bluuebunny/crossref_metadata_2025 huggingface dataset:
A fully implantable bolus injector for biotelemetric studies
Authors: Axelsson Michael , Pitsillides Koullis
Published: June 2021

Pharmacological studies in freely moving animals equipped with telemetric systems is hampered by the lack of exteriorized catheters for administrating pharmacological substances or have to rely on slow infusion‐ or mini‐osmotic pumps. To overcome this limitation a fully implantable bolus injector was developed. The injector can be activated remotely when used in conjunction with the EndoGear (Endosomatic Systems Inc, CA, USA) series of implantable biotelemetry systems. The pressure and flow generating capacity of the injector were measured at 4 different output pressures (0, 5, 10 and 15 kPa). Pressure and flow were recorded using a pressure transducer (DPT‐6100, pvb Medizintechnik, Germany) and an in‐line, flow‐through transit time flow probe with an internal diameter of 1 mm (Transonic Systems Inc, USA).

The results show that the injection time, here defined as the time between the injector activation time and the time when flow dropped back below 0.5 ml min−1 is pressure sensitive and the injection time at 15 kPa is 3.2 seconds longer than at 0 kPa. The injection time at 15kPa output pressure is still just above 10 sec and well within the specifications for safe and acceptable bolus injection times. The remote control of the injector by the EndoGear implantable biotelemetry system makes this a useful tool for pharmacological studies in freely moving animals, without disturbing the animals.
Diastolic Dysfunction in Rats Following Intravenous Injection of HIV gp120
Authors: Chen Fangping , Berzingi Chalak , Huang Aijie , Finkel Mitchell s
Published: June 2021

HIV infection in patients is associated with a surprisingly high frequency of diastolic dysfunction followed by the development of a dilated cardiomyopathy. Potential mechanisms include direct effects of HIV proteins, including gp120. We have previously reported direct inotropic and signaling effects of HIV gp120 on isolated cardiac myocytes in vitro. We now report effects of a single injection of HIV gp120 on cardiac hemodynamics in vivo. HIV gp120 (50ug/kg) was injected intravenously and hemodynamic measurements were made at 1 hr, 24 hrs and 48 hrs in freely ambulatory, awake rats. Rats injected with gp120 demonstrated a blunted diastolic response to increasing doses of intravenous infusion of the beta‐adrenergic agonist, isoproterenol(ISO), compared with similarly instrumented controls at 48 hrs (gp120 vs control, p<0.01; n=6, for each). Pre‐treatment with the p38 MAP kinase inhibitor, SB 203580, prevented the diastolic dysfunction (gp120+ SB vs control, P=NS; n=6, for each). Western analyses confirmed the inhibition of p38 MAP kinase by SB in vivo. There were no differences in ISO dose response curves or p38 MAP kinase phosphorylation between gp 120‐treated and control rats at 1 or 24 hrs. (P=NS; n=6, for each). Thus, evidence of diastolic dysfunction is apparent in rats following a single dose of HIV gp120. These findings lend further support to the hypothesis that the HIV virus, itself, may contribute to myocardial dysfunction in HIV infected patients.
Dilated cardiomyopathy in aged cardiac‐specific EP4 receptor KO mice
Authors: Harding Pamela , Yang Xiao‐Ping , He Quan , Yang James , Shesely Ed , LaPointe Margot C
Published: June 2021

To determine if a cardiac phenotype develops in aged EP4 KO mice, we performed echocardiography to assess ejection fraction (EF) on 23‐33‐week‐old male KO and wild‐type (WT) littermates. Hearts were removed for histology to assess hypertrophy (MCSA), fibrosis (ICF) and macrophage infiltration. WT mice had normal EF ( 80±0.6%, n=70), whereas KO mice had a lower EF (60±2.7%, n=55, p<0.001) coupled with left ventricular dilatation. MCSA and infiltrating macrophages were not different between groups but ICF increased by 35% in KO mice. In the KO mice, half had an EF below 60% and half above [EF in KO‐high function group (77±1.3%, n=28) vs EF in KO‐low group (42.5±2.4%, n=27); p<0.001]. In contrast to male KO mice, female KO mice had no cardiac phenotype through 28 weeks of age. Whole genome gene expression profiling (Illumina Mouse WG‐6 v2 chips) on hearts of 30‐32 week old male WT and KO mice indicated that 1100 genes were significantly differentially expressed. Of the 593 genes over expressed in the KO heart, many were involved in cardiac remodeling, apoptosis, calcium handling and cell signaling, with quantitative differences between KO low vs high function groups. In conclusion, the absence of EP4 in cardiac myocytes in a subgroup of aged male KO mice is associated with increased fibrosis, reduced EF and dilated cardiomyopathy.
Beta‐adrenergic Stimulation Activates Protein Kinase Cε through Epac in Cardiomyocytes
Authors: Duquesnes Nicolas , Li Lin , Morel Eric , Metrich Mélanie , Matéo Philippe , Lucas Alexandre , Fortin Dominique , Lezouac'h Frank , Crozatier Bertrand
Published: June 2021

