Search

Differential expressed gene (DEG) changes in the WM are consistent with a significant response to trauma in the GM and CC+EC WM but not AC Spatial transcriptomics reveals distinct white matter pathology modulated by neutrophil/monocyte signaling following a single, focal cortical contusion injury in mice Savannah K Kounelis-Wuillaume1, Camille Alba1,2, Andrew Frank3, Emilie Goguet4, William Brooks5Gauthaman Sukumar2, Matthew D. Wilkerson1,6, Clifton L. Dalgard1,2, Joseph McCabe1, Martin L. Doughty1. 1Department of Anatomy, Physiology, & Genetics, Uniformed Services University; 2The American Genome Center; 3Henry M. Jackson Foundation; 4Department Microbiology, Uniformed Services University; 5School of Medicine, Uniformed Services University; 6Center for Military Precision Health The Injured Cortex has a Substantial (Sub)Acute Neuroinflammatory Response Disclaimer. The opinions and assertions expressed herein are those of the author(s) and do not reflect the official policy or position of the Uniformed Services University of the Health Sciences or the Department of Defense. The authors have no conflicts of interest. This work was supported by the Brain Injury Association of America (BIAA) & a Cosmos Club Foundation Scholar Award (Kounelis-Wuillaume), a USU Award (Doughty) and the Department of Defense Program Objective Memorandum HU0001-20-20037 (Dalgard). Comparison 2 days post-injury 7days post-injury CCI Cortex vs Sham Cortex 6143 868 CCI CC+EC vs Sham CC+EC 1197 244 CCI AC vs Sham AC 43 34 CCI CC-EC vs CCI AC 632 1086 Sham CC-EC vs Sham AC 292 167  Damage to grey matter (GM) and white matter (WM) in traumatic brain injury (TBI) initiates spatiotemporally distinct pathophysiological processes  An early modulator of GM and WM injury are peripheral immune cells (PICs) that infiltrate damaged brain tissue during the acute post-injury period  We used Visium Spatial Transcriptomics (10x Genomics) to identify: 1. Gene expression changes in the damaged WM and GM of TB, RITM0038957, Damage to grey matter (GM) and white matter (WM) in traumatic brain injury (TBI) initiates spatiotemporally distinct pathophysiological processes ? An early modulator of GM and WM injury are peripheral immune cells (PICs) that infiltrate damaged brain tissue during the acute post-injury period ? We used Visium Spatial Transcriptomics (10x Genomics) to identify: 1. Gene expression changes in the damaged WM and GM of TBI mice 2. How these gene changes are affected by the infiltration of neutrophils and monocytes into the damaged brain

Mousewalker as an application for gait analysis following experimental traumatic brain injury in mice Laura B. Tucker1, Amanda H. Fu1, Yeonho Kim1 & Joseph T. McCabe2 1 Preclinical Behavior and Modeling Core, Department of Laboratory Animal Resources 2 Department of Anatomy, Physiology, and Genetics Uniformed Services University of the Health Sciences, Bethesda MD USA Digigait Mousewalker Walking speed N/A Yes Body stability No Yes Stance duration Yes Yes Swing duration Yes Yes Stride time (Stance + swing) Yes Yes Stance/swing Yes (Yes) Stance width Yes (Yes) Stance instability No Yes Stride length Yes Yes Duty cycle Yes Yes Swing speed No Yes Propel duration Yes (Yes) Brake duration Yes (Yes) Stride frequency Yes Yes Paw angle Yes Yes Gait symmetry (F/H) Yes Yes Gait symmetry (L/R) No Yes Leg index No Yes Hindlimb shared stance Yes (Yes) Stance factor Yes (Yes) MAX and min dA/dt Yes No Overlap distance Yes (Yes) Paw placement position Yes Yes Anterior/posterior extreme positions No Yes Paw drag Yes No Ataxia coefficient Yes (Yes) Forelimb weight support Yes (Yes) Figure 2. The FTIR walkway. The animal is placed in the enclosure and a mirror placed at a 45° angle underneath allows the feet to be filmed from below (inset, left). Interaction between the backlight and the side LEDs creates high contrast when the feet are in contact with the acrylic walkway. Walking is voluntary. Table 1. Quantitative measurements provided by Mousewalker. The Mousewalker software (also applicable to rats and flies) returns a wealth of quantitative data that is comparable to the well-known Digigait system. Numerous values related to stance (portion of the step cycle the paw is on the ground) and swing (part of the step cycle the paw is not in contact with the ground) are provided, as well as measures related to interlimb coordination, step length and paw positioning. Mousewalker also returns all raw data related to body and paw positions as a function of time, allowing the user to create, RITM0038939, Motor testing is a popular domain for evaluating functional deficits following traumatic brain injury (TBI). The analysis of gait, the pattern of walking or sequence of foot movements during locomotion, has become increasingly included in behavioral protocols as it may reveal subtle changes in motor coordination following insults. Here, we apply the open source software Mousewalker and a frustrated total internal reflection walkway (FTIR; commercially available) and explore the utility of the device and software for detecting subtle motor changes following a single mild TBI.

