Lactoylglutathione lyase, a critical enzyme in methylglyoxal detoxification, contributes to survival of Salmonella in the nutrient rich environment

Glyoxalase I which is synonymously known as lactoylglutathione lyase is a critical enzyme in methylglyoxal (MG) detoxification. We assessed the STM3117 encoded lactoylglutathione lyase (Lgl) of Salmonella Typhimurium, which is known to function as a virulence factor, due in part to its ability to detoxify methylglyoxal. We found that STM3117 encoded Lgl isomerises the hemithioacetal adduct of MG and glutathione (GSH) into S-lactoylglutathione. Lgl was observed to be an outer membrane bound protein with maximum expression at the exponential growth phase. The deletion mutant of S. Typhimurium (lgl) exhibited a notable growth inhibition coupled with oxidative DNA damage and membrane disruptions, in accordance with the growth arrest phenomenon associated with typical glyoxalase I deletion. However, growth in glucose minimal medium did not result in any inhibition. Endogenous expression of recombinant Lgl in serovar Typhi led to an increased resistance and growth in presence of external MG. Being a metalloprotein, Lgl was found to get activated maximally by Co2+ ion followed by Ni2+, while Zn2+ did not activate the enzyme and this could be attributed to the geometry of the particular protein-metal complex attained in the catalytically active state. Our results offer an insight on the pivotal role of the virulence associated and horizontally acquired STM3117 gene in non-typhoidal serovars with direct correlation of its activity in lending survival advantage to Salmonella spp.

Rrp1B gene polymorphism (1307T > C) in metastatic progression of breast cancer

Rrp1B (ribosomal RNA processing1 homolog B) is a novel candidate metastasis modifier gene in breast cancer. Functional gene assays demonstrated that a physical and functional interaction existing between Rrp1b and metastasis modifier gene SIPA1 causes reduction in the tumor growth and metastatic potential. Ectopic expression of Rrp1B modulates various metastasis predictive extra cellular matrix (ECM) genes associated with tumor suppression. The aim of this study is to determine the functional significance of single nucleotide polymorphism (SNP) in human Rrp1B gene (1307 T > C; rs9306160) with breast cancer development and progression. The study consists of 493 breast cancer cases recruited from Nizam's Institute of Medical Sciences, Hyderabad, and 558 age-matched healthy female controls from rural and urban areas. Genomic DNA was isolated by non-enzymatic method. Genotyping was done by amplification refractory mutation system (ARMS-PCR) method. Genotypes were reconfirmed by sequencing and results were analyzed statistically. We have performed Insilco analysis to know the RNA secondary structure by using online tool m fold. The TT genotype and T allele frequencies of Rrp1B1307 T > C polymorphism were significantly elevated in breast cancer (chi (2); p = < 0.008) cases compared to controls under different genetic models. The presence of T allele had conferred 1.75-fold risk for breast cancer development (OR = 1.75; 95 % CI = 1.15-2.67). The frequency of TT genotype of Rrp1b 1307T > C polymorphism was significantly elevated in obese patients (chi (2); p = 0.008) and patients with advanced disease (chi (2); p = 0.01) and with increased tumor size (chi (2); p = 0.01). Moreover, elevated frequency of T allele was also associated with positive lymph node status (chi (2); p = 0.04) and Her2 negative receptor status (chi (2); p = 0.006). Presence of Rrp1b1307TT genotype and T allele confer strong risk for breast cancer development and progression.

Co-liposomes of redox-active alkyl-ferrocene modified low MW branched PEI and DOPE for efficacious gene delivery in serum

Herein, we present six new lipopolymers based on low molecular weight, branched polyethylenimine (BPEI 800 Da) which are hydrophobically modified using ferrocene terminated alkyl tails of variable lengths. The effects of degree of grafting, spacer length and the redox state of ferrocene in the lipopolymers on the self assembly properties were investigated in detail by TEM, AFM, DLS and zeta potential measurements. The assemblies displayed an oxidation induced increase in the size of the aggregates. The co-liposomes comprising the lipopolymer and a helper lipid, 1,2-dioleoyl phosphatidyl ethanolamine (DOPE), showed excellent gene (pDNA) delivery capability in a serum containing environment in two cancer cell lines (HeLa and U251 cells). Optimized formulations showed remarkably higher transfection activity than BPEI (25 kDa) and were also significantly better than a commercial transfection reagent, Lipofectamine 2000 as evidenced from both the luciferase activity and GFP expression analysis. Oxidation of ferrocene in the lipopolymers led to drastically reduced levels of gene transfection which was substantiated by reduced cellular internalization of fluorescently labelled pDNA as detected using confocal microscopy and flow cytometry. Moreover, the transfection inactive oxidized lipopolyplexes could be turned transfection active by exposure to ascorbic acid (AA) in cell culture medium during transfection. Endocytosis inhibition experiments showed that gene expression mediated by reduced formulations involved both clathrin and caveolae mediated pathways while the oxidized formulations were routed via the caveolae. Cytotoxicity assays revealed no obvious toxicity for the lipopolyplexes in the range of optimized transfection levels in both the cell lines studied. Overall, we have exploited the redox activity of ferrocene in branched PEI-based efficient polymeric gene carriers whose differential transfection activities could be harnessed for spatial or temporal cellular transfections.