Protein kinase C (PKC) activation is classically considered as independent of the β‐adrenergic pathway. However, the cAMP‐ activated exchange factor Epac was recently shown to activate phospholipase C. βA stimulation is thus likely to stimulate PKC. We evaluated in cardiomyocytes whether βA stimulation could activate PKCε. Rat neonatal cardiomyocytes were subjected to isoproterenol stimulation (ISO). Inositol trisphosphate production was increased by 34% by 1µM ISO (p<0.05) and PKCε was translocated to particulate fractions (PF, western blot) in the perinuclear area (confocal microscopy) in a PKA‐independent manner since it was not inhibited by an infection with an adenovirus encoding a PKA inhibitor. Instead, PKCε activation was Epac dependent since 8‐CPT, an Epac activator, induced the same PKCε translocation as ISO and siRNAs of Epac completetly inhibited PKCε activation. The same translocation of PKCε in PF induced by βA stimulation was found in adult isolated rat hearts perfused by ISO with a sarcolemmmal membrane localization. This was associated with a phosphorylation of connexin‐43 on ser368 that was blocked by the PKC inhibitor BIM. In conclusion, these data demonstrate a new interconnection between β‐adrenergic and PKC pathways via Epac in cardiac cells with a potential role in cell‐to‐cell communications.
Mechanisms of ATP release from 1321N1 cells
Authors: Blum Andrew , Dubyak George
Published: June 2021

Astrocytes are an important source of extracellular ATP in the brain. Regulated release of ATP from astrocytes occurs in response to diverse metabolic, mechanical, or inflammatory stimuli. Non‐junctional hemichannels composed of connexin 43 have been shown to be a major conduit for release of ATP from murine astrocytes in response to hypoxia, but the role of non‐junctional connexin hemichannels in G‐protein coupled receptor (GPCR) mediated ATP release is not determined. Previously, using 1321N1 human astrocytes we found that rapid accumulation of active GTP‐bound Rho in response to GPCR activation greatly enhances Ca2+ dependent ATP release. GPCR induced ATP release from 1321N1 cells is attenuated by carbenoxolone, flufenamic acid, and probenecid. These structurally unrelated compounds are non‐selective inhibitors of non‐junctional hemichannels. Here we investigate the contribution of connexin 43 non‐junctional hemichannels to GPCR induced ATP release from 1321N1 astrocytes by connexin 43 overexpression and connexin 43 knockdown.
Angiogenic Gene Modification Of Skeletal Muscle Cells To Compensate For Age‐related Decline In Function
Authors: Zhan Wang , Delo Dawn , Eberli Daniel , Atala Anthony , Soker Shay
Published: June 2021

INTRODUCTION

In this study, we investigated muscle regenerative ability in different age groups of experimental animals.

METHOD

Muscle precursor cells (MPCs) were cultured from mice. In vitro analysis included cell growth assay, quantitative myotube formation. In vivo studies involved cells transfected or not transfected by adenovirus encoding Vascular Endothelial Growth Factor (VEGF). MPCs were injected subcutaneously into mice. After 2 and 4 weeks, the engineered muscle samples were harvested and analyzed.

RESULTS

The young mice MPC growth rate and myotube formation were greater than mature mice and old mice MPC. Bioengineered muscle tissues were smaller and less contractile in old mice group. With expressing recombinant VEGF, bioengineered muscles in all age groups had a greater mass, increased neovascularization, and contractile function. Histological analysis of the in vivo engineered muscle tissue stained positive for muscle specific proteins.

CONCLUSIONS

This study demonstrates the MPC regenerative possibilities in young and old mice, and augmentation of neovascularization increased muscle mass and strength of contractility of engineered muscle tissues. Funding NIH Ruth L. Kirschstein National Research Service Award Individual Fellowship (F31), the Novartis‐Stiftung, the Roche Research Foundation, GEBERT RÜF STIFTUNG, and CROWN Foundation.
Control Entropy (CE) Identifies Constraints of Treadmill Locomotion in Highly Trained Runners
Authors: McGregor Stephen James , Busa Michael A , Yaggie James A , Skufca Joseph D , Bollt Erik M
Published: June 2021

The purpose of this study was to test if Control Entropy (CE), a novel regularity measure not limited by the requirement of stationarity, could identify differential changes in system constraints of locomotion in highly trained runners using high resolution accelerometry (HRA). Procedures were approved by the local HSRC. Collegiate runners (n=8, 21.4±1.7yr, 65.5±5.7 kg, 70.1±6.2 ml/kg/min) performed two level treadmill tests starting at 2 kph and increasing 2 kph/2 min to exhaustion. Gaits ranged from slow walking to maximal running. Data was streamed at 625 Hz from wireless triaxial HRA (Microstrain, VT) affixed to subject's lower back. CE (CEKS[x(t)] =hKS[sign(dx/dt(t))] was calculated using 8 bit partitions and multi‐scale window lengths and compared using K‐L analysis. Dominant CE modes in K‐L analysis showed that CE initially increased in all axes during the walk phase, then declined during running first in the vertical (V), then lateral (M/L) and anterior/posterior (A/P) axes (p<.05). These results indicate that constraints in locomotion are apparent earliest in the V, and latest in the A/P axes. Accelerations increased most dramatically in the V axis during running to overcome gravity (energetic constraint), while constraints in the A/P axis are most likely due to biomechanical limitations. Therefore, CE may indeed identify differential constraints during treadmill locomotion.
Three Dimensional Finite Element Modeling of Human Knee in response to external loads
Authors: alobaid yaqoob f
Published: June 2021

