Design and expression of peptides with antimicrobial activity against Salmonella typhimurium.

We showed previously that insertion of Synechocystis Δ12 -desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ12 -desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α-antimicrobial peptide (α-AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg2+ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA-Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α-AMPs to a specific MLD generating live non-virulent bacterial strains.

Plant nanobionic materials with a giant temperature response mediated by pectin-Ca2+.

Conventional approaches to create biomaterials rely on reverse engineering of biological structures, on biomimicking, and on bioinspiration. Plant nanobionics is a recent approach to engineer new materials combining plant organelles with synthetic nanoparticles to enhance, for example, photosynthesis. Biological structures often outperform man-made materials. For example, higher plants sense temperature changes with high responsivity. However, these properties do not persist after cell death. Here, we permanently stabilize the temperature response of isolated plant cells adding carbon nanotubes (CNTs). Interconnecting cells, we create materials with an effective temperature coefficient of electrical resistance (TCR) of -1,730% K(-1), ∼2 orders of magnitude higher than the best available sensors. This extreme temperature response is due to metal ions contained in the egg-box structure of the pectin backbone, lodged between cellulose microfibrils. The presence of a network of CNTs stabilizes the response of cells at high temperatures without decreasing the activation energy of the material. CNTs also increase the background conductivity, making these materials suitable elements for thermal and distance sensors.

Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the "Membrane Sensor Hypothesis" which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Antifungal activity of azole compounds CPA18 and CPA109 against azole-susceptible and -resistant strains of Candida albicans.

OBJECTIVES: In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. METHODS: Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. RESULTS: Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. CONCLUSIONS: Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

Can the stress protein response be controlled by 'membrane-lipid therapy'?

In addition to high temperature, other stresses and clinical conditions such as cancer and diabetes can lead to the alteration of heat-shock protein (HSP) levels in cells. Moreover, HSPs can associate with either specific lipids or with areas of special membrane topology (such as lipid rafts), and changes in the physical state of cellular membranes can alter hsp gene expression. We propose that membrane microheterogeneity is important for regulating the HSP response. In support of this hypothesis, when particular membrane intercalating compounds are used to alter membrane properties, the simultaneous normalization of dysregulated expression of HSPs causes beneficial responses to disease states. Therefore, these compounds (such as hydroxylamine derivatives) have the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy'.

Up-regulated expression of desaturase genes of Mucor rouxii in response to low temperature associates with pre-existing cellular fatty acid constituents.

Transcriptional response of desaturase genes to low temperature was investigated in the dimorphic fungus Mucor rouxii. The two morphological forms of M. rouxii, yeast-like and mycelial cells containing different fatty acid profiles were shifted from 30 to 10°C. Both cultures exhibited significantly altered fatty acid composition, whose content in polyunsaturated fatty acids increased as consequence of the temperature shift and was accompanied by a reduction of C18:1Δ(9) about 2 h after the temperature shift. These changes were particularly significant in phosphatidylcholine and phosphatidylethanolamine fractions. Moreover, the fatty acid profiles of monoacylglycerol and diacylglycerol were also modulated in response to the lower temperature of incubation. The changes of membrane lipids of M. rouxii were due to the cold-induced expression of Δ(9)-, Δ(12)- and Δ(6)-desaturase genes. Although the mRNA levels of the three desaturases were transiently induced by lowering the temperature, the pre-existing composition of fatty acid profiles of mycelial and yeast-like forms of M. rouxii may have lead to different expression profiles of desaturase genes that modified their membrane physical state under cold shock. While expression of Δ(12)-desaturase gene contributed mainly to cold acclimation of mycelia, Δ(9)-desaturase expression was the main transcript identified in the yeast-like culture after temperature shift.

Genetic modification of the Salmonella membrane physical state alters the pattern of heat shock response.

It is now recognized that membranes are not simple physical barriers but represent a complex and dynamic environment that affects membrane protein structures and their functions. Recent data emphasize the role of membranes in sensing temperature changes, and it has been shown that the physical state of the plasma membrane influences the expression of a variety of genes such as heat shock genes. It has been widely shown that minor alterations in lipid membranes are critically involved in the conversion of signals from the environment to the transcriptional activation of heat shock genes. Previously, we have proposed that the composition, molecular arrangement, and physical state of lipid membranes and their organization have crucial roles in cellular responses during stress caused by physical and chemical factors as well as in pathological states. Here, we show that transformation of Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) with a heterologous Delta(12)-desaturase (or with its trans-membrane regions) causes major changes in the pathogen's membrane dynamic. In addition, this pathogen is strongly impaired in the synthesis of major stress proteins (heat shock proteins) under heat shock. These data support the hypothesis that the perception of temperature in Salmonella is strictly controlled by membrane order and by a specific membrane lipid/protein ratio that ultimately causes transcriptional activation of heat shock genes. These results represent a previously unrecognized mode of sensing temperature variation used by this pathogen at the onset of infection.

