A Robust Structural PGN Model for Control of Cell-Cycle Progression Stabilized by Negative Feedbacks

The cell division cycle comprises a sequence of phenomena controlled by a stable and robust genetic network. We applied a probabilistic genetic network (PGN) to construct a hypothetical model with a dynamical behavior displaying the degree of robustness typical of the biological cell cycle. The structure of our PGN model was inspired in well-established biological facts such as the existence of integrator subsystems, negative and positive feedback loops, and redundant signaling pathways. Our model represents genes interactions as stochastic processes and presents strong robustness in the presence of moderate noise and parameters fluctuations. A recently published deterministic yeast cell-cycle model does not perform as well as our PGN model, even upon moderate noise conditions. In addition, self stimulatory mechanisms can give our PGN model the possibility of having a pacemaker activity similar to the observed in the oscillatory embryonic cell cycle

A Robust Structural PGN Model for Control of Cell-Cycle Progression Stabilized by Negative Feedbacks

The cell division cycle comprises a sequence of phenomena controlled by a stable and robust genetic network. We applied a probabilistic genetic network (PGN) to construct a hypothetical model with a dynamical behavior displaying the degree of robustness typical of the biological cell cycle. The structure of our PGN model was inspired in well-established biological facts such as the existence of integrator subsystems, negative and positive feedback loops, and redundant signaling pathways. Our model represents genes interactions as stochastic processes and presents strong robustness in the presence of moderate noise and parameters fluctuations. A recently published deterministic yeast cell-cycle model does not perform as well as our PGN model, even upon moderate noise conditions. In addition, self stimulatory mechanisms can give our PGN model the possibility of having a pacemaker activity similar to the observed in the oscillatory embryonic cell cycle

Determination of cell shape in Staphylococcus aureus

"Cell size and morphology are two extremely important characteristics in the adaptation of bacteria to the external environment and are often associated to bacterial survival and growth. In Staphylococcus aureus, a common colonizer of human skin and mucus membranes, the small spherical shape of cells may be an advantage during colonization, helping this pathogen to evade host immune system. The fact that cell shape is maintained over consecutive generations evidences the existence of tightly regulated underlying mechanisms. Bacterial shape is maintained by the existence of an external cell wall mainly composed of peptidoglycan (PGN), a mesh-like molecule made by glycan chains cross-linked by short peptide bridges. Localization of PGN synthesis is dependent on the action of cytoskeletal proteins, which direct the activity of proteins involved in this synthesis, including Penicillin-Binding Proteins (PBPs) and proteins from the shape, elongation, division and sporulation (SEDS) family, to specific regions of the cells. T(...)

Mechanistic Insights into the Anti-angiogenic Activity of Trypanosoma cruzi Protein 21 and its Potential Impact on the Onset of Chagasic Cardiomyopathy

Chronic chagasic cardiomyopathy (CCC) is arguably the most important form of the Chagas Disease, caused by the intracellular protozoan Trypanosoma cruziit is estimated that 10-30% of chronic patients develop this clinical manifestation. The most common and severe form of CCC can be related to ventricular abnormalities, such as heart failure, arrhythmias, heart blocks, thromboembolic events and sudden death. Therefore, in this study, we proposed to evaluate the anti-angiogenic activity of a recombinant protein from T. cruzi named P21 (rP21) and the potential impact of the native protein on CCC. Our data suggest that the anti-angiogenic activity of rP21 depends on the protein's direct interaction with the CXCR4 receptor. This capacity is likely related to the modulation of the expression of actin and angiogenesis-associated genes. Thus, our results indicate that T. cruzi P21 is an attractive target for the development of innovative therapeutic agents against CCC.Univ Fed Sao Paulo, Escola Paulista Med, Departamento Microbiol Imunol Parasitol, BR-05508 Sao Paulo, SP, BrazilUniv Fed Uberlandia, Inst Ciencias Biomed, Dept Imunol, Lab Tripanosomatideos, Uberlandia, MG, BrazilUniv Fed Uberlandia, Inst Genet & Bioquim, Lab Bioquim & Toxinas Animais, Uberlandia, MG, BrazilCeTICS, Inst Butantan, Sao Paulo, BrazilUniv Fed Uberlandia, Fac Med, Centro Referencia Nacl Dermatol Sanitaria Hanseni, Lab Patol Mol & Biotecnol, Uberlandia, MG, BrazilUniv Fed Uberlandia, Inst Ciencias Biomed, Dept Immunol, Lab Osteoimunol & Imunol Tumores, Uberlandia, MG, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Departamento Microbiol Imunol Parasitol, BR-05508 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Departamento Microbiol Imunol Parasitol, BR-05508 Sao Paulo, SP, BrazilWeb of Scienc

