Seeking mTORC1 Inhibitors Through Molecular Dynamics Simulation of Arginine Analogs Inhibiting CASTOR1.

BACKGROUND: Hyperactivity of the mechanistic target of rapamycin complex 1 (mTORC1) is implicated in a variety of diseases such as cancer and diabetes. Treatment may benefit from effective mTORC1 inhibition, which can be achieved by preventing arginine from disrupting the cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1)-GTPase-activating proteins toward RAGS subcomplex 2 (GATOR2) complex through binding with CASTOR1. An attractive idea is to determine analogues of arginine that are as competent as arginine in binding with CASTOR1, but without disrupting the CASTOR1-GATOR2 interaction. MATERIALS AND METHODS: Molecular dynamics simulations were performed for binding of arginine analogues with CASTOR1 and binding free energy, hydrogen bond formation, and root mean squared deviation and root mean square fluctuation kinetics were then calculated. RESULTS: The binding free energy calculations revealed that Nα-acetyl-arginine, citrulline, and norarginine have sufficient binding affinity with CASTOR1 to compete with arginine. The hydrogen bond analysis revealed that norarginine, Nα-acetyl-arginine and D-arginine have proficient H-bonds that can facilitate their entering the narrow binding pocket. CONCLUSION: Norarginine and Nα-acetyl-arginine are the top drug candidates for mTORC1 inhibition, with Nα-acetyl-arginine being the best choice.

The Importance of the Autopsy in Medicine: Perspectives of Pathology Colleagues.

This article presents a perspective on the importance of the autopsy in medical practice and science based on experiences of the authors as physician-scientists involved in autopsy practice. Our perspectives are presented on the seminal contributions of the autopsy in the areas of cardiovascular disease, including congenital heart disease, atherosclerosis, coronary artery disease, and myocardial infarction, and infectious disease, including tuberculosis and viral infections. On the positive side of the future of the autopsy, we discuss the tremendous opportunities for important research to be done by application of advanced molecular biological techniques to formalin-fixed, paraffin-embedded tissue blocks obtained at autopsy. We also note with concern the countervailing forces impacting the influence of pathology in education and clinical practice at our academic medical centers, which also present impediments to increasing autopsy rates. Our challenge as academic pathologists, whose careers have been molded by involvement in the autopsy, is to counter these trends. The challenges are great but the benefits for medicine and society are enormous.

Human Herpesvirus 6 and Malignancy: A Review.

In order to determine the role of human herpesvirus 6 (HHV-6) in human disease, several confounding factors, including methods of detection, types of controls, and the ubiquitous nature of the virus, must be considered. This is particularly problematic in the case of cancer, in which rates of detection vary greatly among studies. To determine what part, if any, HHV-6 plays in oncogenesis, a review of the literature was performed. There is evidence that HHV-6 is present in certain types of cancer; however, detection of the virus within tumor cells is insufficient for assigning a direct role of HHV-6 in tumorigenesis. Findings supportive of a causal role for a virus in cancer include presence of the virus in a large proportion of cases, presence of the virus in most tumor cells, and virus-induced in-vitro cell transformation. HHV-6, if not directly oncogenic, may act as a contributory factor that indirectly enhances tumor cell growth, in some cases by cooperation with other viruses. Another possibility is that HHV-6 may merely be an opportunistic virus that thrives in the immunodeficient tumor microenvironment. Although many studies have been carried out, it is still premature to definitively implicate HHV-6 in several human cancers. In some instances, evidence suggests that HHV-6 may cooperate with other viruses, including EBV, HPV, and HHV-8, in the development of cancer, and HHV-6 may have a role in such conditions as nodular sclerosis Hodgkin lymphoma, gastrointestinal cancer, glial tumors, and oral cancers. However, further studies will be required to determine the exact contributions of HHV-6 to tumorigenesis.

Active HHV-6 Infection of Cerebellar Purkinje Cells in Mood Disorders.

Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD.

Lymphoproliferative Syndromes Associated with Human Herpesvirus-6A and Human Herpesvirus-6B.

Human herpesvirus 6A and 6B (HHV-6A and HHV-6B) have been noted since their discovery for their T-lymphotropism. Although it has proven difficult to determine the extent to which HHV-6A and HHV-6B are involved in the pathogenesis of many diseases, evidence suggests that primary infection and reactivation of both viruses may induce or contribute to the progression of several lymphoproliferative disorders, ranging from benign to malignant and including infectious mononucleosis-like illness, drug induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DIHS/DRESS), and nodular sclerosis Hodgkin's lymphoma. Herein, we discuss the conditions associated with the lymphoproliferative capacity of HHV-6, as well as the potential mechanisms behind them. Continued exploration on this topic may add to our understanding of the interactions between HHV-6 and the immune system and may open the doors to more accurate diagnosis and treatment of certain lymphoproliferative disorders.