1 Center for Military Precision Health, The American Genome Center, Uniformed Services University of the Health Sciences, Bethesda, MD 2 Department of Anatomy Physiology and Genetics, Uniformed Services University, Bethesda MD 3 Henry M. Jackson Foundation for Military Medicine Inc, Bethesda MD 4 Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 5 Pulmonary and Mediastinal Pathology, Department of Defense, Joint Pathology Center, Silver Spring, MD 6 Murtha Cancer Center Research Program, Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD Tumor microenvironment differences between lung cancer subtypes revealed by spatial transcriptomics Matthew D. Wilkerson1,2, Savannah Kounelis-Wuillaume2, Camille Alba1,3, Teri J. Franks5, Martin L. Doughty2, Robert Kortum4, Robert F. Browning, Jr. 5, Clifton L. Dalgard1,2, Craig D. Shriver 6 Introduction Methods: Tissue and Analysis Workflow Lung cancer is a leading cause of cancer deaths worldwide and has complex underlying genetic drivers, subtypes and immune cell types. Molecular analysis of bulk tumors has repeatedly identified key somatic driver genes and subtypes in lung cancer. However, these key molecular strata of bulk lung tumors still contain significant heterogeneity which if characterized in finer detail may reveal new tumor microenvironment factors and lead to improved patient prognostication and therapy options. Here, we sought to compare the tumor microenvironments of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) through spatial transcriptomics. We utilized tumor tissues from a cohort of lung cancers from the U.S., as part of the Applied Proteogenomics OrganizationaL Learning and Outcomes (APOLLO) Research Network1. Immune properties of spatial clusters Spatial expression patterns across replicate sections and lung cancer histologies Summary Disclaimer: The contents of this publication are the sole responsibility, RITM0036121, Lung cancer is a leading cause of cancer deaths worldwide and has complex underlying genetic drivers, subtypes and immune cell types. Molecular analysis of bulk tumors has repeatedly identified key somatic driver genes and subtypes in lung cancer. However, these key molecular strata of bulk lung tumors still contain significant heterogeneity which if characterized in finer detail may reveal new tumor microenvironment factors and lead to improved patient prognostication and therapy options. Here, we sought to compare the tumor microenvironments of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) through spatial transcriptomics. We utilized tumor tissues from a cohort of lung cancers from the U.S., as part of the Applied Proteogenomics OrganizationaL Learning and Outcomes (APOLLO) Research Network1.

Introduction Workflow Summary Detecting differentially expressed genes under different biological conditions is crucial to characterize mechanisms of cancer development and identifying determinants of patient outcome. High throughput technologies such as RNA sequencing and mass spectrometry-based proteomics have been widely used to identify differentially expressed genes (DEG), on a transcript and on a peptide basis, respectively. However, both transcription and translation of genes provide information about gene expression. Thus, leveraging both RNA and protein expression data could potentially produce more accurate results. Various statistical tools have been developed to tackle the differential expression problem for a single platform, such as edgeR, DESeq2, etc. However, a tool that integrates both transcriptome and proteomics data for differential expression analysis has not yet been developed. Meta-analysis can potentially increase statistical power and identify new cancer genes in large sample cohorts and potentially also in small sample cohorts. Here we present DE-Meta, a new tool developed using R, which performs combined meta-analysis of RNA-seq and MS-based proteomics on matched tumor specimens. Lung adenocarcinoma (LUAD) is a primary cause of cancer-related deaths worldwide, despite advances in somatically targeted therapeutics and immune checkpoint therapies. The diagnosis and treatment of LUAD patients pose significant challenges due to the vast morphological and molecular heterogeneity observed both within and between tumors. Two published lung adenocarcinoma datasets were used in this study: APOLLO11,7 and CPTAC2. APOLLO7 is a proteogenomic study seeking to describe the major genome, transcriptome, proteome and phosphoproteome alterations, subtypes, and molecular predictors of patient outcomes. Stratifying LUAD patients using expression subtypes can enhance prediction of clinical outcomes. Expression subtype status of these two cohorts were predicted, RITM0035818, Detecting differentially expressed genes under different biological conditions is crucial to characterize mechanisms of cancer development and identifying determinants of patient outcome. High throughput technologies such as RNA sequencing and mass spectrometrybased proteomics have been widely used to identify differentially expressed genes (DEG), on a transcript and on a peptide basis, respectively. However, both transcription and translation of genes provide information about gene expression. Thus, leveraging both RNA and protein expression data could potentially produce more accurate results. Various statistical tools have been developed to tackle the differential expression problem for a single platform, such as edgeR, DESeq2, etc. However, a tool that integrates both transcriptome and proteomics data for differential expression analysis has not yet been developed. Meta-analysis can potentially increase statistical power and identify new cancer genes in large sample cohorts and potentially also in small sample cohorts. Here we present DE-Meta, a new tool developed using R, which performs combined meta-analysis of RNA-seq and MS-based proteomics on matched tumor specimens.

