High-Resolution Large-Area Image Analysis Deciphers the Distribution of Salmonella Cells and ECM Components in Biofilms Formed on Charged PEDOT:PSS Surfaces.

Biofilms, comprised of cells embedded in extracellular matrix (ECM), enable bacterial surface colonization and contribute to pathogenesis and biofouling. Yet, antibacterial surfaces are mainly evaluated for their effect on bacterial cells rather than the ECM. Here, a method is presented to separately quantify amounts and distribution of cells and ECM in Salmonella biofilms grown on electroactive poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS). Within a custom-designed biofilm reactor, biofilm forms on PEDOT:PSS surfaces electrically addressed with a bias potential and simultaneous recording of the resulting current. The amount and distribution of cells and ECM in biofilms are analyzed using a fluorescence-based spectroscopic mapping technique and fluorescence confocal microscopy combined with advanced image processing. The study shows that surface charge leads to upregulated ECM production, leaving the cell counts largely unaffected. An altered texture is also observed, with biofilms forming small foci or more continuous structures. Supported by mutants lacking ECM production, ECM is identified as an important target when developing antibacterial strategies. Also, a central role for biofilm distribution is highlighted that likely influences antimicrobial susceptibility in biofilms. This work provides yet a link between conductive polymer materials and bacterial metabolism and reveals for the first time a specific effect of electrochemical addressing on bacterial ECM formation.

Periplasmic stress contributes to a trade-off between protein secretion and cell growth in Escherichia coli Nissle 1917.

Maximizing protein secretion is an important target in the design of engineered living systems. In this paper, we characterize a trade-off between cell growth and per-cell protein secretion in the curli biofilm secretion system of Escherichia coli Nissle 1917. Initial characterization using 24-h continuous growth and protein production monitoring confirms decreased growth rates at high induction, leading to a local maximum in total protein production at intermediate induction. Propidium iodide (PI) staining at the endpoint indicates that cellular death is a dominant cause of growth reduction. Assaying variants with combinatorial constructs of inner and outer membrane secretion tags, we find that diminished growth at high production is specific to secretory variants associated with periplasmic stress mediated by outer membrane secretion and periplasmic accumulation of protein containing the outer membrane transport tag. RNA sequencing experiments indicate upregulation of known periplasmic stress response genes in the highly secreting variant, further implicating periplasmic stress in the growth-secretion trade-off. Overall, these results motivate additional strategies for optimizing total protein production and longevity of secretory engineered living systems Graphical Abstract.

Population genetic and biophysical evidences reveal that purifying selection shapes the genetic landscape of Plasmodium falciparum RH ligands in Chhattisgarh and West Bengal, India.

BACKGROUND: Reticulocyte binding protein-like homologs (RHs) are currently being evaluated as anti-erythrocytic stage vaccine targets against Plasmodium falciparum malaria. Present study explores the possible evolutionary drivers shaping the genetic organization of Pfrhs in Indian parasite population. It simultaneously evaluates a putative gain-of-function variant of PfRH5, a keystone member of PfRH family. METHODS: Receptor binding regions of Pfrh1, Pfrh2a/b, Pfrh4 and whole Pfrh5 were amplified using blood samples of P. falciparum malaria patients from Chhattisgarh and West Bengal and sequenced. Assembled sequences were analysed using MEGA7 and DnaSPv6. Binding affinities of recombinant PfRH5 proteins with basigin (BSG) were compared using in silico (CHARMM and AUTODOCK) and in vitro (Circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry) methods. RESULTS: Pfrh1 (0.5), Pfrh2a/b (0.875), Pfrh4 (0.667) and Pfrh5 (0.778) sequence changes corresponded to low frequency (< 0.05) variants which resulted in an overall negative Tajima's D. Since mismatch distribution of none of the Pfrh loci corroborated with the model of demographic expansion, a possible role of natural selection formulating Pfrh sequence diversity was investigated. Among the 5 members, Pfrh5 displayed very high dN/dS (5.7) ratio. Nevertheless, the model of selective sweep due to presence of any advantageous substitutions could not be invoked as polymorphic nonsynonymous sites (17/18) for Pfrh5 exceeded significantly over the divergent (62/86) ones (p = 0.0436). The majority of extant PfRH5 sequences (52/83) differed from the reference Pf3D7 allele by a single amino acid mismatch (C203Y). This non-conservative alteration was predicted to lower the total interaction energy of that PfRH5variant with BSG, compared to PfRH53D7. Biophysical evidences validated the proposition that PfRH5variant formed a more stable complex with BSG. Thermodynamic association constant for interaction of BSG with PfRH5variant was also found to be higher (Kavariant = 3.63E6 ± 2.02E6 M-1 and Ka3D7 = 1.31E6 ± 1.21E6 M-1). CONCLUSIONS: Together, the study indicates that the genetic architecture of Pfrhs is principally shaped by purifying selection. The most abundant and ubiquitous PfRH5 variant harbouring 203Y, exhibits a greater affinity for BSG compared to PfRH53D7 possessing 203C allele. The study underscores the importance of selecting the functional allele that best represents circulating strains in natural parasite populations as vaccine targets.