Mycobacterium tuberculosis Response Regulators, DevR and NarL, Interact in Vivo and Co-regulate Gene Expression during Aerobic Nitrate Metabolism

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and Delta narL mutant strain during exponential growth in broth cultures with or without nitrate defined an similar to 30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis Delta devR, Delta devS Delta dosT, and Delta narL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR similar to P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

Dendron conjugation to graphene oxide using click chemistry for efficient gene delivery

Owing to its large surface area and rapid cellular uptake, graphene oxide (GO) is emerging as an attractive candidate material for delivery of drugs and genes. The inherent sp(2) pi-pi interaction of GO helps to carry drugs and single stranded RNA (ssRNA) but there is no such interaction with double stranded DNA (dsDNA). In this work, a polyamidoamine (PAMAM) dendron was conjugated with nano GO (nGO) through ``click'' chemistry to improve the DNA complexation capability of GO as well as its transfection efficiency. The DNA complexation capability of GO was significantly enhanced after dendronization of GO yielding spherical nanosized (250-350 nm) particles of the dendronized GO (DGO)/pDNA complex with a positive zeta potential. The transfection efficiency of GO dramatically increased after conjugation of the PAMAM dendron. Transfection efficiency of 51% in HeLa cells with cell viability of 80% was observed. The transfection efficiency was significantly higher than that of polyethyleneimine 25 kDa (27% efficiency) and also surpassed that of lipofectamine 2000 (47% efficiency). The uptake of the DGO/pDNA complex by the caveolae mediated endocytosis pathway may significantly contribute to the high transfection efficiency. Thus, dendronized GO is shown to be an efficient gene carrier with minimal toxicity and is a promising candidate for use as a nonviral carrier for gene therapy.

Role of Areca Nut Induced TGF-beta and Epithelial-Mesenchymal Interaction in the Pathogenesis of Oral Submucous Fibrosis

Areca nut consumption has been implicated in the progression of Oral Submucous fibrosis (OSF); an inflammatory precancerous fibrotic condition. Our previous studies have demonstrated the activation of TGF-beta signaling in epithelial cells by areca nut components and also propose a role for epithelial expressed TGF-beta in the pathogenesis of OSF. Although the importance of epithelial cells in the manifestation of OSF has been proposed, the actual effectors are fibroblast cells. However, the role of areca nut and TGF-beta in the context of fibroblast response has not been elucidated. Therefore, to understand their role in the context of fibroblast response in OSF pathogenesis, human gingival fibroblasts (hGF) were treated with areca nut and/or TGF-beta followed by transcriptome profiling. The gene expression profile obtained was compared with the previously published transcriptome profiles of OSF tissues and areca nut treated epithelial cells. The analysis revealed regulation of 4666 and 1214 genes by areca nut and TGF-beta treatment respectively. The expression of 413 genes in hGF cells was potentiated by areca nut and TGF-beta together. Further, the differentially expressed genes of OSF tissues compared to normal tissues overlapped significantly with areca nut and TGF-beta induced genes in epithelial and hGF cells. Several positively enriched pathways were found to be common between OSF tissues and areca nut + TGF-beta treated hGF cells. In concordance, areca nut along with TGF-beta enhanced fibroblast activation as demonstrated by potentiation of alpha SMA, gamma SMA and collagen gel contraction by hGF cells. Furthermore, TGF-beta secreted by areca nut treated epithelial cells influenced fibroblast activation and other genes implicated in fibrosis. These data establish a role for areca nut influenced epithelial cells in OSF progression by activation of fibroblasts and emphasizes the importance of epithelial-mesenchymal interaction in OSF.