The knee is one of the most commonly injured joints in the human body. 3D FE modeling is a useful tool for understanding of the stress ‐ strain distributions within articular cartilage in response to external loads and investigating both the prevention of injury and the pathological degeneration of the joints. This paper propose 3‐D dynamic finite element (3‐DDFE) model based on the digital data set from the human Knee section. The analysis was constructed to demonstrate the effectiveness of the 3‐DDFE model as a potential Knee fracture investigatory technique. The fresh Computerized Topography (CT) scans were used for the Kneel analysis and the Magnetic Resonance Imaging (MRI) images for the brain model. The 3‐DDFE analysis of human Knee would allow the assessment of the injurious effects of different impact conditions and enable the development of enhanced Knee fracture and protection criteria for human Knee.
A Kinematic Model Coupling Cytoskeletal Dynamics with JNK Activation in Response to Matrix Stretching
Authors: Kaunas Roland , Huang Zuyi , Hahn Juergen
Published: June 2021

The role of the actin cytoskeleton in regulating mechanotransduction in response to external forces is complex and incompletely understood. Here, we develop a mathematical model coupling the dynamic disassembly and reassembly of actin stress fibers and associated focal adhesions to the activation of c‐jun N‐terminal kinase (JNK) in cells attached to deformable matrices. The model is based on the assumptions that stress fibers are pre‐extended at a "homeostatic" level under normal, non‐perturbed conditions, and that perturbations in stress fiber length destabilize stress fibers. The subsequent reassembly of fibers upregulates the rate of JNK phosphorylation as a result of the formation of new integrin bonds within the associated focal adhesions, while the rate of JNK dephosphorylation is independent of the stretch signal. Disassembly of overly stretched stress fibers and reassembly of stress fibers at the "homeostatic" level lead to a decrease in the rates of integrin turnover and JNK activation. Numerical solutions of the model equations predict that different patterns of matrix stretch result in distinct temporal patterns in JNK activation that compare well with published experimental results. This model describes a mechanism by which the dynamic properties of the actin cytoskeleton allow cells to adapt to applied forces to modulate intracellular signaling.
Global Analysis of Endocytic Recycling
Authors: Conibear Elizabeth , Burston Helen E. , Maldonado‐Báez Lymarie , Davey Michael , Montpetit Benjamen , Schluter Cayetana , Wendland Beverly
Published: June 2021

Endocytosis of receptors at the plasma membrane is controlled by a complex machinery that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, suggesting yeast endocytosis may be subject to different regulatory mechanisms. We have systematically defined genes required for internalization using a quantitative genome‐wide screen that monitors localization of the yeast VAMP/Synaptobrevin homolog Snc1. Genetic interaction mapping placed these genes into functional modules containing known and novel endocytic regulators and uncharacterized factors. We demonstrate a cargo‐specific requirement for clathrin and the yeast AP180 clathrin adaptor proteins. In addition, we identify a novel conserved component of the endocytic machinery that is recruited to cortical actin patches prior to actin polymerization and is required for normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic recycling.
The Bilateral Nature and Origin of the Medial Circumflex Femoral Artery
Authors: Beckman Michael , Frumberg David , Márquez Samuel
Published: June 2021

The medial circumflex femoral artery (MCFA) largely supplies the posterior aspect of the femoral head while also contributing to the neck of the femur. It thus plays a major role in the healing of hip fractures and prevention of subsequent avascular necrosis in this susceptible area of the human body. The MCFA is commonly described in anatomy and orthopaedic textbooks as typically arising from the deep artery of the thigh close to the femoral artery. In this study, 76 medial circumflex femoral arteries from 38 adult cadavers representing an urban population were surveyed during the first year gross anatomy courses for College of Health Related Professions and College of Medicine students at SUNY Downstate Medical Center. One cadaver was excluded due to a synthetic aortic‐iliac‐femoral arterial system yielding a final sample size of 74 hips from 37 cadavers. Results show a large amount of variation in the origin of the MCFA with the following four general patterns emerging from this study: 1) MCFA branches off the deep artery of the thigh; 2) MCFA branches off the femoral artery proximal to the origin of the deep femoral vessel; 3) MCFA branches off the femoral artery distal to the origin of deep femoral vessel; and 4) MCFA branches off at the bifurcation of the femoral and deep arteries of the thigh in a "trifurcation" pattern. The low statistical finding of only 37.8% of our sample with the MCFA branching off the deep femoral artery is surprising given the high frequency reported in the literature. The fact that about 50% of cadavers from this study showed bilateral asymmetry emphasizes not only the highly variable nature of MCFA branching patterns but decreases the predictive power of symmetry in any given individual.

Grant Funding Source

Do Not Have Funding
Clavicular length asymmetries in primates indicative of handedness in daily activity
Authors: Grisotti Gabriella , Gannon Patrick J. , Marquez Samuel
Published: June 2021

Handedness is well understood in humans but not for other primates. Some studies in non‐human primates have focused on behaviors such as reaching for food correlated with hemispheric specialization. Other studies reported asymmetry in upper limb bones of Pan troglodytes to support handedness. Given relations of the upper limb and clavicle, we hypothesized that evidence of handedness would be manifest in measurements of clavicular length. We collected data from chimpanzees (n=8), rhesus (n=2), and spider (n=2) monkeys to represent great apes and Old and New World monkeys from the Division of Mammalogy at the American Museum of Natural History. Preliminary results showed a non‐significant but 62% rightward asymmetry in chimpanzees but essentially symmetry in the monkeys. Clearly, a larger sample size is required to explore these findings further. The relationship of the clavicle between the axial and appendicular skeletons means that it is responsible for transmitting a great deal of force. Activities of the handed upper limb may well have a differential effect on the clavicle. Thus, the clavicle may offer a unique approach to recognize handedness even in extinct primates.