Non-synonymous amino acid alterations in PfEBA-175 modulate the merozoite ligand's ability to interact with host's Glycophorin A receptor.

The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins.

Time-dependent enhancement of fluorescence from Rhodobacter capsulatus SB1003 and its critical dependence on concentration temperature and static magnetic field.

Continuous exposure of 395 nm light increases the fluorescence emission intensity of photosynthetic purple non-sulphur bacteria, Rhodobacter capsulatus (SB1003). We show that such an increase in fluorescence emission of extracellular pigment complexes (PC) from these photosynthetic bacteria depends on the concentration of the pigment and temperature and can also be modulated by the static magnetic field. The time-dependent enhanced emission disappears either at or below 300 K or below a threshold sample concentration (0.1 mg/ml). The enhanced emission reappears at this condition (T < 278 K) if a static magnetic field (395 mT) is introduced during fluorescence measurement. The time dependence of emission is expressed in terms of a first-order rate constant, k = dF/(Fdt). The sign of k shifts from positive to negative as PC concentration is lowered than a threshold value, implying onset of fluorescence decay (k < 0) rather than amplification (k > 0). At PC concentration higher than a threshold, k becomes negative if the temperature is lowered. But, surprisingly, at low temperature, a static magnetic field reverts the k value to positive. We explain the logical nature of k-switching and photo-dynamics of the aforesaid microbial fluorescence emission by aggregation of protoporphyrin rings present in the PC. While the simultaneous presence of decay in fluorescence and susceptibility to static magnetic field suggests the dominance of triplet states at low temperatures, the process is reversed by SMF-induced removal of spin degeneracy. At higher temperatures, the optical excitability and lack of magnetic response suggest the dominance of singlet states. We propose that the restructuring of the singlet-triplet distribution by intersystem crossing may be the basis of this logical behaviour. In context with microbial function, time-dependent enhancement of fluorescence also implies relay of red photons to the neighbouring microbes not directly exposed to the incident radiation, thus serving as an indirect photosynthetic regulator.

Probiotics as cheater cells: parameter space clustering for individualized prescription.

Clinicians often perform infection management administering probiotics along with antibiotics. Such probiotics added to an infecting population showing antibiotic resistance can be compared to a dynamical system composed of cheaters and workers. The presence of cheater strains is known to modulate the fitness of the infecting population. We propose a model where probiotics as cheater strain re-establishes the susceptibility of a resistant population towards an antibiotic. Control parameters must assume optimal values in order to attain minimum worker number within a finite time-scale feasible in a clinical set-up. The problem is made non-trivial by the complicated interplay between parameters. The model is an extension of a logistic framework, where a pay-off function has been included to account for the effect of instantaneous worker number on death rates of each species. The outcomes for a randomized set of parameter values and initial conditions are utilized in partitioning the set and desired clusters were identified. For a test case, one can take random combinations of controllable parameters and combine them with fixed parameters and find out the closeness of the points to the desired cluster centroids. This process leads to the identification of optimum antibiotic versus probiotic dosage range leading to elimination or limited existence of the genetically resistant population.

KLF15 negatively regulates estrogen-induced epithelial cell proliferation by inhibition of DNA replication licensing.