Oxidative stress caused either due to overproduction of reactive oxygen species (ROS) or failure of endogenous antioxidants leads to damage of cellular macromolecules and intracellular organelles, which if remain uncontrolled may lead to cell death. Lysosomes play a vital role in maintaining cellular homeostasis by acting as the terminal degradative organelles in autophagic process. Autophagy is a highly regulated pro-survival pathway that delivers the toxic cellular debris and damaged organelles to the highly acidic lysosomes for digestion. Any impairment in lysosomal function can lead to inhibition of autophagic process and protein accumulation. Importantly, due to the presence of potent hydrolyzing enzymes within its lumen, any compromise in lysosomal membrane integrity can lead to severe cellular damage and cell death. Several factors including ROS has been shown to contribute to lysosomal membrane permeabilization (LMP); however, the molecular mechanisms involved in the induction of cell death by LMP during uncontrolled oxidative stress is poorly understood. In this thesis, I demonstrate that excessive oxidative stress induced by inactivation of endogenous thiol antioxidants such as thioredoxin (Trx) and glutathione (GSH) systems promote oxidative damage of lysosomes and results in activation of apoptotic and non-apoptotic cell death mechanisms. Inactivation of Trx system led to blockade of autophagic process and protein accumulation, which was attributed to loss of lysosomal proteolytic ability and acidity. On further exploration, I discovered that inhibition of Trx system caused oxidative inactivation of cathepsin L, a pro-autophagic lysosomal protease and LMP resulting in the induction of apoptotic pathway. Using small molecule inhibitor screening, cathepsin B was found to be the executioner of apoptotic cell death in this model. Complementary studies in another model of a recently identified non-apoptotic cell death mechanism known as ferroptosis further co

Over the past decade, researchers have utilized novel technologies to improve the lives of the elderly population. Virtual Reality (VR) is among the most promising platforms that could help the elderly stay cognitively active; However, the extent to which this population is willing to seek out and engage in VR activities remains unclear. In this document, we have discussed our two studies regarding VR and seniors. Our first study is a pilot project including three senior residents of Winnipeg. In this study, we assessed the impact of VR game name “DoVille” on the cognitive capacities of the elderly. We designed a two-week procedure with 20 minutes of VR sessions per day. While the comparison of pre-DoVille and post-DoVille test scores were statistically insignificant, we have gained valuable information about the feasibility and possible challenges of similar projects in the future. Among these issues, we have discussed the eligibility criteria, VR sessions’ setting and length of training sessions for the seniors. The second study is a survey project assessing the attitudes of the elderly toward VR during the COVID-19 pandemic. All senior residents of Manitoba between the ages of 65 and 90 were eligible to participate in the study. The survey was administered online and by phone, and 103 individuals responded to our questionnaire. Our results suggest that a large proportion of elderly individuals have become interested in VR technology as a result of the COVID-19 pandemic. We developed two models for VR use based on the responses. Our model of VR use for communication/interaction could account for approximately 50% of the variance in interest levels in VR, and our model of VR use for cognitive benefits accounted for 35% of the variance. These models included variables such as previous experience with technology, age and gender. In conclusion, these two studies provide us with a better understanding of the elderly’s interest in technology and how we could implement n