Changes in membrane fluid state and heat shock response cause attenuation of virulence.

So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Delta(12)-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MPhi) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Delta(9)-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MPhi along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MPhi of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.

The significance of lipid composition for membrane activity: new concepts and ways of assessing function.

In the last decade or so, it has been realised that membranes do not just have a lipid-bilayer structure in which proteins are embedded or with which they associate. Structures are dynamic and contain areas of heterogeneity which are vital for their formation. In this review, we discuss some of the ways in which these dynamic and heterogeneous structures have implications during stress and in relation to certain human diseases. A particular stress is that of temperature which may instigate adaptation in poikilotherms or appropriate defensive responses during fever in mammals. Recent data emphasise the role of membranes in sensing temperature changes and in controlling a regulatory loop with chaperone proteins. This loop seems to need the existence of specific membrane microdomains and also includes association of chaperone (heat stress) proteins with the membrane. The role of microdomains is then discussed further in relation to various human pathologies such as cardiovascular disease, cancer and neurodegenerative diseases. The concept of modifying membrane lipids (lipid therapy) as a means for treating such pathologies is then introduced. Examples are given when such methods have been shown to have benefit. In order to study membrane microheterogeneity in detail and to elucidate possible molecular mechanisms that account for alteration in membrane function, new methods are needed. In the second part of the review, we discuss ultra-sensitive and ultra-resolution imaging techniques. These include atomic force microscopy, single particle tracking, single particle tracing and various modern fluorescence methods. Finally, we deal with computing simulation of membrane systems. Such methods include coarse-grain techniques and Monte Carlo which offer further advances into molecular dynamics. As computational methods advance they will have more application by revealing the very subtle interactions that take place between the lipid and protein components of membranes - and which are so essential to their function.

Biomimetic temperature-sensing layer for artificial skins.

Artificial membranes that are sensitive to temperature are needed in robotics to augment interactions with humans and the environment and in bioengineering to improve prosthetic limbs. Existing flexible sensors achieved sensitivities of <100 millikelvin and large responsivity, albeit within narrow (<5 kelvin) temperature ranges. Other flexible devices, working in wider temperature ranges, exhibit orders of magnitude poorer responses. However, much more versatile and temperature-sensitive membranes are present in animals such as pit vipers, whose pit membranes have the highest sensitivity and responsivity in nature and are used to locate warm-blooded prey at distance. We show that pectin films mimic the sensing mechanism of pit membranes and parallel their record performances. These films map temperature on surfaces with a sensitivity of at least 10 millikelvin in a wide temperature range (45 kelvin), have very high responsivity, and detect warm bodies at distance. The produced material can be integrated as a layer in artificial skin platforms and boost their temperature sensitivity to reach the best biological performance.

Deployable micro-traps to sequester motile bacteria.

The development of strategies to reduce the load of unwanted bacteria is a fundamental challenge in industrial processing, environmental sciences and medical applications. Here, we report a new method to sequester motile bacteria from a liquid, based on passive, deployable micro-traps that confine bacteria using micro-funnels that open into trapping chambers. Even in low concentrations, micro-traps afford a 70% reduction in the amount of bacteria in a liquid sample, with a potential to reach >90% as shown by modelling improved geometries. This work introduces a new approach to contain the growth of bacteria without chemical means, an advantage of particular importance given the alarming growth of pan-drug-resistant bacteria.

Sudden origins: a general mechanism of evolution based on stress protein concentration and rapid environmental change.

A major theme in Darwinian evolutionary theory is that novelty arises through a process in which organisms and their features are gradually transformed. Morgan provided Darwinism and the evolutionary synthesis with the idea that minor mutations produce the minuscule morphological variations on which natural selection then acts, and that, although mutation is random, once a process of gradual genetic modification begins, it becomes directional and leads to morphological, and consequently organismal, transformation. In contrast, studies on the role of cell membrane physical states in regulating the expression of stress proteins in response to environmental shifts indicate the existence of a downstream mechanism that prevents or corrects genetic change (i.e., maintains "DNA homeostasis"). However, episodic spikes in various kinds of environmental stress that exceed an organism's cells' thresholds for expression of proper amounts of stress proteins responsible for protein folding (including stochastically occurring DNA repair) may increase mutation rate and genetic change, which in turn will alter the pattern of gene expression during development. If severe stress disrupts DNA homeostasis during meiosis (gametogenesis), this could allow for the appearance of significant mutational events that would otherwise be corrected or suppressed. In evolutionary terms, extreme spikes in environmental stress make possible the emergence of new genetic and consequent developmental and epigenetic networks, and thus also the emergence of potentially new morphological traits, without invoking geographic or other isolating mechanisms.