Microbial and epigenetic factors in the pathogenesis of nasopharyngeal carcinoma

While uncommon in most of the world, nasopharyngeal carcinoma (NPC) shows an unusual geographic and ethnic distribution, being highly prevalent in Southern China and Southeast Asia. Genetic susceptibility, Epstein-Barr virus (EBV) infection and additional environmental exposures are well established risk factors for NPC in endemic areas. However, the detailed molecular mechanisms of NPC pathogenesis remain largely unknown. In this thesis, several novel pathogenic mechanisms of NPC development and progression are presented. The interaction of EBV encoded latent membrane protein 2A (LMP2A) with cellular proteins promoting invasiveness of NPC cells is described in paper I. Spleen tyrosine kinase (Syk) interacts with integrin β4 subunit (ITGβ4) in epithelial cells through an ITAM-like motif, and concurrent LMP2A expression interferes with this interaction by competitive binding to Syk. Both Syk and LMP2A affect cell surface expression of ITGβ4. Particularly, ITGβ4 concentrates at cellular protrusions in LMP2A expressing cells, which may contribute to the migration property of NPC-cells. Paper II and paper III focus on the epigenetic alteration of candidate tumor suppressor genes (TSGs) and their possible role in NPC tumorigenesis. Cadherin 4 (CDH4) and ubiquitin-conjugating enzyme 2L6 (UBE2L6) are downregulated due to promoter hypermehtylation in NPC. Both genes suppress the proliferation and colony formation of NPC-cells. CDH4 impedes cell migration and elicits cell communication; UBE2L6 induce apoptosis of NPC cells and counteracts degradation of adipocyte triglyceride lipase (ATGL) through ISG15-conjugation of valosin-containing protein (VCP). CDH4 and UBE2L6 could be involved in both initiation and progression of NPC. Suppression of UBE2L6 encoded protein UbcH8 correlated with poor outcome in NPC patients. In paper IV, we compared the response of NPC and normal nasopharyngeal epithelial (NNE) cell lines to bacteria and bacterial cell wall components. Strong nuclear translocation of NF-κB and significant induction of proinflammatory factors IL6, IL8, IL1α and CXCL2 were observed in NNE cells, but not in NPC cells upon exposure to Gram-positive bacteria streptococci and peptidoglycan (PGN). We identified three different mechanisms by which the activation of NF-κB in NPC cells could be hampered. It could be trapped by an enhanced accumulation of cytoplasmic lipids. I-κB degradation could be impaired due to downregulation of UBE2L6. We also showed that overexpression of lysine-specific demethylase-1 (LSD1) blocked the transcriptional activation of proinflammatory genes. Together these mechanisms might contribute to decreased immune reactivity in NPC and thus affect tumor progression

Role of Bovine Ileal Sub-epithelial Myofibroblasts and Epithelial Cells in Innate Immunity

