K12 Cells Research Articles

UtPCR: A Strategy for the Highly Specific and Absolutely Quantitative Detection of Single Molecules within Only Minutes.

Bloodstream infection is a major health problem worldwide, with extremely high mortality. Detecting infection in the early stage is challenging due to the extremely low concentration of bacteria in the blood. Digital PCR provides unparalleled sensitivity and can achieve absolute quantification, but it is time-consuming. Moreover, the presence of unavoidable background signals in negative controls poses a significant challenge for single-molecule detection. Here, we propose a novel strategy called "Ultrafast flexible thin tube-based droplet digital PCR (utPCR)" that can shorten the digital PCR process from 2 h to only 5 min, with primer annealing/extension time reduced from minutes to only 5 s. Importantly, the ultrafast PCR eliminates nonspecific amplification and thus enables single-molecule detection. The utPCR enabled the sensitive detection and digital quantification of E. coli O157 in the high background of a 106-fold excess of E. coli K12 cells. Moreover, this method also displayed the potential to detect rare pathogens in blood samples, and the limit of detection (LOD) could be as low as 10 CFU per mL of blood without false positive results. Considered ultrafast (<5 min) and highly sensitive (single-molecule detection), the utPCR holds excellent prospects in the next generation of molecular diagnosis.

Phagocytosis and oxidative activity of neutrophils after interaction with uropathogenic &lt;i&gt;Escherichia coli&lt;/i&gt; biofilms

Recurrent urinary tract infections (UTIs) are associated primarily with the ability of Escherichia coli to form biofilms. The interaction of neutrophils, factors of innate immunity, with microorganisms in biofilms is difficult compared to planktonic forms due to the lack of direct contact, as well as due to the antiphagocytic action of the extracellular matrix of biofilms. The purpose of this study was evaluation of neutrophils phagocytic and oxidative activity during interaction with biofilms of uropathogenic E. coli (UPEC) DL82 and R44. Peripheral blood neutrophils from healthy men were isolated using ficoll-urographin double gradient, incubated for 1 h with bacterial cells from biofilms or their supernatants, then leukocytes functional activity was evaluated. Phagocytic activity of neutrophils was determined by the degree of bioluminescence inhibition of bioluminescent strain E. coli K12 TG1 lux+ (pXen) upon their absorption by neutrophils. Production of extracellular reactive oxygen species (ROS) was analyzed by the intensity of luminol-dependent chemiluminescence in spontaneous and stimulated by E. coli K12 variants. Significance of differences was determined using Student’s t-test at p &lt; 0.05. It was found that neutrophils interaction with UPEC biofilm cells or supernatants did not affect the phagocytic activity. E. coli DL82 supernatants reduce neutrophils spontaneous ROS production compared to control and biofilm cells. E. coli R44 supernatants with a low virulence potential did not affect ROS production, while biofilm cells stimulated it. When assessing stimulated ROS production, exposure to R44 strain supernatants did not cause a decrease in neutrophils activation in response to an external stimulus (E. coli K12 cells). Preliminary contact of neutrophils with E. coli R44 bacteria resulted in a high and prolonged level of ROS production compared to the control. Neutrophils interaction with DL82 cells resulted in a higher level of ROS compared to supernatants, however a subsequent rapid depletion of neutrophils oxidative potential was observed. Thus, cells and supernatants of UPEC biofilms can determine the activation of neutrophils.

Oxidative Stress Response and E. coli Biofilm Formation under the Effect of Pristine and Modified Carbon Nanotubes.