Metabolic specific myogenic precursors in the splanchnic, somatic mesoderm, and the neural crest give rise to specialized cell types such as cardiac, skeletal, and smooth muscle cells. This initiation of muscle cell lineage is coordinated by a reinforcing networking of transcription factors that regulate gene expression during cell proliferation and differentiation. Myocardin is a transcriptional coactivator that binds to transcription factors to regulate gene expression specific to both cardiac and smooth muscle cells. It is previously shown that Myocardin interacts with transcription factors of the MADS-Box family proteins such as myocyte enhancer factor-2 (MEF2) and serum response factor (SRF), that are known regulators of cellular differentiation and metabolism. Conversely, the role of Myocardin as well as its regulation of MADS-Box transcription factors and mitochondrial function during development and disease is not well understood. Therefore, we chose to investigate a Myocardin-regulated genetic pathway that regulates mitochondrial function in cardiac muscle that becomes dysregulated during disease. My thesis summarizes our evaluation of the hypothesis in two studies. In the first study we characterize a mechanistic pathway involving MEF2 and SRF, that regulates mitochondrial function in all three muscle lineages. This initial study demonstrates a genetic pathway in which Nix is a direct target of miR-133a, its role in regulating insulin sensitivity and metabolic dysfunction in myocytes. These novel findings lead to my second study that examines the role of Myocardin in regulating miR-133a and Nix in cardiac cells. I provide evidence that miR−133a is a direct transcriptional target of Myocardin that regulates mitochondrial permeability transition to preserve cell survival during cardiac development and following myocardial ischemic injury. iii Together, these findings inform researchers about the vital role of a Myocardin-regulated pathway that maintains mito

Several brainstem regions are necessary to provide transmission of a descending signal to motor networks within the lower the limbs. The Parapyramidal region (PPR) within the ventrolateral medulla appears to play an important but yet unspecified role in locomotion. The recent advent of DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) has allowed for non-invasive in vivo manipulation of neurons. DREADDs were stereotaxically injected into the PPR using adeno-associated viral vectors (AAVs). After a recovery period, rats were tested in both voluntary (open field) and fictive locomotion experiments to evaluate motor activity after administration of the DREADD actuator. Immunohistochemical detection confirmed DREADDs within the PPR. Locomotor activity increased significantly (172%) after DREADD activation during open field locomotor testing. With electrical stimulation, burst duration was significantly increased (174%) in flexor hindlimb nerves. Chemogenetic activation of the PPR seems to facilitate hindlimb motor output but specific effects on fictive locomotion were inconclusive.

Rhabdomyosarcoma (RMS) is a muscle-derived tumor and is the most common pediatric soft tissue sarcoma representing 5% of all childhood cancers. Statistically, RMS is a major clinical problem in pediatric oncology. Treatment of RMS with the oral alkylating agent temozolomide (TMZ), alone or in combination with other drugs, has recently received considerable interest. However, the mechanism of action of TMZ remains unclear. The aim of this investigation was to determine if autophagy modulates TMZ-induced cell death in an RMS cell line (RH30 cells), and determine if the cellular response to TMZ is different in RMS cells compared to non-transformed mouse myoblast cell line (C2C12 cells). We show that TMZ decreased the viability of RMS cells in a dose- and time-dependent manner and induced accumulation of sub-G1 cell population, representing apoptotic cells. Interestingly, TMZ induced apoptosis by 17-fold in the RH30 cells, (2.11% vs 36.94%; p<0.05), but only by 3-fold in C2C12 cells (10.95% vs 29.64%; not significant). In RH30 cells, TMZ decreased the expression of antiapoptotic proteins BCL-XL and MCL-1. Moreover, we show that TMZ induced biochemical markers of autophagy, such as LC3 lipidation and P62 degradation (Immunoblotting), and induced morphological evidence autophagy, including accumulation of autophagosomes and autophagolysosome in both cell lines, determined by transmission electron microscopy. Treatment of RMS cells with the autophagy inhibitor Bafilomycin A1 significantly increased TMZ-induced cell death in RMZ cell line. We have demonstrated that autophagy inhibition increased TMZ induced apoptosis. Our investigation showed that TMZ induced simultaneous autophagy and apoptosis in both RH30 and C2C12s; however, the regulation of apoptotic cell death induction by TMZ appears to be dependent on autophagy processes.

A lysosomal acid phosphatase 2 (Acp2) mutant mouse (naked-ataxia, nax) shows severe cerebellar cortex defect with a significant reduction in granule cells in the cerebellum. The sonic hedgehog (SHH) and N-Myc pathways are important for granule cell precursor proliferation and differentiation. The project tests the hypothesis that the proliferation defect of granule cells is associated with SHH - N-Myc pathway impairment in the nax mutant cerebellum. To investigate morphological changes in the nax cerebellum and alteration in the SHH - N-Myc pathway, in vivo and in vitro immunohistochemistry and Western blotting were used. My data showed a strikingly reduced N-Myc expression in the nax cerebellum, which was accompanied by an increase in proteasome activity, suggesting that the significant reduction of granule cells in nax cerebellum is potentially due to the dysregulation of N-Myc via the abnormal ubiquitin-proteasome system activity.