Grant Funding Source

Do not have funding
An anatomical exploration of femoral angle of inclination in multi‐regional populations: clinical implications
Authors: Charubhumi Daniel , Schachar Jeffrey S , Chaudhri Ali , Márquez Samuel
Published: June 2021

The angle of inclination (AOI) is clinically relevant because of a predisposition for congenital femoral dysplasia. While AOI of the femur varies with age, gender, and ontogenetic growth patterns, abnormal AOI conditions increase stress at the hip joint and significantly affect gait. This study examined AOI in three ethnic/racial populations from European, African, and Asian ancestry. An Inuit group dated 500 BCE ‐ 500 CE was sampled from archaeological remains of Ipiutak populations from the osteological collections of the American Museum of Natural History. To measure AOI, a line was drawn from the midpoint of the intercondylar fossa to the greater trochanter projecting proximally through the shaft of the femur, and a second line was drawn from the femoral head to the femoral shaft through the center of the femoral neck; the AOI was defined by the intersection of these two lines. Preliminary results showed significant AOI differences (p < 0.01) found only between the Inuit and European populations. Developmentally, the AOI is widest at infancy and gradually narrows, ending its decline in adulthood. Inuit femurs presented with the greatest AOI of 139 degrees, demonstrating the orthopaedic condition of coxa valga. The fact that these findings suggest a possible increased prevalence of congenital femoral dysplasia in these populations has considerable clinical implications and will require further study.

Grant Funding Source

N/A
Femoral neck anteversion: an orthopaedic evaluation between multi‐regional populations
Authors: Schachar Jeffrey Scott , Charubhumi Daniel , Marquez Samuel
Published: June 2021

Femoral neck anteversion (FNA) varies with age, sex, and ontogenetic growth pattern. This study examined African (Af), European (E), and two Asian groups, one of which, an Inuit (I) population from osteological collections of the American Museum of Natural History. FNA is the angular difference between proximal and distal ends of the bone. A line was drawn in the coronal plane through the lateral and medial femoral condyles. A second line was drawn from the greater trochanter to the head of the femur, through the center of the femoral neck. The intersection forms the anteversion angle. Selecting only male and right femurs controlled for the confounding variables of sex and asymmetries. Statistically significant differences (p < 0.05) were found between E and I groups and between Af and I groups. Average anteversion angle for the Af group was 15.7 degrees, 11.1 for the E group, 33.4 for the I group, and 23.0 for the Asian group. Developmentally, the amount of FNA is 40 degrees at birth, decreasing to ~15 degrees at maturity. The study revealed that the I sample presented with the largest angle, displaying excessive femoral torsion. Femoral torsion is viewed symptomatically as in‐toeing, which usually corrects without treatment as the child matures. Our results suggest that the Inuit sample may not have undergone the physiologic torsion reduction of 25 degrees as seen in the other representative samples.

Grant Funding Source

N/A
MORPHOLOGYCAL TYPES OF INTRAPARENCHYMAL SPATIAL DISTRIBUTION OF BILE DUCTS OF CAUDATE LOBE. STUDY ON CORROSION CASTS
Authors: MATUSZ Petru L , HORDOVAN Eniko‐Christine , PUSZTAI Agneta Maria
Published: June 2021

Terminologia Anatomica (1998) homologates 2 ducts of caudate lobe (CLD): right (RCLD) and left (LCLD), each originating from the homonym hepatic duct (right ‐ RHD and left ‐ LHD). Our previous work evidenced variability both in origin and in number of the CLD. We analyzed on 150 hepatic corrosion casts the types of spatial distribution of CLD according to the number of ducts and the drainage level. The corrosion casts were made by injecting with plastic (AGO II paste and TECHNOVIT 7143) of the vasculo‐ductal systems, followed by parenchyma corrosion with hydrochloric acid. At first we analyzed the formation of the intrahepatic bile ducts system (IHBDS) and found 3 distinct aspects: Type I (modal, 84.67%) confluence of RHD with LHD; Type II (6%) confluence of LHD with the anterior branch (ABr) and the posterior branch (PBr); Type III (9.33%) confluence of LHD with ABr. We found 307 CLD, their number varying between 1‐3/cast (1 CLD in 13.33%; 2 in 68.67% and 3 in 18%). According to the number and drainage level of CLD we found 21 morphological types. RHD collects 32.90% of all CLD and LHD 54.72%. We found also CLD that drain into: lateral branch (1.30%) medial branch (0.32%), anterior branch (0.98%) and posterior branch (9.77%). In cases when the posterior branch drains into LHD, the number of collected CLD increases. Knowledge of CLD drainage in other elements of IHBDS than RHD and LHD is useful when planning and performing caudate lobe parenchyma surgery (Supported by CNMP 41092/2007).
Looking the river Horse in the Mouth: A Reexamination of the Dentitions of the Living Hippopotami
Authors: Naples Virginia L. , Gramarye Justin
Published: June 2021