Copy Number Heterogeneity, Large Origin Tandem Repeats, and Interspecies Recombination in Human Herpesvirus 6A (HHV-6A) and HHV-6B Reference Strains.

Quantitative PCR is a diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of human herpesvirus 6A/B (HHV-6A/B) is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies Inc. Eleven of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1,254 bp and 983 bp, respectively. The average copy number measured was between 5 and 10 times the number of copies of the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and a >5.5-kb region from HHV-6B, from U41 to U43, that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-to-U24 region and the U94/U95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different laboratories on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate that large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae.IMPORTANCE Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very accuracy through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6A/B biology and the standard selection process.

Human herpesvirus 6-induced inflammatory cardiomyopathy in immunocompetent children.

Over the last decade, human herpesvirus 6 (HHV-6) has been implicated in the etiology of pediatric myocarditis and subsequent dilated cardiomyopathy (DCM). This review provides an overview of recent literature investigating the pathophysiological relevance of HHV-6 in inflammatory cardiomyopathy. We examined 11 cases of previously published pediatric myocarditis and/or DCM associated with HHV-6 and also our experience of detection of virus particles in vascular endothelium of HHV-6 positive endomyocardial biopsy tissue by electron microscopy. The exact role of the presence of HHV-6 and its load remains controversial as the virus is also found in the heart of healthy controls. Therefore, the question remains open whether and how cardiac HHV-6 may be of pathogenetic importance. Quantitative polymerase chain reaction or mRNA testing allows differentiation between low-level latent virus found in asymptomatic myocardium and active HHV-6 infection. Although only a small number of pediatric cases have been reported in literature, HHV-6 should be considered as a causative agent of inflammatory cardiomyopathy, especially in children under three who might be experiencing a primary infection. Future studies are needed to establish a threshold for determining active infection in biopsy samples and the role of coinfections other cardiotropic viruses.

Anti-herpesviral effects of a novel broad range anti-microbial quaternary ammonium silane, K21.

We have created a novel quaternary ammonium silane, K21 through sol-gel chemistry, using an ethoxylated version of an organosilane quaternary ammonium compound and TetraEthyl Ortho Silicate (TEOS) as precursors. Previous studies using the precursor molecule quaternary ammonium compounds (QACs) and a methacryloxy version of K21, primarily designed for use in dental healthcare, have shown inhibited growth properties against several types of gram-positive and gram-negative bacteria including Escherichia coli, Streptococcus mutans, Actinomyces naeslundii and Candida albicans etc. Here we tested the effect of K21 on HSV-1, HHV-6A and HHV-7 in in vitro cell culture infection models. Our results show growth inhibitory effect of K21 on HSV-1, HHV-6A and HHV-7 infection.

Altered Micro-RNA Degradation Promotes Tumor Heterogeneity: A Result from Boolean Network Modeling.

Cancer heterogeneity may reflect differential dynamical outcomes of the regulatory network encompassing biomolecules at both transcriptional and post-transcriptional levels. In other words, differential gene-expression profiles may correspond to different stable steady states of a mathematical model for simulation of biomolecular networks. To test this hypothesis, we simplified a regulatory network that is important for soft-tissue sarcoma metastasis and heterogeneity, comprising of transcription factors, micro-RNAs, and signaling components of the NOTCH pathway. We then used a Boolean network model to simulate the dynamics of this network, and particularly investigated the consequences of differential miRNA degradation modes. We found that efficient miRNA degradation is crucial for sustaining a homogenous and healthy phenotype, while defective miRNA degradation may lead to multiple stable steady states and ultimately to carcinogenesis and heterogeneity.

Chromosomally integrated human herpesvirus 6 in heart failure: prevalence and treatment.