In the epithelial compartment of the uterus, estradiol-17ß (E(2)) induces cell proliferation while progesterone (P(4)) inhibits this response and causes differentiation of the cells. In this study, we identified the mechanism whereby E(2) and P(4) reciprocally regulate the expression of minichromosome maintenance (MCM)-2, a protein that is an essential component of the hexameric MCM-2 to 7 complex required for DNA synthesis initiation. We show in the uterine epithelium that Kruppel-like transcription (KLF) factors, KLF 4 and 15, are inversely expressed; most importantly, they bind to the Mcm2 promoter under the regulation of E(2) and P(4)E(2), respectively. After P(4)E(2) exposure and in contrast to E(2) treated mice, the Mcm2 promoter displays increased histone 3 (H3) methylation and the recruitment of histone deacetylase 1 and 3 with the concomitant deacetylation of H3. This increased methylation and decreased acetylation is associated with an inhibition of RNA polymerase II binding, indicating an inactive Mcm2 promoter following P(4)E(2) treatment. Using transient transfection assays in the Ishikawa endometrial cell line, we demonstrate that Mcm2 promoter activity is hormonally stimulated by E(2) and that KLF15 inhibits this E(2) enhanced transcription. KLF15 expression also blocks Ishikawa cell proliferation through inhibition of MCM2 protein level. Importantly, in vivo expression of KLF15 in an estrogenized uterus mimics P(4)'s action by inhibiting E(2)-induced uterine epithelial MCM-2 expression and DNA synthesis. KLF15 is therefore a downstream physiological mediator of progesterone's cell cycle inhibitory action in the uterine epithelium.

Cooperative control via lymphoid enhancer factor 1/T cell factor 3 and estrogen receptor-alpha for uterine gene regulation by estrogen.

Accumulating evidence indicates that estrogen regulates diverse but interdependent signaling pathways via estrogen receptor (ER)-dependent and -independent mechanisms. However, molecular relationship between these pathways for gene regulation under the direction of estrogen remains unknown. To address this possibility, our uterine analysis of Wnt/beta-catenin downstream effectors revealed that lymphoid enhancer factor 1 (Lef-1) and T cell factor 3 (Tcf-3) are up-regulated temporally by 17beta-estradiol (E2) in an ER-independent manner. Lef-1 is abundantly up-regulated early (within 2 h), whereas Tcf-3 is predominantly induced after 6 h, and both are sustained through 24 h. Interestingly, activated Lef-1/Tcf-3 molecularly interacted with ERalpha in a time-dependent manner, suggesting they possess a cross talk in the uterus by E2. Moreover, dual immunofluorescence studies confirm their colocalization in uterine epithelial cells after E2. Most importantly, using chromatin immunoprecipitation followed by PCR analyses, we provide evidence for an interesting possibility that ERalpha and Tcf-3/Lef-1 complex occupies at certain DNA regions of estrogen-responsive endogenous gene promoters in the mouse uterus. By selective perturbation of activated Lef-1/Tcf-3 or ERalpha signaling events, we provide in this study novel evidence that cooperative interactions, by these two different classes of transcription factors at the level of chromatin, direct uterine regulation of estrogen-responsive genes. Collectively, these studies support a mechanism that integration of a nonclassically induced beta-catenin/Lef-1/Tcf-3 signaling with ERalpha is necessary for estrogen-dependent endogenous gene regulation in uterine biology.

Increased level of cellular Bip critically determines estrogenic potency for a xenoestrogen kepone in the mouse uterus.

Xenoestrogen mimics estrogen-like activities primarily based on alterations of gene expression and interactions with estrogen receptor (ER)-alpha and -beta. However, the requirement for large concentrations to induce estrogenic phenotypes and low affinity for ERs has challenged the notion that prevailing xenoestrogens are significant health hazards. Here in this study, we show that under certain conditions, exposure of xenoestrogen could be potentially harmful in respect to enhanced uterine estrogenicity. Previously, we have demonstrated that estradiol-17beta up-regulates uterine Bip, a stress-related endoplasmic reticulum protein, via an ER-independent mechanism in mice. Moreover, this protein essentially involves in estradiol-17beta-mediated uterine growth response and ERalpha-dependent gene transcription. Here, we demonstrate that among three tested xenoestrogens, only kepone (>15-30 mg/kg) exerts sustained inductive response for uterine Bip expression. Interestingly, this kepone-induced Bip strongly correlates with ERalpha-dependent growth and gene expressional responses in the mouse uterus. Furthermore, these effects were strongly suppressed after knockdown of uterine Bip, via the adenovirus approach. Although kepone at 7.5 mg/kg was not effective, it was strongly stimulatory by the adenovirus-driven forced expression of uterine Bip. In contrast, the control green fluorescence protein virus was not effective in the aforementioned responses. Furthermore, the induction of uterine Bip by stress-related signals also revealed the onset of uterine growth in mice when exposed to a sublethal dose of kepone. Collectively, studies provide novel molecular evidence that Bip acts as a critical regulator to amplify estrogenic potency for a weak xenoestrogen kepone.