Dentitions of the two living species of hippopotamus, Hippopotamus amphibius and Choeropsis liberiensis were reexamined with regard to tooth numbers and types, and patterns of eruption, occlusion, and replacement. Dental formulae of both species of hippo were found to have been incorrectly interpreted by previous authors. In addition to thegosis, attrition caused by foraging behavior and tongue wear was noted on the incisors. Hippos have ever‐growing incisors and canines, and selection for the presence of unusually large teeth, i. e., tooth and tooth root space, orientation and positional requirements, have shaped the anterior portion of the skull in both species, for foraging and intraspecific competition. A diastema is produced by lateral displacement of the canine and elongation of the anterior rostrum to compensate for elongation of its root in H. amphibius. The latter factor has drastically reduced the available space for the first premolar, resulting in its elimination from the adult dentition, although the deciduous first premolar often persists into adulthood. An increased time interval between successive molar eruptions in the common hippo reflects a possible adaptation to a lifespan of up to sixty years.
Forelimb anatomy of the aardvark, Orycteropus afer (Mammalia, Tubulidentata)
Authors: Voegele Georgina McKusick
Published: June 2021

Soft tissue anatomy, especially myology, has been largely neglected by systematic biologists reconstructing the phylogeny of Mammalia. This is due to inadequate description and illustration of structures in many mammalian orders, and conflicting or inaccurate terminology in the existing descriptions. While anatomical terminology has been standardized for the domestic animals and their myology is well known, many rare mammals have not been dissected in decades and the available descriptions lack standardization and often compare taxa now known to be genetically unrelated. This leaves systematic biologists unable to verify muscle character states in the exotic mammals whose evolutionary relationships are most controversial. For example, little morphological evidence has been found to support the clade Afrotheria, which unites several mammalian orders thought to be unrelated until the advent of genetic sequencing.

Here I present the forelimb anatomy of the aardvark, Orycteropus afer, the one living member of the order Tubulidentata and a member of the clade Afrotheria, based on my dissections of two alcohol‐preserved museum specimens. These dissections are part of a dissertation project to document the limb anatomy of several mammalian orders, particularly the little known smaller afrotheres, for use in systematics research. This research was supported by the Field Museum of Natural History Visiting Scholar Fund.

Grant Funding Source

National Science Foundation
Statistical analysis and mathematical modeling of collective cell motion in endothelial and epithelial cultures
Authors: Szabo Andras , Unnep Renata , Czirok Andras
Published: June 2021

Cell migration within confluent monolayers is instrumental in several morphogenic processes. The phenomenon was studied with time‐lapse microscopy of in vitro monolayer cultures of endothelial and epithelial cells. Statistical analysis of cell trajectories reveals that motile confluent cultures may behave either as vortical fluids or as deforming elastic sheets. The type of behavior is determined by the duration of the cell‐cell contacts: trajectory analysis of adjacent cell pairs reveals that endothelial cells diverge easily, while other epithelial cells adhere to each other for an extended time period. The observed flow fields can be explained by our generalized cellular Potts model; in the model we represent cell polarization and the resulting self‐propulsion, as well as links between the cytoskeleton of adjacent cells.

The work was funded by the NIH (R01 HL87136), and the Hungarian National Office for Research and Technology (NKTH).
A method to quantify apoptosis in mouse embryos using flow cytometry
Authors: Mallela Murali K , Gutierrez Juan Claudio , Hrubec Terry C
Published: June 2021

Cell proliferation, differentiation and apoptosis play a major role in morphogenesis and organogenesis of the developing embryo. Teratogens can cause alteration in apoptotic patterns in the developing embryo which may lead to fetal malformations; however, no studies have quantified apoptosis in early embryos. We developed a protocol to quantify apoptosis in CD‐1 mouse embryos using flow cytometry. Valproic acid (VA) and diabetes mellitus were then used to induce neural tube defects and cardiac malformations respectively to evaluate apoptotic changes induced by known teratogens. Apoptotic levels were quantified in head regions of VA exposed embryos and hearts of hyperglycemic fetuses. Embryonic cells were disassociated enzymatically followed by differential staining with Annexin‐V and propidium iodide to differentiate apoptotic, dead and live cells. Apoptosis levels increased in the head regions of VA exposed embryos (18.1± 2.2 %) than the controls (12.7 ± 1.9 %). Increased levels were also observed in the hearts of hyperglycemic fetuses compared to controls. TUNEL assay, used to localize apoptotic regions, showed increased apoptosis along the open neural folds of the VA exposed embryos. This is the first report using flow cytometry to evaluate apoptosis in early embryos, and the results indicate this may be a useful method to quantify relative changes in apoptotic levels in teratogen exposure.

Grant Funding Source

NIH grant K01RR16241‐01e
N‐cadherin and actin are present at desmosomes in the seminiferous epithelium of the adult testis
Authors: de Asis Marc Aristaeus Kaw , Young J'Nelle S. , Vogl A. Wayne
Published: June 2021

Desmosomes in the seminiferous epithelium of the adult mammalian testis are not as well defined morphologically as those in most other epithelia. Moreover, the plasma membrane related plaques in Sertoli cells are linked to vimentin filaments, not to keratin filaments. Previous studies have indicated that these desmosomes contain classical actin‐related cadherins, thereby raising the interesting possibility that these junctions may have characteristics both of actin and of intermediate filament based junctions. To explore this possibility, we probed the seminiferous epithelium of adult male Spraque Dawly rats for N‐cadherin, actin, and plectin. In fixed frozen sections, N‐cadherin labeled sites at the base of the epithelium that only partly appeared to colocalized with the intensely labeled actin in ectoplasmic specializations. In fragmented epithelium, the actin probe reacted with plectin positive desmosomes. At the ultrastructural level, the probe for N‐cadherin clearly labeled desmosomes. Significantly, an antibody to actin also reacted above background with desmosomes and was localized close to the cytoplasmic face of the membrane related plaques. Our results are consistent with the conclusion that desmosomes in the seminiferous epithelium have characteristics both of actin and of intermediate filament based attachment junctions.