Homology, disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection.

During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.

Differential gene expression between squamous cell carcinoma of esophageus and its normal epithelium; altered pattern of mal, akr1c2, and rab11a expression.

AIM: To identify the altered gene expression patterns in squamous cell carcinoma of esophagus (ESCC) in relation to adjacent normal esophageal epithelium. METHODS: Total RNA was extracted using SV total RNA isolation kit from snap frozen tissues of ESCC samples and normal esophageal epithelium far from the tumor. Radio-labeled cDNA were synthesized from equal quantities of total RNAs of tumor and normal tissues using combinations of 24 arbitrary 13-mer primers and three different anchoring oligo-dT primers and separated on sequencing gels. cDNA with considerable different amounts of signals in tumor and normal tissue were reamplified and cloned. Using southern blot, the clones of each band were controlled for false positive results caused by probable heterogeneity of cDNA population with the same size. Clones that confirmed differential expression by slot blot selected for sequencing and northern analysis. Corresponding full-length gene sequences was predicted using human genome project data, related transcripts were translated and used for various protein/motif searches to speculate their probable functions. RESULTS: The 97 genes showed different levels of cDNA in tumor and normal tissues of esophagus. The expression of mal gene was remarkably down regulated in all 10 surveyed tumor tissues. Akr1c2, a member of the aldo-keto reductase 1C family, which is involved in metabolism of sex hormones and xenobiotics, was up-regulated in 8 out of 10 inspected ESCC samples. Rab11a, RPL7, and RPL28 showed moderate levels of differential expression. Many other cDNAs remained to further studies. CONCLUSION: The mal gene which is switched-off in all ESCC samples can be considered as a tumor suppressor gene that more studies in its regulation may lead to valuable explanations in ESCC development. Akr1c2 which is up-regulated in ESCC probably plays an important role in tumor development of esophagus and may be proposed as a potential molecular target in ESCC treatments. Differential display technique in spite of many disadvantages is still a valuable technique in gene function exploration studies to find new candidates for improved ones like gene chips.

Insertion of a 59 amino acid peptide in Salmonella Typhimurium membrane results in loss of virulence in mice.

We demonstrated previously that expression of a single trans-membrane region of the Δ(12) -desaturase gene of Synechocystis sp. PCC 6803 in Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) altered the membrane physical state of this pathogen, induced a significant change in the pattern of mRNA transcription of major heat shock genes, and inhibited pathogen growth inside murine macrophages. In this study, we demonstrate that injection of the modified Salmonella strain [Stm(pBAD200)] into C57Bl6j mice is safe. Survival of mice was associated with bacterial clearance, an increased number of splenic leukocytes, and high levels of interleukin-12, interferon Î³ and tumor necrosis factor α in spleens as well as in sera. Furthermore, Stm(pBAD200)-injected mice developed a Salmonella-specific antibody and Th1-like responses. Mice challenged with Stm(pBAD200) are protected from systemic infection with Salmonella wild-type. Similarly, mice infected with Stm(pBAD200) by the oral route survived when challenged with an oral lethal dose of Salmonella wild-type. The avirulent Stm(pBAD200) phenotype is associated with a remarkable change in the expression of the hilC, hilD, hilA, invF and phoP genes, among others, whose products are required for invasion and replication of Salmonella inside phagocytic cells. These data demonstrate the use of trans-membrane peptides to generate attenuated strains, providing a potential novel strategy to develop vaccines for both animal and human use.

Mapping intravascular ultrasound controversies in interventional cardiology practice.