Myofibrillar adenosine triphosphatase (ATPase) activity was demonstrated in sections of masseter and temporalis muscles and of selected limb muscles of adult rhesus monkeys. Incubations were performed either with no pre-treatment or after prior incubation in alkaline media (pH 10.2???10.4) or acidic media (pH 3.8???4.6). Without pre-treatment, fibres having high or low ATPase activity were observed in limb and masticatory muscles. Following alkaline pre-incubation the difference between high and low ATPase of limb muscle fibres is accentuated, whereas pre-incubation in acidic media (pH 4.3) results in inhibition of high and potentiation of low ATPase activities (???acid reversal???). While pre-incubation of masticatory muscle sections at pH 10.2 accentuates differences in ATPase activity, pre-incubation at pH 10.4 abolishes ATPase activity. In contrast, masticatory muscle fibres showed no reversal of ATPase activity following acidic pre-incubation (pH 4.3). Pre-incubation at pH 3.8 abolished the ATPase activity of both limb and masticatory muscle fibres. The biochemical basis for the differences in ATPase histochemistry between masticatory and limb muscles is not known.

One aspect of tissue engineering of skeletal muscle involves the transposition and transplantation of whole muscles to treat muscles damaged by injury or disease. The transposition of whole muscles has been used for many decades, but since 1970, the development of techniques for microneurovascular repair has allowed the transplantation of muscles invariably result in structural and functional deficits. The deficits are of the greatest magnitude during the first month, and then a gradual recovery results in the stabilization of structural and functional variables between 90 and 120 days. In stabilized vascularized grafts ranging from 1 to 3 g in rats to 90 g in dogs, the major deficits are ∼25% decrease in muscle mass and in most grafts ∼40% decrease in maximum force. The decrease in power is more complex because it depends on both the average shortening force and the velocity of shortening. As a consequence, the deficit in maximum power may be either greater or less than the deficit in maximum force. Tenotomy and repair are the major factors responsible for the deficits. Although the data are limited, skeletal muscle grafts appear to respond to training stimuli in a manner no different from that of control muscles. The training stimuli include traditional methods of endurance and strength training, as well as chronic electrical stimulation. Transposed and transplanted muscles develop sufficient force and power to function effectively to: maintain posture; move limbs; sustain the patency of sphincters; partially restore symmetry in the face; or serve as, or drive, assist devices in parallel or in series with the heart.

This study investigated whether mixture extract of Pterocarpus indicus, Momordica charantia, Phaseolus vulgaris and Andrographis paniculata could lower plasma glucose in Type 2 Diabetes Mellitus (T2DM) patients. 43 T2DM patients, consist of 10 male and 33 female patients participated in this possitive-controlled, double-blind, and crossover clinical study with administration of either mixture extract (22 mg/kg BW) or glibenclamide 5 mg daily in breakfast time. Treatment of extract or glibenclamide was administered for a month, then medication was changed after a week of washout period and finally combination therapy was administered after wash out period as well. The efficacy of mixture extracts was measured by using Fasting Plasma Glucose (FPG) and two-hour Postprandial Plasma Glucose (PPG). Forty-one subjects completed the study. Data was analyzed using SPSS 19 with Student's t test, and p<0.05 was considered significant. FPG level significantly decreased 16.07 mg/dl after extract, 43.34 mg/dl after glibenclamide and 50.16 mg/dl after combination treatment. Two-hour PPG level decreased 25.88 mg/dl after extract, 66.61 after glibenclamide and 58.93 after combination treatment. We concluded that extract administration could lower FPG and PPG although not as good as glibenclamide treatment did and combination treatment was the best to lower FPG.

High Mobility Group AT-hook protein 2 (HMGA2) is a non-histone chromatin binding protein expressed in stem cells, cancer cells but not in normal human somatic cells. The presence of HMGA2 in cancer correlates with advanced neoplastic disease and poor prognosis. HMGA2 plays important roles in Base Excision Repair (BER) and at replication forks. HMGA2 is present at mammalian metaphase telomeres and its loss induces chromosomal aberrations. However, the functional role of HMGA2 at telomeres remains elusive. We hypothesized a protective role of HMGA2 that guards telomeres and modulates DNA damage repair signaling pathways. Employing different HMGA2+ human tumor cell models, we investigated the HMGA2-mediated functions that contribute to chemoresistance in glioblastoma (GB). This study presents a novel interaction of HMGA2 with telomeric protein TRF2 (Telomere Repeat-Binding Factor 2). This interaction retains TRF2 at telomeres, thus capping the telomeres and reducing telomere-dysfunction induced foci despite induced telomere stress. Loss of HMGA2 coincides with increased phosphorylation of TRF2, decreased TRF2 retention at telomeres and increased formation of telomeric aggregates, anaphase bridges and micronuclei. These findings provide new evidence for a unique role of HMGA2 at telomeres as a novel contributor of telomeric integrity. We show that upon DNA damage, HMGA2 causes increased and sustained phosphorylation of Ataxia Telangiectasia and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1). Prolonged presence of pCHK1Ser296 coincides with prolonged G2/M block and increased tumor cell survival. The relationship between (ATR)-CHK1 DNA damage response pathway and HMGA2 identifies a novel mechanism by which HMGA2 can alter DNA repair function in cancer cells. We identified HMGA2 as a novel factor contributing to temozolomide (TMZ) resistance in GB. HMGA2 knockdown sensitizes the GB cells to TMZ. We propose a specific combination of FDA-approved drugs, TMZ and Dov