Grant Funding Source

NSERC
Endothelial mechanosensing by primary cilia
Authors: Hierck Beerend P , Van der Heiden Kim , Egorova Anastasia D , Van de Pas Simone , Poelmann Robert E
Published: June 2021

Primary cilia are microtubule containing membrane protrusions which are associated with fluid sensing in endothelial cells (EC). We show that ciliated EC are present at atherosclerotic predilection sites in adult mice under athero‐prone flow conditions, but not in athero‐protected areas. Upon plaque formation in Apoe−/− mice, as well as after experimental induction of flow disturbance, the number of ciliated EC is increased. The aim of this study was to relate the presence of a primary cilium on EC to the flow profile to which these cells are exposed to, in vitro and in vivo, and to decipher its role in the mechanosensor for shear stress. We analyzed EC under steady, pulsatile and oscillatory flow and show that ciliation is indeed induced by oscillatory fluid flow. EC respond to shear with an acute intracellular calcium transient, and with a prolonged change in gene expression. The latter is mediated through the microtubular part of the cytoskeleton to which the primary cilium is connected. Experimental de‐ciliation of EC largely abolishes the shear response, which shows that the primary cilium sensitizes EC for fluid shear stress.

The mechanosensor for shear forces facilitates a two step response in endothelial cells: (1) an acute and transient increase in calcium and release of stored vasoactive substances, and (2) a prolonged phenotypic adaptation which is reflected by altered morphology and gene expression.
In Vivo reorganization of alpha 1 integrin in developing skeletal muscle
Authors: Pereira Lindsay R , Mattioli Patrisia M , DiLullo Camille
Published: June 2021

Integrins play an important role in muscle development. In developing chick skeletal muscle In Vitro, the integrin subunit alpha 1 has been shown to reorganize from a punctate to a periodic distribution along the AI junction subsequent to sarcomere assembly. Also subsequent to sarcomere assembly, laminin, a major component of the basement membrane, has been observed with a periodicity along the myofibril. Analysis In Vitro has shown that the periodic distribution of laminin and alpha 1 integrin are correlated both spatially and temporally and suggests a role for alpha 1 integrin in the establishment of the basement membrane. To confirm the distribution of these proteins in muscle development In Vivo, sectioned skeletal muscle from embryonic chick days 16‐20 was examined. Initially muscle sections were immunofluorescently labeled with antibodies to alpha 1 integrin and muscle specific proteins. Data indicated that alpha 1 integrin reorganized from a punctate to a periodic distribution beginning at day 16. By day 18 all myofibers examined were observed to have alpha 1 integrin periodically distributed along the sarcomeres. The temporal and spatial distribution of laminin in relation to alpha 1 integrin at embryonic days 16‐18 will be examined to determine the correlation of these two proteins in In Vivo muscle development.
Role of KLF2 in atrioventricular cushion development
Authors: Haar Jack L. , Lung Tina K. , Chiplunkar Aditi R. , Lloyd Joyce A.
Published: June 2021

Krüppel like factors (KLFs) are transcription factors with important roles in development. KLF2 KO mice die by embryonic day 14.5 (E14.5) and exhibit heart failure and hemorrhaging. KLF2 is expressed in multiple tissues including erythroid and endothelial cells. Our preliminary studies have shown that, KLF2 KO mice have abnormal development of the atrioventricular (AV) cushions of the heart. Light microscopy revealed that the E9.5 WT (n = 3) AV cushion is lined by a single layer of endothelial cells, with underlying mesenchymal cells. In E9.5 KLF2 KO mice (n = 3), there is 2‐fold increase of cells in the AV canal; these cells are not in a single layer. There was no significant difference in the mesenchymal cell count, but KLF2‐/‐ have a decreasing trend, compared to WT. Electron microscopy revealed that the WT AV canal is straight with no recesses; the endothelial cells lining the canal are squamous with no cell processes extending toward the lumen. In KLF2‐/‐ embryos, the canal is open with many recesses. It is lined by endothelial cells having numerous cytoplasmic processes extending toward the lumen. Based on these observations, the KLF2 gene is required for normal AV cushion development. The findings in this study further suggest the testable hypothesis that there may be abnormal endothelial to mesenchymal transformation in the AV cushions of KLF2 KO embryos. (Supported by NIH R01DK074694)
Analysis of Tissue Layer Stratification in Cells over Expressing Cdk4 and Long‐time Cultured Cells with Green Fluorescence Protein
Authors: Terefe Eyuel Solomon , Vaughan Melville Bailey
Published: June 2021