Effects of paraquat on anti-oxidant system in rats.

The toxic effects of paraquat on the anti-oxidant defense system of male albino rats were evaluated, after administering either a single dose (1.5 and 7.5 mg/kg of body weight) or continuous daily doses (same as above, i.e., 1.5 mg/kg and 7.5 mg/kg of body weight) for 3 and 7 days. Glutathione levels in blood cells, liver, lung and kidney tissues decreased in a dose and time dependent manner. Glutathione reductase and glucose-6-phosphate dehydrogenase activity decreased, whereas the activity of glutathione-S-transferase, glutathione peroxidase, catalase and superoxide dismutase increased in paraquat exposure. Malondialdehyde formation also increased in a dose and time dependent manner. The alterations of anti-oxidant system particularly glutathione can be utilized as biomarkers during management of paraquat poisoning.

Chromatin immunoprecipitation assay detects ERalpha recruitment to gene specific promoters in uterus.

Chromatin immunoprecipitation (ChIP) technique allows detection of proteins that bind to chromatin. While this technique has been applied extensively in cell-based studies, its tissue-based application remains poorly explored. We are specifically interested in examining estrogen-dependent transcriptional mechanism in respect of recruitment of estrogen receptor-alpha (ERalpha), a ligand-activated transcription factor, to uterine gene promoters in mice. Recent gene-array studies, utilizing ERalpha knock-out vs. wild-type mice, have revealed that estrogen regulates numerous uterine genes temporally and most importantly via ERalpha during the phase-II response, including three well characterized genes viz., lactoferrin (Ltf), progesterone receptor (Pgr) and cyclinD1 (Ccnd1). Here, utilizing systematic ChIP studies, we demonstrate endogenous recruitment of ERalpha to above uterine gene promoters following estradiol-17beta (E(2)) injection in mice.

Environmental contaminants in pathogenesis of breast cancer.

This review is an attempt to comprehend the diverse groups of environmental chemical contaminants with a potential for pathogenesis of breast cancer, their probable sources and the possible mechanisms by which these environmental contaminants act and interplay with other risk factors. Estrogens are closely related to the pathogenesis of breast cancer. Oxidative catabolism of estrogen, mediated by various cytochrome P450 enzymes, generates reactive free radicals that can cause oxidative damage. The same enzymes of estrogenic metabolic pathways catalyze biological activation of several environmental (xenobiotic) chemicals. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals. Breast tissue can be a target of several xenobiotic agents. DNA-reactive metabolites of different xenobiotic compounds have been detected in breast tissue. Many phase I and II xenobiotic metabolizing enzymes are expressed in both normal and cancerous breast tissues. These enzymes play a significant role in the activation/detoxification of xenobiotic and endogenous compounds including estrogens. More than 30 carcinogenic chemicals are present in tobacco smoke; many of them are fat-soluble, resistant to metabolism and can be stored in breast adipose tissue. Similarly, pesticides are also known to cause oxidative stress; while some act as endocrine disruptor, some are shown to suppress apoptosis in estrogen sensitive cell lines. Reports have shown an association of smoking (both active and passive) and pesticides with breast cancer risk. However, the issues have remained controversial. Different mutagenic substances that are generated in the cooking process e.g., heterocyclic amines and polycyclic aromatic hydrocarbons (PAHs) can be a threat to breast tissue. PAHs and dioxins exert their adverse effects through the aryl hydrocarbon receptor (AhR), which activates several genes involved in the metabolisms of xenobiotic compounds and endogenous estrogens. These chemicals also induce AhR-dependent mitochondrial dysfunction. Many of the environmental pollutants suppress the immune system, which are implicated to risk. A better understanding about the biological effects of different environmental carcinogenic compounds and determination of their impact on rising incidence of breast cancer will be beneficial in improving preventive policy against breast cancer.

Bip is a molecular link between the phase I and phase II estrogenic responses in uterus.