Traumatic brain injury (TBI) has a 20% mortality rate and a 10-15% rate of resultant permanent disability. The consequences of TBI range from brief loss of consciousness, to prolonged coma or death. Mild TBI is amongst the common causes of admission to trauma centers all over the world. Future technologies such as magnetic resonance elastography and robotic surgery demand information about the physical properties of brain tissue. Walsh and Schettini described the mechanical behavior of brain tissue under normal status as nonlinear viscoelastic behavior and defined the associated biomechanical changes and responses in a quantitative measurement of the material changes. Yet, there is still a lack of data concerning time-dependent deformation and mechanical property changes associated with TBI. My goal in this project was to describe these mechanical responses and to create a system for measuring and evaluating the mechanical response of brain tissue in vivo. This was to be achieved by inducing cortical contusions with a calibrated weight-drop method in seventy-four young adult male Sprague-Dawley rats. Instrumented indentation was performed on control brains and 1 hour to 3 weeks after contusion with intact dura using a 4-mm-diameter flat punch indenter to a maximum depth of 1.2 mm at loading. Loading rates did not exceed 0.34 N/min and 1.2 mm/min. In order to obtain force displacement data, we studied the elastic response of the traumatized brain tissue and the deformation process (creep) during the loading and unloading of indenter. After euthanasia, the brain was removed and evaluated histologically with different methods to reveal acute and chronic changes related to the contusion. The results revealed that the biomechanical properties of the brain tissue were changed after cortical contusion. Brain tissue elasticity decreased in the edematous brain at one day following the contusion and increased at 3 weeks, in association with reactive astroglial changes. This e

The sodium-calcium exchanger (NCX) is a countertransporter of Na+ and Ca2+ across most cell membranes. It has been identified as an essential component of Ca2+ homeostasis in physiological and disease conditions in both cardiovascular and neurological settings. The exchanger not only transports Na+ and Ca2+, but is also regulated by these ions. Although ionic regulatory profiles differ between NCX isoforms, similar regulatory domains have been identified. Previous structure-function studies have determined key residues within these domains, particularly in the eXchanger Inhibitory Peptide region (XIP) and the Ca2+ binding domains (CBD1/2), which have a direct impact on ionic regulation of the outward exchange currents. Recent structural studies of the Ca2+ binding domains of NCX1 suggest a mechanism by which Ca2+ binding would not only be essential for activation of current but may also influence Na+-dependent inactivation. The alternative splice region is located within the Ca2+ binding domain and may play a role in mediating these regulatory phenotypes. Previous studies have demonstrated that specific combinations of the mutually-exclusive and cassette exons are associated with profound effects on ionic regulation in NCX1. This study focuses on examining the mechanisms by which the alternative splice region, in combination with specific regulatory domains, modulates exchange activity in two isoforms, NCX1 and NCX2. Chimaeric and mutant constructs in the alternative splice region were expressed in Xenopus oocytes and outward Na+-Ca2+ exchange activity was assessed using the giant, excised patch clamp technique. Substitution of the region corresponding to the mutually exclusive exon in either exchanger greatly reduced the extent of Na+-dependent inactivation, independently of intracellular Ca2+ concentrations. However, replacement of both the region corresponding to the mutually exclusive exon A and the XIP region reestablishes a wild-type profile in NCX2. The first

We have developed and employed a stereotaxic coordinate system for the pig brain based on external skull landmarks. Sagittal, coronal, and horizontal planes were defined. Based on histological maps and ventricular casts, the coordinates for locating the lateral ventricles were described. Guide tubes leading to the lateral ventricles have been chronically implanted. This access route to the ventricular system has been used for stimulation of the dipsogenic response with angiotensin and for withdrawal of cerebrospinal fluid. Solved and unsolved problems arising with these procedures have been defined.