Gastro-intestinal (GI) tract harbors largest number of microbiota as well as the largest number of immune cells for a given tissue. The host needs to mount an effective immune response against invading pathogens and tolerance against commensals. Thus, regulatory mechanism and barrier function of the GI tract are of utmost importance for appropriate host microbe interaction and gut homeostasis. Intestinal epithelial cells (IECs) act as the first line of defense against invading pathogens. IECs recognize pathogens and commensals and mount an effective innate immune response. Such recognition of pathogens is mediated through germ line encoded pattern recognition receptors (PRRs). Intestinal sub-epithelial myofibroblasts (ISEMFs) reside just beneath the surface epithelium and are involved in maturation and differentiation of epithelium. ISEMFs protect from pathogens that breach surface epithelium by expressing PRRs. Lack of stable intestinal epithelial and sub-epithelial myofibroblast cell lines has slowed down scientific studies on these cells. In this study, we established and characterized ISEMF cells from the ileum of a 2-day old calf. We also had generated stable bovine ileal epithelial cell (BIEC-c4) cultures in our lab. On real time-quantitative polymerase chain reaction (RT-qPCR) analysis both these cell types expressed Toll-like receptors (TLRs) 1-9. To investigate their responses to various pathogen-associated molecular patterns (PAMPs), we stimulated both cell types for 3 hours and 24 hours with various PAMPs. The RT-qPCR assay was used to investigate changes in TLR gene expression and in cytokine genes following stimulation. Lipopolysaccharide, peptidoglycan, and flagellin were used as bacterial ligands of surface PRRs. Similarly, γ-D-Glu-mDAP, muramyl dipeptide, polyinosonic:polycytidylic acid, poly I:C complexed with lyovec, and imiquimod were used as ligands of cytosolic and endosomal PRRs. Bovine ileal ISEMFs responded to bacterial PAMPs and to ligands of cytosolic and endosomal PRRs by significantly altering TLR gene expression. Unlike bovine ISEMFs, BIEC-c4 cells responded only to bacterial ligands. Thus, we conclude that bovine ileal ISEMF can be a good model to study innate immune responses and signaling pathways occurring at subepithelial compartment. However, BIEC-c4 cells may serve as a good in-vitro model to study enteric infectious disease pathogenesis and innate immune responses associated with them

Development of a Streptococcus pneumoniae system to control exposure of immature peptidoglycan

Streptococcus pneumoniae is a Gram-positive bacterial pathogen capable of causing from mild infections to more severe conditions such as pneumonia and meningitis. Pneumococcal virulence is dependent on the production of different virulence factors, such as particular peptidoglycan-associated surface proteins or the production of a capsular polysaccharide structure surrounding the bacteria. Peptidoglycan, a major cell wall component, offers structural stability to the cell and is dynamically tailored by hydrolases, such as LytA, during physiological mechanisms like cell elongation, division and septation. Capsule production, which is strictly regulated, requires expression of several genes present in the cps locus, including genes with a regulatory role such as wzd and wze. The interaction between proteins Wzd and Wze, in the presence of ATP, is thought to play a central role in the regulation of capsule synthesis. The aim of this dissertation was to develop a S. pneumoniae system capable of controlling the exposure of immature peptidoglycan to enzymes present in the surrounding medium. To achieve this goal, pneumococcal strains were constructed where the Escherichia coli LacI repressor (encoded by the lacI gene) is constitutively expressed and the capsule regulatory genes pneumococcal wzd and wze are placed under the control of an IPTG-inducible promoter (Plac). This was performed in the background of S. pneumoniae wzd and wze deletion mutants, where immature peptidoglycan is exposed at the surface of the septal region due to absence of capsule at the division septum. One of the constructed strains, 6314Δwze-lacI-Plac-wze, was analyzed by immunofluorescence microscopy to assess if septal capsule expression was restored at the division septum upon expression of IPTG-inducible wze. This analysis showed that the constructed mutant, in the presence of IPTG, presented a seemingly intermediate phenotype between absence of capsule in the division septum and full encapsulation

Characterization of Porcine Respiratory Epithelial Cells and Their Innate Immune Responses to Bacterial and Viral Ligands

