea , King Jonathan Alan
Published: June 2021

It has been difficult to identify the features of partially folded proteins that are recognized by the Group II chaperonins. A partially folded intermediate of the two‐domain all β‐sheet human γ‐D crystallin has been well characterized. We have investigated the interactions of Methanococcus maripaludis chaperonin (Mm‐Cpn) with Human γ‐Crystallin substrates. A homologue of human chaperonins, Mm‐Cpn binds to and inhibits the aggregation of several closely related members of the Human γ?Crystallin protein family including HγD‐ and HγC‐crystallins. Although members of this protein family share a high degree of homology, Mm‐Cpn suppression of HγD aggregation is twice as efficient as the suppression of HγC aggregation. It remains unclear whether this difference in suppression efficiency is linked to sequence‐determined substrate binding kinetics or to differences in substrate aggregation kinetics. To better understand how sequence difference may affect aggregation and chaperone binding, we describe the chaperone‐induced aggregation suppression for the isolated N‐terminal and C‐terminal domains of both HγD and HγC. Supported by an NIH Roadmap Award to the Center for Protein Folding Machinery
Hsp90 and p23 facilitate steroid hormone receptor signaling in yeast
Authors: Tan Xiaobing , Wilson Mark , Miller Charles
Published: June 2021

Steroid hormone receptors (SHRs) are among the best‐characterized "client" proteins of Hsp90 complexes. However, effects of Hsp90 and its co‐chaperone on SHR signal transduction have not been examined in a common system. We used a yeast system engineered to express human androgen, estrogen αand ?, glucocorticoid, mineralocorticoid, or progesterone receptors along with relevant reporter genes and tested whether Hsp90 isoproteins and p23 affected SHR signaling. Deletion of the endogenous HSC82 (homolog of human Hsp90 ?) or SBA1 (homolog of human p23) genes in this system strongly inhibited ligand‐mediated signaling of all SHRs in reporter gene assays. HSC82 gene deletion had greater impact on SHR signaling than did SBA1 gene deletions. Deletion of the HSP82 gene (homolog of human Hsp90α) did not affect SHR signaling, suggesting that the Hsc82p‐Sba1p complex specifically supports SHR signaling in this system. Effects of a lack of Hsc82p or Sba1p were restricted to SHR signaling because both cell growth and signaling by an unrelated reporter construct were unaffected by chaperone status. Geldanamycin and radicicol (Hsp90 inhibitors) strongly reduced SHR signaling at doses that did not affect cell growth. Collectively these results indicate that all SHRs, including estrogen receptorβ, are dependent on the Hsp90 chaperone system for proper function. Funded by NCI 1R33CA101622‐01A2.
Expression and characterization of Francisella tularensis fatty acid biosynthetic genes FabD and FabH in E. coli
Authors: Chmielowski Jennifer N , Bohn Kelsey C , Wen Lisa , Huang Jenq‐Kuen , Timsina Yam N , Chand Phani C , Pathak Ashish K
Published: June 2021

Francisella tularensis is an extremely infectious airborne pathogen, and has long been considered as a potential biological weapon. The intent of this research is to study a key regulatory enzyme of type II FAS of Francisella tularensis, namely FabH, as a novel drug target against tularemia. FabH, also known as 3‐oxoacyl‐acyl carrier protein synthase III, catalyzes the initial condensation and elongation reactions in fatty acid synthesis. Genes encoding the Francisella tularensis of FabD and FabH were custom synthesized and inserted in pET15b expression vector by Celtek Genes. Each recombinant protein was overexpressed by IPTG induction. Both recombinant FabD and FabH proteins were purified by affinity chromatography on a Ni‐NTA column. FabH activity was measured by monitoring the conversion of [14C]‐acetyl‐CoA and malonyl‐ACP substrates (produced by FabD in the coupled assay) to a radiolabeled acetoacetyl‐ACP. The active enzyme has been successfully used in screening a class of 46 newly synthesized indole‐2‐carboxylic acid derivatives for its inhibition. Three compounds have shown IC50 values in the range of 2‐6 µM against Francisella tularensis FabH.

Supported in part by the University Research Council, Western Illinois University.