Intravascular ultrasound is a catheter-based imaging modality that was developed to investigate the condition of coronary arteries and assess the vulnerability of coronary atherosclerotic plaques in particular. Since its introduction in the clinic 20 years ago, use of intravascular ultrasound innovation has been relatively limited. Intravascular ultrasound remains a niche technology; its clinical practice did not vastly expand, except in Japan, where intravascular ultrasound is an appraised tool for guiding percutaneous coronary interventions. In this qualitative research study, we follow scholarship on the sociology of innovation in exploring both the current adoption practices and perspectives on the future of intravascular ultrasound. We conducted a survey of biomedical experts with experience in the technology, the practice, and the commercialization of intravascular ultrasound. The collected information enabled us to map intravascular ultrasound controversies as well as to outline the dynamics of the international network of experts that generates intravascular ultrasound innovations and uses intravascular ultrasound technologies. While the technology is praised for its capacity to measure coronary atherosclerotic plaque morphology and is steadily used in clinical research, the lack of demonstrated benefits of intravascular ultrasound guided coronary interventions emerges as the strongest factor that prevents its expansion. Furthermore, most of the controversies identified were external to intravascular ultrasound technology itself, meaning that decision making at the industrial, financial and regulatory levels are likely to determine the future of intravascular ultrasound. In light of opinions from the responding experts', a wider adoption of intravascular ultrasound as a stand-alone imaging modality seems rather uncertain, but the appeal for this technology may be renewed by improving image quality and through combination with complementary imaging modalities.

Cloning and characterization of a Δ9-desaturase gene of the Antarctic fish Chionodraco hamatus and Trematomus bernacchii.

Chionodraco hamatus and Trematomus bernacchii are perciforms, members of the fish suborder Notothenioidei that live in the Antarctic Ocean and experience very cold and persistent environmental temperature. These fish have biochemical and molecular features that allow them to live at these extreme cold temperatures. Fine tuning of the level of unsaturated fatty acids content in membrane is a key mechanism of living organisms to adapt to cold and high temperatures. Desaturases are key enzymes that synthesize unsaturated fatty acyl-CoAs from saturated fatty acids. We cloned and sequenced a Δ(9)-desaturase gene and its cDNA of C. hamatus, and the cDNA of T. bernacchii. The coded proteins are virtually identical and share homology to other Δ(9)-desaturase fish sequences. These proteins contain, in the first trans-membrane domain, two cysteine residues that may form a disulfur bond present in the corresponding membrane region of Δ(9)-desaturase proteins of other Antarctic fish but not in Eleginops maclovinus that experiences higher environmental temperatures and in all other Δ(9)-desaturase genes of mammals present in data bases. C. hamatus Δ(9)-desaturase gene complements a Saccharomyces cerevisiae mutant lacking Δ(9)-desaturase (Ole1) gene. Analysis of sequence homology of the trans-membrane domains of Δ(9)-desaturase and the cytoplasmic region of the same proteins of Antarctic fish, non-Antarctic fish and mammals suggest that the significant differences found in the homologous sequences of the first trans-membrane domain may be due to the specific lipid content of their membrane.

New nucleotide-competitive non-nucleoside inhibitors of terminal deoxynucleotidyl transferase: discovery, characterization, and crystal structure in complex with the target.

Terminal deoxynucletidyl transferase (TdT) is overexpressed in some cancer types, where it might compete with pol µ during the mutagenic repair of double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. Here we report the discovery and characterization of pyrrolyl and indolyl diketo acids that specifically target TdT and behave as nucleotide-competitive inhibitors. These compounds show a selective toxicity toward MOLT-4 compared to HeLa cells that correlate well with in vitro selectivity for TdT. The binding site of two of these inhibitors was determined by cocrystallization with TdT, explaining why these compounds are competitive inhibitors of the deoxynucleotide triphosphate (dNTP). In addition, because of the observed dual localization of the phenyl substituent, these studies open the possibility of rationally designing more potent compounds.

Toward highly potent cancer agents by modulating the C-2 group of the arylthioindole class of tubulin polymerization inhibitors.

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC50 = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes.

Design, synthesis, and cytotoxic evaluation of acyl derivatives of 3-aminonaphtho[2,3-b]thiophene-4,9-dione, a quinone-based system.

A series of 3-acyl derivatives of the dihydronaphtho[2,3-b]thiophen-4,9-dione system were studied with respect to cytotoxicity and topoisomerase II inhibitory activity. These analogues were designed as electron-deficient anthraquinone analogues with potential intercalation ability. Derivatives 3-(diethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11m) and 3-(2-(dimethylamino)ethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11p) showed a high efficacy in cell lines that were highly resistant to treatment with doxorubicin, such as MDA-MB435 (melanoma), IGROV (ovarian), and SF-295 (glioblastoma) human cell lines. Both compounds inhibit topoisomerase II mediated relaxation of DNA, while only 11p incites arrest at the S phase in Caco-2 cells, inducing a delay of cell cycle progression and an increase of cell differentiation. The ability of these derivatives to modulate small heat shock proteins and cardiotoxicy effects was also explored. In addition, the DNA-binding properties of these compounds were investigated and discussed.