I examined whether the stratification of keratinocytes, the primary cells of skin epithelium, that over expressed the Cdk4 and telomerase (hTER)‐which made them into immortal cells, had an influence on whether the keratinocytes tissue layer was organized as normal keratinocytes. These immortal keratinocytes were engineered to overexpress GFP so they could be used in wound healing models to monitor keratinocytes migration in real time as they healed a wound. The goal was to determine if GFP affected their ability to stratify and differentiate similar to normal keratinocytes. I arrived at my results by examining immunoflourcense stains for the presence of proteins within the epithelial layers/ dermal compartment in their proper location and orientation. The staining was for the proteins: Involucrin, Keratin 14, and basement membrane proteins collagen IV and laminin‐5. I found that the organization of the epithelial layers of the over expressing keratinocytes to be similar as those of non‐over expressing cells, which means that keratinocytes maintained epithelial cellular organization although they were going through abnormal proliferation. I also analyzed the epithelium layer organization of keratinocytes that were cultured for prolonged period. I also found that skin equivalents that were maintained for long period of time were similar to those that weren't maintained for such a long period.

Grant Funding Source

LS‐OKAMP
Keratinocyte senescence effects on differentiation and migration in a skin equivalent
Authors: Vaughan Melville Bailey , Andrews Capri M , Wright Woody E , Shay Jerry W
Published: June 2021

Keratinocyte telomere shortening has been demonstrated in autologous skin grafts from burn patients. Telomere shortening leads to keratinocyte replicative senescence in vitro. Wound healing is clearly delayed in telomerase knockout mice with telomere dysfunction. Our goal was to test the effect of keratinocyte replicative senescence on differentiation and migration using a human skin equivalent model. 14 day old skin equivalents were wounded using an excimer laser, then cultured for another 14 days. Tissues were harvested at various time points after wounding. Paraffin sections were stained to determine morphology and protein expression. Results were compared to early‐passage keratinocyte controls. Skin equivalents with aged keratinocytes showed reduced stratification, proliferation, and laminin‐5 staining in the basement membrane. Stratification proteins were unchanged. Interestingly, keratinocyte migration was accelerated. Decreased basement membrane staining and accelerated keratinocyte migration might be explained by increased matrix metalloproteinase expression, which will be determined later. Our previous studies suggest these changes can be rescued by telomerase.
Atypical PKC isoforms are expressed in regions of interest to respiratory motor control
Authors: Guenther Courtney H. , Baker‐Herman Tracy L. , Windelborn James A. , Mitchell Gordon S.
Published: June 2021

Atypical protein kinase C (PKC) isoforms play important roles in many neural processes, including synaptic plasticity and neurodegenerative disease. However, the role of atypical PKCs in respiratory motor control has not been explored. Since preliminary data from our laboratory suggest that atypical PKC inhibition alters respiratory nerve activity in anesthetized rats, we explored the neuroanatomical distribution of atypical PKCs in brainstem and spinal cord regions known to play critical roles in respiratory control. Specific areas examined include neurons associated with: rhythm generation (preBötzinger Complex), pattern formation (Bötzinger Complex, ventral respiratory group), chemosensitivity (retrotrapezoid nucleus, nucleus tractus solitarius), and respiratory motor nuclei (phrenic and hypoglossal). Brainstem and spinal cords from perfused Lewis rats were sectioned (40μm slices) and stained with an antibody targeting the C‐terminus of atypical PKCs. Results: atypical PKCs are expressed in phrenic and hypoglossal motoneurons, nucleus tractus solitarius, and the Bötzinger complex. Identifying the neuroanatomical distribution of atypical PKCs in regions critical for respiratory motor control is a first step towards understanding their potential role in the control of breathing (Supported by NIH HL 80209).

Grant Funding Source

NIH HL 80209
Comparative analysis of early axon tracts in the embryonic vertebrate brain
Authors: ware michelle , Schubert Frank
Published: June 2021

Initial nerve connections in the vertebrate brain form an array of longitudinal tracts, transversal tracts and commissures. These tracts will act as a scaffold for later, follower axons that allow more complex connections to be set up in the brain. For correct development of the early axon scaffold, neurones are specified to their specific fate by homeobox genes such as Sax1, Emx2, Six3, Pax6 and Nkx1.1 at the correct place and time. The early axon scaffold has been identified in a number of species, and many of the tracts appear remarkably conserved between all vertebrates analysed. However, a direct comparison of early tracts between different species is lacking, and many of the tracts are poorly characterised. By using a range of pan‐neural antibodies the early axon scaffold can be mapped in zebrafish, Xenopus, catshark, chick and mouse embryos. The aim is to find one antibody which labels all the tracts and provide a comparative description of early neurons and their tracts in the embryonic vertebrate brain. The first time series of axon tracts has been analysed in chick embryos using Tuj1 antibody to demonstrate the formation of neurones and tracts in the early axon scaffold. Xenopus and mouse embryos have also been labelled with a range of antibodies to describe the formation of the early axon scaffold with the aim of completing a time series in these species. The pan‐neural staining is combined with retrograde labelling using DiI to highlight and further characterise specific tracts.
An Immunohistochemical Study of Human Thoracic Intervertebral Discs
Authors: Smith Michael P , Myren Richard L , Wood Aaron T , Dedrick Gregory , Sizer Phillip S , Brismee Jean‐Michele , Sawyer Barbara G
Published: June 2021