A specific immune serum against frog rhodopsin has been developed in rabbits. The specificity of the antirhodopsin serum was established using the indirect Coon's antibody technique. Absorption of the serum with electrophoretically purified rhodopsin abolished its ability to stain sections of retina or isolated retinal receptor disk membranes. Using fluorescein-labeled sheep anti-rabbit [gamma]-globulin on sections of retina treated with antirhodopsin [gamma]-globulin, fluorescence was observed in all retinal receptor outer segments and in the myoids and ellipsoids of retinal receptor inner segments. In addition, it was localized in cytoplasmic structures of the pigmented epithelium. Dissected retinas with intact outer segments showed fluorescence in the outer segments when the retinas were treated with antirhodopsin immune serum, washed thoroughly, incubated in fluorescein-labeled sheep anti-rabbit [gamma]-globulin and again washed thoroughly. This is suggestive of a diffusion pathway into the outer segments. Isolated whole rod outer segments and isolated disk membranes stained uniformly by the indirect technique. From these observations we conclude that the photopigment is indeed part of the disk membranes of the outer segments and that the external membrane of the outer segment also contains photopigment. Use of the specific antirhodopsin immune serum has allowed the description of the molecular arrangement of photopigment in retinal receptor disk membranes (Blasie, Worthington & Dewey, 1968; Blasie & Worthington, 1969).

Whole 3 g extensor digitorum longus (EDL) muscles of cats were autotransplanted. The EDL muscles were either transplanted without denervation prior to transplantation (normal transplants) or denervated 3 to 4 weeks prior to transplantation (pre-denervated transplants). A few peripheral skeletal muscle fibers survived transplantation but most fibers degenerated and then regenerated as the transplant became revascularized. Both normal and pre-denervated muscles regenerated successfully and by 50 days after transplantation fibers which had reinnervated showed high and low myofibrillar ATPase activity. Compared to controls, the smaller mean fiber cross-sectional area of the transplants was due to the large number of small fibers, but some fibers in the transplant were larger than any fibers observed in the controls. Transplants regained 57 percent of the muscle mass of the controls. Contraction and half relaxation times of transplanted muscles were slower than controls, but peak isometric tetanus tension per cm2 of muscle was nearly normal. Fifty to 170 days after transplantation, muscles showed low oxidative capacity and fatigued rapidly.

Greater understanding of the growth factor regulation of Leydig cells during their postnatal differentiation has been achieved. Increasingly, attention will shift toward the goal of isolating Leydig stem cells because of their therapeutic potential in the treatment of male hypogonadism and the syndrome of androgen deficiency of aging males (ADAM). Markers of Leydig stem cells such as Kit receptor can be exploited in harvesting enriched fractions from the testis for amplification in vitro. When subsequently implanted back into the same testis, we expect the amplified stem cells to undergo differentiation into steroidogenic Leydig cells.

The endocrine disruptor hypothesis asserts that exposures to environmental agents, which have the ability to perturb the hormonal milieu, exert adverse effects on cellular differentiation and function in affected tissues. These compounds exhibit a predilection for steroid hormone receptors [androgen and estrogen receptors (AR, ER)]. Because ARs and ERs are highly expressed in reproductive tissues, it is not surprising that analysis of reproductive activity following toxicant exposures has received a great deal of attention. We have obtained data describing the effects of an antiandrogen di(2-ethlhexyl) phthalate (DEHP) on the reproductive tract of the male rat. DEHP is the most abundant phthalate in the environment, and phthalates are used to impart flexibility to infant toys, building and food-packaging products, and biomedical devices. DEHP action appears to depend on the time of exposure because prenatal exposures decreased serum luteinizing hormone (LH) and testosterone (T) levels in prepubertal rats while chronic exposures of weanling rats to DEHP caused simultaneous elevations in serum LH and T levels. The pituitary gonadotropin LH is the primary regulator of androgen biosynthesis by Leydig cells in the testis. Taken together, these observations imply that the male reproductive axis is subject to regulation by DEHP. Given the sensitivity of developing organ systems to the action of hormonally active agents, implying increased vulnerability of children to the effects of DEHP, ourfindings suggest a decrease in the levels of DEHP and other phthalates in consumer products.