Experimental Investigations on Interaction of Two Drops by Thermocapillary-Buoyancy Migration

Experiments were performed, in a terrestrial environment, to study the migration and interaction of two drops with different diameters in matrix liquid under temperature gradient field. Pure soybean oil and silicon oil were used as matrix liquid and the drop liquid, respectively. The information on the motions of two drops was recorded by CCD camera system in the experiments to analyze the trajectories and velocities of the drops. Our experiments showed that, upon two drops approaching each other, the influence of the larger drop on the motion of the smaller one became significant. Meanwhile the smaller drop had a little influence on the larger one all the time. The oscillation of migration velocities of both drops was observed as they were approaching. For a short period the smaller drop even moved backward when it became side by side with the larger one during the migration. Although our experimental results on the behavior of two drops are basically consistent with the theoretical predictions, there are also apparent differences. 2006 Elsevier Ltd. All rights reserved. Keywords: Thermocapillary migration; Drop; Interaction; Oscillation 1. Introduction A bubble or drop will move when placed in another fluid with temperature gradient. This motion happens as a consequence of the variation of interfacial tension with temperature. Such a phenomenon is already known as Marangoni migration problem. With the development of microgravity science, bubble dynamics and droplet dynamics became a hot point problem of research because this investigation is very important for basic research as well as for applications in reduced gravity environment, such as space material science, chemical engineering and so on. Young et al. first investigated the thermocapillary migration of

Planar Thermocapillary Migration of Two Bubbles in Microgravity Environment

A theoretical investigation is performed on the thermocapillary motion of two bubbles in arbitrary configuration in microgravity environment under the assumption that the surface tension is high enough to keep the bubbles spherical. The two bubbles are dr

Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

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Molecular mechanisms of interleukin-2 gene inducibility: developmental control and combinatorial action of transcription factors

Interleukin-2 is one of the lymphokines secreted by T helper type 1 cells upon activation mediated by T-cell receptor (TCR) and accessory molecules. The ability to express IL-2 is correlated with T-lineage commitment and is regulated during T cell development and differentiation. Understanding the molecular mechanism of how IL-2 gene inducibility is controlled at each transition and each differentiation process of T-cell development is to understand one aspect of T-cell development. In the present study, we first attempted to elucidate the molecular basis for the developmental changes of IL-2 gene inducibility. We showed that IL-2 gene inducibility is acquired early in immature CD4- CD8-TCR- thymocytes prior to TCR gene rearrangement. Similar to mature T cells, a complete set of transcription factors can be induced at this early stage to activate IL-2 gene expression. The progression of these cells to cortical CD4^+CD8^+TCR^(1o) cells is accompanied by the loss of IL-2 gene inducibility. We demonstrated that DNA binding activities of two transcription factors AP-1 and NF-AT are reduced in cells at this stage. Further, the loss of factor binding, especially AP-1, is attributable to the reduced ability to activate expression of three potential components of AP-1 and NF-AT, including c-Fos, FosB, and Fra-2. We next examined the interaction of transcription factors and the IL-2 promoter in vivo by using the EL4 T cell line and two non-T cell lines. We showed an all-or-none phenomenon regarding the factor-DNA interaction, i.e., in activated T cells, the IL-2 promoter is occupied by sequence-specific transcription factors when all the transcription factors are available; in resting T cells or non-T cells, no specific protein-DNA interaction is observed when only a subset of factors are present in the nuclei. Purposefully reducing a particular set of factor binding activities in stimulated T cells using pharmacological agents cyclosporin A or forskolin also abolished all interactions. The results suggest that a combinatorial and coordinated protein-DNA interaction is required for IL-2 gene activation. The thymocyte experiments clearly illustrated that multiple transcription factors are regulated during intrathymic T-cell development, and this regulation in tum controls the inducibility of the lineage-specific IL-2 gene. The in vivo study of protein-DNA interaction stressed the combinatorial action of transcription factors to stably occupy the IL-2 promoter and to initiate its transcription, and provided a molecular mechanism for changes in IL-2 gene inducibility in T cells undergoing integration of multiple environmental signals.

Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo

Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a potent growth factor that mature T lymphocytes use as a proliferation signal and the production of IL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response. IL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate multichain receptor. IL-2 expression is induced only upon stimulation and transcriptional activation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible and constitutive trans-acting factors. Over the past several years, thousands of papers have been published regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The vast majority of these reports describe work that has been carried out in vitro. However, considerably less is known about control of IL-2 gene expression and IL-2 function in vivo.

To gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and developmental patterns of IL-2 gene expression in the mouse were established by employing in situ hybridization and immunohistochemical staining methodologies to tissue sections generated from normal mice and mutant animals in which T -cell development was perturbed. Results from these studies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2 gene expression and protein synthesis in the thymus, the primary site of T-cell development in the body, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the extrathymic expansion of mature T cells in particular tissues, independent of an acute immune response to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the mucosal immune system, and (5) potential function of IL-2 in early events associated with hematopoiesis.

Extensive analysis of IL-2 mRNA accumulation and protein production in the murine thymus at various stages of development established the existence of two classes of intrathymic IL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal thymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2 expression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12 hours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the presence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the generation of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was also detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2 producers in the fetal thymus) but persist in the thymus during later stages of development and after birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region and cortex, indicating that these cells reside in the same areas where immature T cells are consigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low, indicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation event. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in the postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination of intrathymic IL-2 production in mutant postnatal mice unable to produce either αβ or γδ T cells showed that postnatal IL-2 producers in the thymus belong to both αβ and γδ lineages. Additionally, further studies indicated that IL-2 synthesis by immature αβ -T cells depends on the expression of bonafide TcR αβ-heterodimers. Taken altogether, IL-2 production in the postnatal thymus relies on the generation of αβ or γδ-TcR^+ cells and induction of IL-2 protein synthesis can be linked to an activation event mediated via the TcR.

With regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body sections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2 mRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are associated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2 mRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder. Additional analysis of isolated tissues taken from older animals revealed that IL-2 expression was undetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that extrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to particular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.

Based on the neonatal in situ hybridization results, a detailed investigation into possible induction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that IL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell proliferation of resident cells and/or participate in immune function in these tissues. Pertaining to IL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were observed when resident Vγ_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+ cells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this age a significant number of CD3ε^+ cells were also found in association with follicles. The colocalization of IL-2 expression and CD3ε^+ cells suggests that IL-2 expression is induced when T cells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were readily detected once T-cell density in the skin reached steady-state proportions. At this point, T cells were no longer found associated with hair follicles but were evenly distributed throughout the epidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T cells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of a specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin indicate that IL-2 may play an additional role in controlling mature T-cell proliferation by participating in the extrathymic expansion of T cells, particularly those associated with the epidermis.

Finally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells were found associated with the lamina propria of neonatal animals and gut-associated IL-2 production persisted throughout life. In older animals, the frequency of IL-2 producing cells in the small intestine was not identical to that in the large intestine and this difference may reflect regional specialization of the mucosal immune system in response to enteric antigen. Similar to other instances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an abrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the neonatal gut suggested that these cells may be generated during fetal development. Examination of the fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a tenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to that observed four days earlier and there was little difference between the frequency of IL-2 producing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing cells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of IL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as the fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of future functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum raises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2 producing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results provide increased understanding of the nature of IL-2 producing cells in the gut and how the absence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute pathology observed in IL-2 deficient animals.

Role of feedback and dynamics in a gene regulatory network

Cells exhibit a diverse repertoire of dynamic behaviors. These dynamic functions are implemented by circuits of interacting biomolecules. Although these regulatory networks function deterministically by executing specific programs in response to extracellular signals, molecular interactions are inherently governed by stochastic fluctuations. This molecular noise can manifest as cell-to-cell phenotypic heterogeneity in a well-mixed environment. Single-cell variability may seem like a design flaw but the coexistence of diverse phenotypes in an isogenic population of cells can also serve a biological function by increasing the probability of survival of individual cells upon an abrupt change in environmental conditions. Decades of extensive molecular and biochemical characterization have revealed the connectivity and mechanisms that constitute regulatory networks. We are now confronted with the challenge of integrating this information to link the structure of these circuits to systems-level properties such as cellular decision making. To investigate cellular decision-making, we used the well studied galactose gene-regulatory network in \textit{Saccharomyces cerevisiae}. We analyzed the mechanism and dynamics of the coexistence of two stable on and off states for pathway activity. We demonstrate that this bimodality in the pathway activity originates from two positive feedback loops that trigger bistability in the network. By measuring the dynamics of single-cells in a mixed sugar environment, we observe that the bimodality in gene expression is a transient phenomenon. Our experiments indicate that early pathway activation in a cohort of cells prior to galactose metabolism can accelerate galactose consumption and provide a transient increase in growth rate. Together these results provide important insights into strategies implemented by cells that may have been evolutionary advantageous in competitive environments.

Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.

Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits

Synthetic biology combines biological parts from different sources in order to engineer non-native, functional systems. While there is a lot of potential for synthetic biology to revolutionize processes, such as the production of pharmaceuticals, engineering synthetic systems has been challenging. It is oftentimes necessary to explore a large design space to balance the levels of interacting components in the circuit. There are also times where it is desirable to incorporate enzymes that have non-biological functions into a synthetic circuit. Tuning the levels of different components, however, is often restricted to a fixed operating point, and this makes synthetic systems sensitive to changes in the environment. Natural systems are able to respond dynamically to a changing environment by obtaining information relevant to the function of the circuit. This work addresses these problems by establishing frameworks and mechanisms that allow synthetic circuits to communicate with the environment, maintain fixed ratios between components, and potentially add new parts that are outside the realm of current biological function. These frameworks provide a way for synthetic circuits to behave more like natural circuits by enabling a dynamic response, and provide a systematic and rational way to search design space to an experimentally tractable size where likely solutions exist. We hope that the contributions described below will aid in allowing synthetic biology to realize its potential.

Análise de polimorfismos e de expressão do gene p16INK4a em neoplasias cervicais

O câncer de colo do útero é o segundo carcinoma mais frequente em mulheres no mundo e um dos cânceres femininos mais incidentes no Brasil. Em lesões pré-malignas e malignas do colo uterino, a proteína p16INK4a, que participa do controle do ciclo celular, apresenta um aumento considerável de sua expressão, devido possivelmente à presença de oncoproteínas do papilomavírus humano (HPV). Dois polimorfismos no gene p16INK4a, p16 500C>G e p16 540C>T, estão localizados na região 3 não traduzida (3UTR), que está envolvida na regulação pós-transcricional da expressão gênica. O objetivo deste estudo foi avaliar possíveis associações entre os polimorfismos p16 500C>G e p16 540C>T e o desenvolvimento de neoplasias cervicais e/ou a severidade das lesões, considerando os níveis de expressão da proteína p16INK4a nas lesões cervicais e certos fatores de risco clássicos para o câncer cervical, incluindo a infecção pelo HPV. Para isso, foram selecionadas 567 mulheres residentes no Rio de Janeiro, 319 com citologia cervical alterada (grupo de casos) e 248 sem história prévia de alteração citológica do colo uterino (grupo de comparação). Amostras de sangue periférico de todas as participantes foram utilizadas na análise molecular dos polimorfismos p16 500C>G e p16 540C>T através da técnica de PCR-RFLP (reação em cadeia da polimerase - polimorfismo de comprimento de fragmento de restrição), usando as enzimas de restrição MspI e HaeIII, respectivamente. A expressão da proteína p16INK4a em 137 biópsias de mulheres pertencentes ao grupo de casos foi avaliada por imunohistoquímica. A detecção de DNA do HPV em células cervicais foi feita em todas as amostras do grupo de comparação e em 194 amostras do grupo de casos pela técnica de PCR, usando dois pares de oligonucleotídeos, MY09/MY11 e GP05+/GP06+. Os dois grupos de estudo se encontram em equilíbrio de Hardy-Weinberg. As distribuições genotípicas para p16 500C>G e p16 540C>T e as distribuições de combinações haplotípicas nos dois grupos não apresentaram diferenças significativas. A análise do subgrupo HSIL+câncer (casos com lesão intraepitelial de alto grau ou carcinoma invasivo) em comparação com o subgrupo LSIL (casos com lesão intraepitelial de baixo grau) revelou diferença significativa entre as distribuições das combinações haplotípicas (p = 0,036) e diferenças marginais entre as distribuições genotípicas para p16 500C>G (p = 0,071) e p16 540C>T (p = 0,051). O alelo p16 540G, em heterozigose ou homozigose (OR = 1,91, IC 95% = 1,08-3,37), e a combinação haplotípica p16 500C-540C 500G-540C (OR = 2,34, IC 95% = 1,202-4,555) mostraram-se associados com a severidade da lesões cervicais. Já o genótipo p16 540T/T (OR = 0,25, IC 95% = 0,08-0,79), e a combinação haplotípica p16 500C-540T 500C-540T (OR = 0,27, IC 95% = 0,088-0,827) exibiram papel protetor contra o desenvolvimento de lesões mais severas. As análises de interação entre os polimorfismos de p16INK4a e a expressão de p16 ou a infecção pelo HPV foram comprometidas pelo número reduzido de amostras analisadas. Não se observou qualquer interação entre os polimorfismos estudados e os fatores de risco clássicos para o câncer de colo uterino. Nossos resultados apontam para a importância dos polimorfismos do gene p16INK4a como marcadores de severidade da neoplasia cervical.