In response to a pathogenic attack, the host produces a series of defense mechanisms through various intracellular signaling pathways. The byproduct of these signaling pathways helps tackle the invading pathogen and protects the body from getting into a diseased state. This system is called the immune system. The immune system can be divided into two branches namely the innate immune system and adaptive immune system. The groups of immune cells that provide protection regardless of the pathogen specificity constitute the innate immune system. The system that acts according to the pathogen specificity is called the adaptive immune response. The production of antibodies by B cells is a prime example of adaptive immune responses. Macrophages, neutrophils, and monocytes are a few examples of innate immune cells. Besides them, mucosal epithelial cells of the intestinal and respiratory systems are crucial in generating innate immune responses. Invading pathogens and their recognition by the host is pivotal in preventing the subsequent infection and diseases. Epithelial cells express various Pathogen Recognition Receptors (PRRs). These PRRs recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). The binding of PAMPs and/or DAMPs to epithelial cells initiates intracellular signaling pathways that lead to the generation of innate immune responses through the regulation of gene expression. Porcine respiratory epithelial cells and their expression of PRRs render them vital not only for the regulation of innate immune responses but also to study respiratory disease pathogenesis. Ironically, only a handful of studies can be found on these cells and the limited number of studies have hindered our understanding of the role of porcine respiratory cells in innate immunity. In this study, we have characterized previously established porcine primary respiratory epithelial cells from nasal turbinate and trachea followed by their immortalization using hTERT and SV40 large T-antigen. We also studied their innate immune responses to various bacterial and viral ligands. Both the primary and immortalized cells showed typical epithelial cobblestone morphology with a heavy expression of cytokeratin indicating epithelial origin. Cells did not change their morphological characteristics even after immortalization. Immortalization was confirmed by immunofluorescence assay for SV40 and immunocytochemistry for hTERT. However, they did look more granulated than the primary cells. Growth curve analysis showed a faster growth rate of immortalized cells in comparison to the primary cells of both nasal and tracheal origin. Finally, we stimulated the primary cells with various bacterial and viral ligands. Upon stimulation, porcine primary respiratory cells mounted innate immune responses through modulation of the expression of various PRRs and the production of cytokines/chemokines. Modulation of gene expression on mRNA level was measured using ΔΔct method. The research findings may be vital in studying the role of respiratory epithelial cells in the pathogenesis of various respiratory diseases and innate immune responses in pigs

Characterization of translationally controlled tumour protein from the sea anemone Anemonia viridis and transcriptome wide identification of cnidarian homologues

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis. These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP (AvTCTP) after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses

STER1, a novel receptor-like kinase, functions in MAMP signalling in Arabidopsis

S. epidermidis has long been recognized as an important opportunistic pathogen accounting for the majority of nosocomial infections alongside S. aureus. However, in spite of this, our understanding of the S. epidermidis virulence mechanisms is still limited. Previous studies have emphasized various analogies in innate immunity against pathogens in plant, invertebrate and mammalian hosts. When compared to in vivo animal models, plant models are an attractive alternative and experiments using the S.aureus-Arabidopsis pathosystem have shown potential for this approach with S. epidermidis. In this study, an Arabidopsis−S.epidermidis system was established aiming to identify possible bacterial virulence traits. As S. epidermidis is not a true plant pathogen it fails to multiply in planta; however, most S. epidermidis strains tested generated a salycilic acid (SA)-dependent necrotic phenotype in Arabidopsis 5 days−post inoculation. Additionally, inoculation with boiled bacteria generated the same visual response as live cells, suggesting a pre−existent, heat stable molecule underlies the plant visual response. Taken together, this data suggests the necrotic response is a visual expression of MAMP perception. Subsequent exploitation of the Arabidopsis natural variation through QTL analysis, resulted in the isolation of the STER1 (Staphylococcus elicitor response 1) gene. This gene is essential for the visual response to S. epidermidis 18888 and encodes a membrane localized DUF26−containing receptor−like kinase. The ster1-1 mutant remained asymptomatic following inoculation with Gram−positive S. epidermidis 18888 and Gram−negative B. ambifaria, suggesting STER1’s likely role in the recognition of a common molecule. Peptidoglycan (PGN), an essential bacterial membrane component was considered a likely candidate and isolated from both species. Inoculation with B. ambifaria PGN generates a plant response that mirrores the one seen with bacterial suspensions of the same organism. By contrast, pure S. epidermidis 18888 PGN does not trigger a visual response in Arabidopsis. Instead, an S. epidermidis 18888 membrane fraction (MF), consisting of PGN, teichoic acid (TA) and an uncharacterized capsular polysaccharide (CPS), was found to generate a necrotic response similar to live cells. Treatment with S. epidermidis MF and B. ambifaria PGN triggered stereotypical defence responses, such as PR1 up−regulation and cell death in wild−type plants, but not in the ster1-1 mutant. Additionally, pre-treatment with S. epidermidis MF and B. ambifaria PGN also restricted Pst DC3000 growth in wild−type plants only, thus emphasizing a likely role for STER1 in basal resistance and PGN perception. In conclusion, the data obtained in this study implicate STER1 in PGN and possibly specialized CPS recognition, either as a receptor, co-receptor or essential signalling component