AIMS: Human herpesvirus 6 (HHV-6) A and B are two betaherpesviruses that are associated with many conditions including roseola, drug-induced hypersensitivity syndrome, limbic encephalitis, and myocarditis. HHV-6 is integrated in the germline [chromosomically integrated HHV-6 (ciHHV-6)] in ∼0.8% of the human population. To date, the prevalence, species distribution, and treatment responses of ciHHV-6 are unknown for cardiac patients. METHODS AND RESULTS: We determined the prevalence of HHV-6 and ciHHV-6 genotypes in 1656 endomyocardial biopsies of patients with persisting unexplained symptoms of heart failure. Infection of cardiac tissue was identified by nested PCR, electron microscopy, and immunohistochemistry. Virus load and mRNA levels were followed in ciHHV-6 patients treated with ganciclovir. HHV-6 was detected in 273 of 1656 cardiac tissues (16.5%; HHV-6B, 98.2%, HHV-6A, 1.8%) by PCR. Nineteen of the 1656 patients (1.1%) presented with persistently high HHV-6 copy numbers indicative of ciHHV-6. Sequencing confirmed ciHHV-6A in seven patients (36.8%) which was considerably higher than detected in non-ciHHV-6 patients. Inheritance was demonstrated in three selected families, confirming ciHHV-6 chromosomal integration by PCR and fluorescence in situ hybridization. HHV-6 reactivation and chromosomal integration were confirmed in peripheral blood mononuclear cells and heart tissue. Virus particles were identified in degenerating myocytes and interstitial cells. Antiviral treatment abolished viral mRNA and ameliorated cardiac symptoms. CONCLUSION: Virus replication in cardiac tissue of ciHHV-6 heart failure patients suggests that ciHHV-6 reactivation causes persistence of unexplained heart failure symptoms. We demonstrated that antiviral treatment, effective in decreasing viral transcripts and clinical complaints of cardiomyopathies, is a new therapeutic option for ciHHV-6-associated diseases.

Certain surfactants show promise in the therapy of pulmonary tuberculosis.

BACKGROUND/AIM: The aim of the present study was to develop the basis for the use of surfactants in the treatment of pulmonary tuberculosis (TB). Bacteria are surrounded by a thick lipid coat primarily consisting of trehalose dimycolate (TDM) and, consequently, are well shielded from the immune system's response and antibiotics. This protective barrier was removed by exposing the bacteria to certain surfactants. MATERIALS AND METHODS: Dodecyl maltoside (DDM) and octyl glucoside (OG) were utilized as non-toxic surfactants. RESULTS: Electron microscopy (EM) studies revealed that aggregated bacteria were also covered with excessive TDM which exacerbate the treatment efforts. Light and EM studies demonstrated that DDM and OG disperse the aggregated bacteria and are bactericidal. CONCLUSION: The studies presented here establish that certain surfactants are proficient in removing MTB's shield and, because they are well known as cell permeabilizing agents, they may also enhance the effectiveness of antibiotics and the immune system's response in the treatment of pulmonary TB.

Computational analysis of mTOR signaling pathway: bifurcation, carcinogenesis, and drug discovery.

Molecularly targeted therapeutics provides potentially more reliable performance while significantly reducing toxicity in comparison with chemotherapy. For cancer signaling networks which are usually complex, multiple molecules should be simultaneously targeted in order to stay in tune with the control mechanisms of the network and to achieve the maximum synergistic effects. Mathematical modeling and computer simulation are important in reproducing the dynamics of the network, some of which may correspond to healthy or cancer phenotypes. More importantly, the effects of multiple molecules can be simulated by perturbing many parameters in the model. In this paper, through the example of the mTOR signaling pathway, we demonstrate that computational analysis can provide great insights into cancer pathogenesis and the possible therapeutic interventions. In particular, we discovered a composite parameter which summarizes the synergistic effects of four different parameters. The geometry of the parameter space could be helpful in the development of a low dosage, minimal toxicity drug to cure cancer.

The impact of declining clinical autopsy: need for revised healthcare policy.

In Western countries, autopsy rates for patients deceased in hospitals have dropped to record lows, while the average frequency of major errors in clinical diagnoses has more than doubled during the same time period. Meanwhile, the Institute of Medicine and the U.S. Department of Health and Human Services have called attention to the high frequency of errors affecting patient safety, bringing the issue of public safety to the forefront of public health concerns. Although autopsies represent a vital tool for the acquisition of new medical knowledge and for medical quality assurance, health care professionals, insurers, and politicians apparently have not chosen the right approach to solve the problem of declining autopsy rates. The present article reviews the current status of clinical autopsies and addresses causes and consequences of their neglect and appeal the urgent need to revise the policy for clinical autopsy.

Complement C2 receptor inhibitor trispanning: a novel human complement inhibitory receptor.

The complement system presents a powerful defense against infection and is tightly regulated to prevent damage to self by functionally equivalent soluble and membrane regulators. We describe complement C2 receptor inhibitor trispanning (CRIT), a novel human complement regulatory receptor, expressed on hemopoietic cells and a wide range of tissues throughout the body. CRIT is present in human parasites through horizontal transmission. Serum complement component C2 binds to the N-terminal extracellular domain 1 of CRIT, which, in peptide form, blocks C3 convertase formation and complement-mediated inflammation. Unlike C1 inhibitor, which inhibits the cleavage of C4 and C2, CRIT only blocks C2 cleavage but, in so doing, shares with C1 inhibitor the same functional effect, of preventing classical pathway C3 convertase formation. Ab blockage of cellular CRIT reduces inhibition of cytolysis, indicating that CRIT is a novel complement regulator protecting autologous cells.