Documented cases of upper back pain are becoming more prevalent in the literature while many sections of the thoracic region are still a mystery. Past studies have examined neurotransmitters in lumbar and cervical disc, but little is known about the innervation of thoracic discs and the correlation of the innervation pattern with upper back pain. Using qualitative immunocytochemical techniques that are specific for known pain‐inducing neurotransmitters, neuropeptide Y (NPY) and Substance P (SP), thoracic intervertebral disc (IVD) specimens from one male and one female cadaver with a mean age of 88 years were stained and examined with a light microscope. Sections were taken from levels of thoracic disc beginning with T3 and ending with T11. In each section at every level, thin fibers immunostained with Substance P were noted at the edges of the disc section, while qualitatively fewer NPY‐immunoreactive axonal fibers were observed in the same areas. Substance P‐stained fibers extended into the disc annulus, but not into the nucleus. NPY was observed within the disc annulus, but not extending from the annular edge into the annular midsubstance. Additionally, regions of both SP and NPY cross‐sectioned axons were noted at the edges of the thoracic IVDs. These findings confirm that pain‐inducing neurotransmitters are present within thoracic intervertebral discs.
The Origin of Inputs to the Monkey Central Mesencephalic Reticular Formation (cMRF)
Authors: Warren Susan , Zhou Lan , May Paul J.
Published: June 2021

The central mesencephalic reticular formation (cMRF) is a midbrain region reciprocally connected to the superior colliculus (SC) where stimulation elicits horizontal saccades. Here we sought to identify its sources of input, by injecting retrograde tracers into the cMRF of M. fascicularis monkeys. In the diencephalon, labeled neurons were observed in the zona incerta, hypothalamus and the ventral lateral geniculate, ipsilaterally. The main source of midbrain input to the cMRF outside the SC was the ipsilateral substantia nigra pars reticulata, and contralateral cMRF. Within the pons, the paramedian reticular formation displayed a few cells, mostly ipsilaterally. Labeled neurons were found bilaterally in the nucleus prepositus hypoglossi and in the interposed and fastigial nuclei. A few neurons were present at medullary levels. It has been suggested that the cMRF might provide a conduit to the SC for ascending information on saccade duration and direction. While some feedback was observed for most of the cMRF's downstream targets, extensive connections to support this proposed function were not found. Instead, the cMRF seems to receive its greatest input from motor, as opposed to sensory, structures that also supply the SC. If the cMRF reticulotectal projection is inhibitory, then the GABAergic cells in the nigra and zona incerta are in position to release this inhibition via the cMRF.

Support: NIH Grant EY014263
The Macaque Midbrain Reticular Formation Sends Different Types of Feedback to Each Side of the Superior Colliculus
Authors: Wang Niping , Warren Susan , May Paul J.
Published: June 2021

The central mesencephalic reticular formation (cMRF) likely plays a role in gaze control, as cMRF neurons receive tectal input and provide a bilateral projection back to the superior colliculus (SC). Whether this feedback is excitatory or inhibitory is unknown. In this study, biotinylated dextran amine (BDA) was injected into the cMRF of M. fascicularis monkeys to anterogradely label reticulotectal terminals and retrogradely label tectoreticular neurons. BDA labeled profiles in the ipsi‐ and contralateral intermediate gray layer (SGI) were examined electron microscopically. Postembedding GABA immunochemistry was used to identify putative inhibitory profiles. Nearly all (98%) of the ipsilateral BDA labeled terminals were GABA‐positive, but profiles postsynaptic to these terminals were exclusively GABA‐negative. Most (81 %) of the retrogradely labeled tectoreticular profiles in ipsilateral SGI were GABA−. In addition, BDA labeled terminals were observed contacting BDA labeled dendrites, indicating the presence of a monosynaptic feedback loop connecting the cMRF and ipsilateral SC. In contrast, within contralateral SGI, 54% of the BDA labeled terminals were GABA+, while 46% were GABA−. Most of the postsynaptic profiles were again GABA−. These results indicate the cMRF inhibits the side of the SC that activates it during a saccade, but it has more complex effects on the contralateral SC.

Support EY014263
Sry gene expression in the sympathetic nervous system of the rat
Authors: Salisbury Ronald Lee , Dunphy Gail , Boehme Shannon , Turner Monte , Milsted Amy , Ely Daniel
Published: June 2021

The Sry gene has a well established role in testes determination. However, we have reported Sry gene expression in the adult adrenal gland and that blood pressure, plasma norepinephrine and tyrosine hydroxylase content increased following exogenous delivery of the Sry gene into the adrenal medulla. We have observed changes in Sry expression in stress responsive areas of the brain, and have co‐localized Sry and tyrosine hydroxylase (Th) in both the adrenal medulla and various brain areas. The objective of this experiment was to evaluate the involvement of the androgen receptor on Sry and Th expression in sympathetic ganglia of the rat. We measured gene expression in celiac ganglia of males lacking functional androgen receptors (testicular feminized males, Tfm) and sibling males having normal receptors. Male WKY rats were crossed to King‐Holtzman Tfm carrier females. Litters yielded normal males and Tfm siblings. All animals were terminated at 12 weeks of age and gene expression was measured by quantitative RT‐PCR. Expression of Sry and Th genes in both groups was measured. In all samples expression of Sry paralleled expression levels of Th which suggests that Th expression is regulated by Sry and not through an androgen receptor‐mediated mechanism. (Supported by NIH 1RO1HL71579‐01A3)