The Center for Nonlinear Dynamics of the Brain (CNDB) studies nonlinear dynamics of large masses of nerve cells in animal and human brains as the basis for explaining the self-organization of goal-directed cognitive behaviors. We believe that this approach to the study of mass neural action will be a basic method of the neurophysiology of the twenty-first century. Our research has application to clinical neurology and psychiatry, to the measurement and enhancement of human mental capabilities and to the design of self-organizing, pattern recognition computer systems. By extending techniques and models derived from the paleocortex to animal and human neocortex, deeper understanding of the neural basis of goal-directed higher brain functions will emerge. The practical consequences of this enhanced knowledge will be better diagnosis of neurologic and psychiatric diseases. It also will result in the ability to predict decrements in higher brain functions consequent to illness, fatigue or drugs. The new models for globally parallel, self-organizing systems which result from this research also will be directly relevant to the design of VLSI architectures for pattern recognition. Keywords: Mathematical modeling; Anatomical modeling.

Effects of hydrostatic pressures, ranging from 1 to 1000 atmospheres, on the transport and ion binding characteristics of human red cells were studied. A high pressure stopped-flow device was designed for rapid mixing and subsequent spectrophotometric study was designed and built with the following characteristics: operating pressures up to 1200 bar, minimum average flow velocities to 2.0m/sec, dead time ranging from 5 to 25 msec. Experimental results with red cells showed that (1) normal potassium ion leakage increases dramatically at pressure 6000 psi, (2) butanol induced potassium ion leakage decreases dramatically at pressures 6000 psi, (3) the Gardos response (potassium ion leakage induced by A23187 + potassium ion) is reduced by pressure, and divalent cation selectivity sequences (Ca Mg Ba Sr) do not change with 1000 atm pressure indicating that ionic radii are of predominant importance in binding, (4) loose potassium ion binding are measured by chlorotetracycline does not change with pressure, (5) both val + potassium ion and butanol diffusion in red cell membranes have apparent activation volumes of 40 ml/mol in agreement with data on liposomes, and (6) perturbations in osmotic water flow through red cells by either high pressures or deuterium oxide substitution can be predicted from the bulk properties of pure water.

The goal of this work is to characterize mathematically the essential mechanisms and principles of operation of the mamalian olfactory neural network and evaluate its computation and pattern recognition capabilities. The intent is to explicate novel design principles that may underly the superior performance of biological systems in pattern recognition through detailed study of a particular system. This research will be for the purpose of establishing a theoretical framework for the evaluation of architectures and algorithms for parallel computation - with particular emphasis on neural networks.

The impairment in the performance of men wearing the protective clothing was determined by measuring the respiratory and thermal responses of the subjects walking on a treadmill at 3 mph, zero grade at 21 and 40 C and 10 mm Hg vapor pressure. The major cause of the discomfort was found to be thermo- regulatory in nature. The M6 hood added considerably to the heat stress of the subjects. Similarly, as compared to subjects with the heads kept uncovered, wearing the M-17 protective mask also resulted in greater discomfort in the heat. Since a high body temperature seems to be essential for a rise in ventilation, further experiments need to be carried out to determine whether in subjects working in the heat, wearing mask and hood after the attainment of a rectal temperature of 39 C or more results in hyperventilation. The subjects in this series reported severe thermal discomfort prior to the attainment of any hyperventilation with only one exception.

The following conclusions are considered warranted by the investigators: (1) There is an increase in the sensitivity of the thermoregulatory centers in the hypothalmus following heat acclimation; (2) there is no increase in the peripheral sensitivity of the reflex sweating mechanism with heat acclimation; (3) the human body undergoes significant physiological adaptation during heat acclimation; (4) the heat acclimation process is limited by the amount of work and heat stress imposed giving only partial adaptation to more severe conditions; and (5) there were training effects associated with the heat acclimation process. The interactive human performance model written at the Department of Human Physiology, University of California, Davis, and originally implemented on a Burroughs B6700 has been adapted to run interactively on the CDC 6600 at Indiana University., See also report dated 13 Dec 72, AD-753 913.

Work is reported in the following areas of research: Construction of a complete thermoregulatory profile in Mucaca mulatta to quantitate the importance of total body sweat gland function in overall thermal balance in the rhesus monkey; quantitation and mathematical modeling of local sweating activity in Mucaca mulatta; development of the surgical techniques and instrumentation associated with a chronic hypothalamic perfusion system in the rhesus monkey resulting in what appears to be a durable and reproducible system; and modification and evaluation of the U.C., Davis, predictive model of human performance to make it compatible with the Indiana University computer. (Modified author abstract)

The report covers the entire scientific effort sustained by Project Themis Contract F44620-68-C-0014 over the 5-year period under the direction of Robert W. Bullard and the year following his death on June 24, 1971. With it are furnished the scientific publications by year, the meetings attend, the abstracts and the review article by Robert W. Bullard. The report also includes the advantages the project has given the department in creating a biophysical approach to physiology, and the advantage to general recruitment of physiologists. The 40 published scientific journal articles adequately summarize the scientific results of this project. (Author)