Immunologic aspects of chronic fatigue syndrome. Report on a Research Symposium convened by The CFIDS Association of America and co-sponsored by the US Centers for Disease Control and Prevention and the National Institutes of Health.

Chronic fatigue syndrome (CFS) is a serious health concern affecting over 800,000 Americans of all ages, races, socioeconomic groups and genders. The etiology and pathophysiology of CFS are unknown, yet studies have suggested an involvement of the immune system. A symposium was organized in October 2001 to explore the possibility of an association between immune dysfunction and CFS, with special emphasis on the interactions between immune dysfunction and other abnormalities noted in the neuroendocrine and autonomic nervous systems of individuals with CFS. This paper represents the consensus of the panel of experts who participated in this meeting. Data suggest that persons with CFS manifest changes in immune responses that fall outside normative ranges, but current research does not provide definitive evidence on whether these immune abnormalities are a cause or result of the illness. It has become clear that CFS cannot be understood based on single measurements of immune, endocrine, cardiovascular, or autonomic nervous system dysfunction. This panel encourages a new emphasis on multidisciplinary research into CFS.

Feed-forward and feedback mechanisms in a dynamic model of human T cell development and regulation.

We describe a computational model of human T cell regulatory dynamics. We used this model to simulate changes in T cell pool numbers and for studying feedback and feed-forward responses in and among these pools. The pools identified were the bone marrow stem cell compartment, early and late thymocyte compartments and the peripheral compartment of mature T lymphocytes. Simulated data showed variable intercompartmental strengths indicative of a range of sensitivities to feedback regulation and respective variable feed-forward responses. The results compare well to known clinical and experimental data, rendering the computational model a good basis for further research in T cell development and regulation.

A simple model to simulate cellular changes in the T cell system following HIV-1 infection.

A new mathematical model is presented to simulate various changes of cell pools in the T cell immune system with validation procedures imitating viral infections. The present paper focuses on changes during the course of an acute progressive HIV-1 infection. Parameters are optimized by a direct search method and the stability of the model is studied. Mathematical modeling supports the hypothesis that the differentiation blockade is one major reason for the depletion of CD4 cells and the proliferation of CD38 cells in HIV-1 infection. The model appears to be a useful basis for further simulating disturbances of the T cell immune system in other viral infections as well as to elucidate the pathogenesis of various immunological diseases including the development of malignant lymphomas.

A continuous model studying T cell differentiation and lymphomagenesis and its distinction with discrete models.

The development of T-lymphocytes (T cells) constitute one of the basic and most vital processes in immunology. Conventional mathematical models, being based on the systems theory, fail to sufficiently distinguish the constituents of thymocytes and are thus of limited significance. On the basis of some well thought-out definitions and concepts, a continuous model was designed to describe T cell maturation in the thymus. A partial differential equation was first derived through the analysis of an infinitesimal element of the flow of thymocytes. A computation scheme was designed to determine the growth field in the thymus based upon available experimental data. The corresponding algorithm proved quite simple. A numerical example is given that focuses on the DN stage of the T cell development. The model opens a window for investigating the thymic microenvironment. The potential of the model in studying lymphomagenesis is discussed.

A general mathematical method for investigating the thymic microenvironment, thymocyte development, and immunopathogenesis.

T-lymphocyte (T-cell) development constitutes one of the basic and most vital processes in immunology. The process is profoundly affected by the thymic microenvironment, the dysregulation of which may be the pathogenesis or the etiology of some diseases. On the basis of a general conceptual framework, we have designed the first biophysical model to describe thymocyte development. The microclimate within the thymus, which is shaped by various cytokines, is first conceptualized into a growth field lambda and a differentiation field mu, under the influence of which the thymocytes mature. A partial differential equation is then derived through the analysis of an infinitesimal element of the flow of thymocytes. A general method is presented to estimate the two fields based on experimental data obtained by flow cytometric analysis of the thymus. Numerical examples are given for both normal and pathologic conditions. Our results are quite good, and even the time varying fields can be accurately estimated. Our method has demonstrated its great potential for the study of immunopathogenesis. The plan for implementation of the method is addressed.

Human herpesvirus-6: a short review of its biological behavior.

HHV-6 shows a widespread distribution with life-long persistence. The virus is frequently reactivated, yet remains clinically inapparent unless the patient is immunodeficient in some way. Even then, HHV-6 reactivation may simply enhance the pathogenicity of other viruses or existing autoimmune disorders rather than becoming a pathogen itself. Future clinical studies need to focus on such indirect viral influences mediated through molecular mimicry and interference with cell receptor expression, and cytokine and chemokine network regulation. Nevertheless, such disturbances may afford therapeutic intervention to disrupt herpesvirus interference and improve certain disease processes. There are only a few diseases for which an immediate causal relationship to HHV-6 infection has been suggested.