The article investigates the expression of oxyR and soxS oxidative stress genes in E. coli under the effect of pristine multi-walled carbon nanotubes (MWCNTs) and pristine single-walled carbon nanotubes (SWCNTs), MWCNTs and SWCNTs functionalized with carboxyl groups (MWCNTs-COOH and SWCNTs-COOH, respectively), SWCNTs functionalized with amino groups (SWCNTs-NH2) and SWCNTs functionalized with octadecylamine (SWCNTs-ODA). Significant differences were found in the expression of the soxS gene, while no changes were observed in the expression level of the oxyR gene. The pro-oxidant effect of SWCNTs, SWCNTs-COOH, SWCNTs-NH2, and SWCNTs-ODA is presented, and the contrary antioxidant effect of pristine MWCNTs and MWCNTs-COOH in the presence of methyl viologen hydrate (paraquat) is shown. The article shows that SWCNTs-COOH, SWCNTs-NH2, and SWCNTs-ODA added to the medium generate the production of reactive oxygen species (ROS) in bacterial cells. SWCNTs-COOH intensified the E. coli biofilm formation, and the biofilm biomass exceeded the control by 25 times. Additionally, it is shown that the rpoS expression increased in response to MWCNTs-COOH and SWCNTs-COOH, and the effect of SWCNTs-COOH was more significant. SWCNTs-COOH and SWCNTs-NH2 initiated an increase in ATP concentration in the planktonic cells and a decrease in the biofilm cells. The atomic force microscopy (AFM) method showed that the volume of E. coli planktonic cells after the exposure to carbon nanotubes (CNTs) decreased compared to that without exposure, mainly due to a decrease in cell height. The absence of a strong damaging effect of functionalized SWCNTs on E. coli K12 cells, both in suspension and in biofilms, is shown. Contact with functionalized SWCNTs initiated the aggregation of the polymeric substances of the biofilms; however, the cells did not lyse. Among the studied CNTs, SWCNTs-COOH caused an increased expression of the soxS and rpoS, the formation of ROS, and stimulation of the biofilm formation.

Rcs phosphorelay affects the sensitivity of Escherichia coli to plantaricin BM-1 by regulating biofilm formation.

Introduction: Plantaricin BM-1 is a class IIa bacteriocin produced by Lactobacillus plantarum BM-1 that exerts significant antibacterial activity against many foodborne bacteria. Studies have shown that class IIa bacteriocins inhibit Gram-positive bacteria via the mannose phosphotransferase system; however, their mechanism of action against Gram-negative bacteria remains unknown. In this study, we explored the mechanism through which the Rcs phosphorelay affects the sensitivity of Escherichia coli K12 cells to plantaricin BM-1. Methods and Results: The minimum inhibitory concentrations of plantaricin BM-1 against E. coli K12, E. coli JW5917 (rcsC mutant), E. coli JW2204 (rcsD mutant), and E. coli JW2205 (rcsB mutant) were 1.25, 0.59, 1.31, and 1.22 mg/ml, respectively. Growth curves showed that E. coli JW5917 sensitivity to plantaricin BM-1 increased to the same level as that of E. coli K12 after complementation. Meanwhile, scanning electron microscopy and transmission electron microscopy revealed that, under the action of plantaricin BM-1, the appearance of E. coli JW5917 cells did not significantly differ from that of E. coli K12 cells; however, cell contents were significantly reduced and plasmolysis and shrinkage were observed at both ends. Crystal violet staining and laser scanning confocal microscopy showed that biofilm formation was significantly reduced after rcsC mutation, while proteomic analysis identified 382 upregulated and 260 downregulated proteins in E. coli JW5917. In particular, rcsC mutation was found to affect the expression of proteins related to biofilm formation, with growth curve assays showing that the deletion of these proteins increased E. coli sensitivity to plantaricin BM-1. Discussion: Consequently, we speculated that the Rcs phosphorelay may regulate the sensitivity of E. coli to plantaricin BM-1 by affecting biofilm formation. This finding of class IIa bacteriocin against Gram-negative bacteria mechanism provides new insights.

Effect of biofilm formation on different types of plastic shopping bags: Structural and physicochemical properties

Plastics and biofilms have a complicated relationship that has great interest. Bacterial cell attachment and biofilm formation is considered to cause health and environmental risks from plastic waste accumulation. In water, plastic waste could serve as a new substrate for bacteria. In our study, the attachment of Escherichia coli K12, to four types of plastic shopping bags (biodegradable polylactic acid and the non-biodegradable polypropylene, polyethylene and polyvinyl chloride) was investigated. The change in physicochemical phenomena of each plastic, such as reduced hydrophobicity and higher exopolysaccharide concentrations (total extractable protein and carbohydrate) resulted in increased biofilm content on the plastic surfaces. The bacterial colonization of different plastic surfaces controls the ionic strength of the nutrition sources. The adhesion of Escherichia coli K12 cells on the surfaces were revealed by SEM images. The finding shows that increases surface roughness, besides favor for adhesion of bacterial cells due to hydrophobicity leading to a rapid attachment of Escherichia coli K12 on the surfaces. In addition, we used Derjaguin-Landau-Verwey-Overbeek theory to predict the attachment of Escherichia coli K12, which gave result of adhesion due to the high energy barrier. This present study added to our knowledge of the possible consequences of plastics acting as a new habitat for microbes in different aquatic condition.