Uterine estrogenic actions are biphasic, early (phase I) and late (phase II) responses. However, the molecular linkage between these phases is not known. Although certain phase I responses are considered estrogen receptor (ER)alpha and ERbeta independent, the phase II responses are ERalpha dependent. We previously observed that among several genes Bip is induced by estrogen in the mouse uterus in an ER-independent manner as a phase I response. Bip is a member of the chaperone family and plays roles in protein processing and confers cellular protection. However, its role in estrogen-dependent uterine biology is unknown. We show here a new function of Bip in regulating estrogen signaling in the uterus. Bip, induced during the phase I responses, molecularly interacts with ERalpha required for estrogen-mediated phase II growth responses. Utilizing in vivo and in vitro model systems, we found that adenovirus-driven suppression of Bip antagonizes ERalpha-mediated uterine gene transcription. Importantly, down-regulation of Bip compromises estrogen-dependent phase II growth responses with sustained phase I responses. In conclusion, Bip is critical for coordinating estrogen-elicited biphasic responses and serves as a molecular link between ERalpha-independent and -dependent estrogenic responses in the uterus.

The serum glycoprotein fetuin-A promotes Lewis lung carcinoma tumorigenesis via adhesive-dependent and adhesive-independent mechanisms.

Fetuin-A is a serum glycoprotein in the cystatin family associated with the regulation of soft tissue calcification. We tested the role of systemic fetuin in tumor cell growth and metastasis by injecting Lewis lung carcinoma (LLC) cells into fetuin-A null and their wild-type (WT) littermate control C57BL/6 mice via the tail vein, s.c., and intrasplenic routes. In the experimental metastasis assay, the lungs of the WT mice were filled with metastatic nodules, whereas the lungs of the fetuin-A null mutant mice were virtually free of colonies at the end of 2 weeks. Lung colonization responded to the levels of serum fetuin-A in a dose-dependent manner, as observed by the formation of half as many colonies in mice heterozygous for the fetuin-A locus compared with homozygous WT mice and restoration of lung colonization by the administration of purified fetuin-A to fetuin-A-null mice. Serum fetuin-A also influenced the growth of LLC cells injected s.c.: fetuin-A-null mice developed small s.c. tumors only after a substantial delay. Similarly, intrasplenic injection of LLC cells resulted in rapid colonization of the liver with metastasis to the lungs within 2 weeks in the WT but not fetuin-A null mice. To examine the mechanism by which fetuin-A influences LLC colonization and growth, we showed that LLC tumor cells adhere to fetuin-A in a Ca(2+)-dependent fashion, resulting in growth of the tumor cells. These studies support the role of fetuin-A as a major growth promoter in serum that can influence tumor establishment and growth.

Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A.

Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media. We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion. Interestingly, the cells can also divide and attain confluency under these conditions. Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca2+ ions. Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes. Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion. Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against beta1 integrins substantially reduced the adhesion. Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.

Members of the cystatin superfamily interact with MMP-9 and protect it from autolytic degradation without affecting its gelatinolytic activities.

Matrix metalloproteinases (MMPs), like other proteinases, can undergo autolytic degradation once activated in vivo. Whereas the activities of these enzymes are tightly regulated by tissue inhibitors of matrix metalloproteinases (TIMPs), it is not clear mechanistically how these enzymes are protected from autolysis in their active state. We previously reported that MMPs particularly MMP-9 and MMP-2 interact with the serum glycoprotein fetuin-A [Arch. Biochem. Biophys. (1995) 322, 250], a member of the cystatin superfamily. In the present analyses, we demonstrate that this interaction protects MMP-9 from autolytic degradation without interfering with its enzymatic activity, allowing it to efficiently digest gelatin. Our data demonstrate that MMP-9 binds to members of the cystatin family with K(diss) ranging from 25 to 58 nM for fetuin-A and 1.5-1.9 microM for cystatin C. The ability of fetuin-A to protect MMP-9 from autolysis requires a molar ratio of at least 8:1 (fetuin-A/MMP-9). More interestingly, our data show that the other members of the cystatin also have the ability to protect MMP-9 from autolysis, provided they are in molar excess relative to MMP-9. Taken together, our data suggest that cystatins, particularly fetuin-A, in any cellular compartment including the circulatory system, efficiently protect MMP-9 and possibly other MMPs from autolysis. This mechanism ensures the digestion of the preferred substrate for MMP-9 without sacrificing the enzyme in the process.