Photocatalytic Bactericidal Performance of LaFeO3 under Solar Light: Kinetics, Spectroscopic and Mechanistic Evaluation

Lanthanum orthoferrites are a versatile class of catalysts. Here, the photocatalytic bactericidal performance of LaFeO3 (LF) to inactivate pathogenic microorganisms, i.e., Escherichia coli (E. coli), in water under simulated solar irradiation conditions was investigated. Various competing and contributing factors were covered to visualize the reaction medium consisting of E. coli K12 cells, organic sub-fractions formed by cell destruction, and LF surface. LF solar photocatalytic inactivation (SPCI) kinetics revealed the highest inactivation rate in ultrapure water as expected, followed by distilled water (DW), aqueous solution containing anions and cations (WM) and saline solution (SS). Characterization of the released organic matter was achieved by UV-vis and fluorescence spectroscopic techniques as well as organic carbon contents (DOC). Upon SPCI, significant amounts of K+ along with released protein contents were detected expressing cell wall destruction and lysis. Under the specified experimental conditions, in the presence of released intracellular organic and inorganic components via cell lysis, a significant count of E. coli was still present in SS, whereas almost all bacteria were removed in other matrices due to various challenging reasons. Based on the presented data, SPCI of E. coli using LF as a novel photocatalyst was successfully demonstrated as an alternative and promising method for disinfection purposes.

The Effect of Oral Probiotic Streptococcus Salivarius K12 on Candida Albicans Biofilm Formation

Introduction: Oral cancer is the sixth most common cancer worldwide with Candida albicans infection being one of the aetiological factors for the disease. Meanwhile, Streptococcus salivarius K12 is an oral probiotic that is beneficial to the oral cavity. The objective of the present study is to determine the effect of S. salivarius K12 on C. albicans biofilm-forming ability with the hypothesis that S. salivarius K12 inhibits biofilm formation of C. albicans Materials and method: To assess the effect of S. salivarius K12 on C. albicans biofilm formation, S. salivarius K12, lab strain C. albicans MYA-4901 and clinical isolates from oral cancer, ALC2 and ALC3 were grown in both nutrient broth (NB) and RPMI. In a mono-species biofilm, 105 of C. albicans cells and 106 of S. salivarius K12 cells were grown separately in a 96-well plate. In contrast, both microorganisms were combined for polymicrobial biofilms with similar cell numbers as in mono-species. The biofilms were incubated for 72 hours at 37°C and the media were replenished every 24 hours. Finally, the crystal violet assay was conducted, and the optical density was measured at OD620nm. Results: Polymicrobial biofilms of C. albicans (MYA-4901 and ALC3) with S. salivarius K12 when grown in NB, exhibited a decrease by 64.5 ± 25.8% and 83.7 ± 5.4%, respectively when compared to the expected biofilms which were predominated by yeast form. Furthermore, polymicrobial biofilms of C. albicans (ALC2 and ALC3) with S. salivarius K12 showed a decrease by 62.5 ± 25.6% and 55.9 ± 17.1 %, respectively when compared to the expected biofilms when grown in RPMI that were predominated by hyphal form. Conclusion: S. salivarius K12 inhibited polymicrobial biofilms formation of C. albicans yeast and hyphal forms, thus supported the hypothesis that S. salivarius K12 inhibits biofilm formation of C. albicans.

MazF activation causes ACA sequence-independent and selective alterations in RNA levels in Escherichia coli.

Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.

ArdB Protective Activity for Unmodified λ Phage Against EcoKI Restriction Decreases in UV-Treated Escherichia coli.

Anti-restriction proteins ArdB/KlcA specifically inhibit restriction (endonuclease) activity of restriction-modification (RM) type I systems. Molecular mechanisms of ArdB/KlcA-based anti-restriction remain unknown. In this study, we quantitate effects of ArdB on protection of unmodified λ phage DNA from EcoKI restriction. After UV irradiations, which produce significant amounts of unmodified chromosomal DNA in Escherichia coli K12 cells, the protective activity of ArdB decreases. Unlike ArdB, DNA-mimicking protein Ocr retains its ability to protect the unmodified λ phage regardless of UV dose. We hypothesize that the observed decrease in ArdB protective activity in UV-treated cells is due to its binding to unmodified chromosomal DNA, which decreases effective concentrations of free ArdB molecules available for λ phage protection against type I restriction enzymes.

Combination Therapy with Disulfiram, Copper, and Doxorubicin for Osteosarcoma: In Vitro Support for a Novel Drug Repurposing Strategy

Although many cancer cells have significantly higher copper concentrations compared with normal cells and tissues, the role of copper in cancer biology and metastatic disease remains poorly understood. Here, we study the importance of copper in osteosarcoma, which frequently metastasizes to the lungs and is often chemoresistant. K12 and K7M2 are murine OS cells with differing metastatic phenotypes: K7M2 is highly metastatic, whereas K12 is much less so. Intracellular copper levels were determined using atomic absorption. Copper transporters were quantified by qPCR. Cytotoxicity of doxorubicin, disulfiram, and copper(II) chloride was assessed with a cell viability fluorescence stain. Additionally, K7M2 viable cell counts were determined by trypan blue exclusion staining after 72 hours of treatment. Copper levels were found to be significantly higher in K12 OS cells than in K7M2 cells. qPCR showed that K12 cells upregulate the copper influx pump CTR1 and downregulate the copper efflux pump ATP7A compared to K7M2 OS cells. Combination treatment of copper chloride (50 nM) with disulfiram (80 nM) was only cytotoxic to K12 cells. Triple treatment with doxorubicin, disulfiram, and copper displayed potent and durable cytotoxicity of highly metastatic K7M2 cells. We demonstrate here that murine OS cell lines differing in metastatic potential also vary in endogenous copper levels and regulation. Additionally, these differences in copper regulation may contribute to selective cytotoxicity of K12 cells by extremely low doses of copper-potentiated disulfiram. The combination of doxorubicin, disulfiram, and copper should be explored as a therapeutic strategy against OS metastases.

Label-free counting of Escherichia coli cells in nanoliter droplets using 3D printed microfluidic devices with integrated contactless conductivity detection

This study describes for the first time the development of 3D printed microfluidic devices with integrated electrodes for label-free counting of E. coli cells incorporated inside droplets based on capacitively coupled contactless conductivity detection (C4D). Microfluidic devices were fully fabricated by 3D printing in the T-junction shape containing two channels for disperse and continuous phases and two sensing electrodes for C4D measurements. The disperse phase containing E. coli K12 cells and the continuous phase containing oil and 1% Span® 80 were pumped through flow rates fixed at 5 and 60 μL min−1, respectively. The droplets with incorporated cells were monitored in the C4D system applying a 500-kHz sinusoidal wave with 1 Vpp amplitude. The generated droplets exhibited a spherical shape with average diameter of 321 ± 9 μm and presented volume of 17.3 ± 0.5 nL. The proposed approach demonstrated ability to detect E. coli cells in the concentration range between 86.5 and 8650 CFU droplet−1. The number of cells per droplet was quantified through the plate counting method and revealed a good agreement with the Poisson distribution. The limit of detection achieved for counting E. coli cells was 63.66 CFU droplet−1. The label-free counting method has offered instrumental simplicity, low cost, high sensitivity and compatibility to be integrated on single microfluidic platforms entirely fabricated by 3D printing, thus opening new possibilities of applications in microbiology.

Bacterial electroactivity and viability depends on the carbon nanotube-coated sponge anode used in a microbial fuel cell

The anode material is vital to improve the power generation of a microbial fuel cell (MFC). In this study, a carbon nanotube (CNT)-coated sponge with macro-porous structure, high surface area, and high conductivity was constructed as an anode to encapsulate Escherichia coli K12 (E. coli K12) cells. To achieve high power generation of the MFC, the optimal concentration of the CNT coating the sponge was found to be 30mgmL−1. At this concentration, a maximum power density of 787Wm−3 and a chemical oxygen demand (COD) removal of 80.9% were obtained with a long stable electricity generation process in batch mode. This indicates that the biofilm on the CNT (30mgmL−1)-coated sponge possessed excellent electroactivity and stability. Scanning electron microscope (SEM) images confirmed that the CNT-coated sponge provided a suitable microenvironment for E. coli K12 cells to maintain their attachment and colonization. Additionally, a CNT-dependent viability phenomenon of the E. coli K12 cells was discovered after electricity generation. This CNT-dependent viability of the E. coli K12 cells was stable and sustainable after storage at −20°C in a milk tube for one year.

Differential imaging of the metabolism of bacteria and eukaryotic cells based on light-addressable potentiometric sensors

A light-addressable potentiometric sensor (LAPS) is a field-effect-based potentiometric sensor with an electrolyte/insulator/semiconductor (EIS) structure, which is able to monitor analyte concentrations of (bio-)chemical species in aqueous solutions in a spatially resolved way. Therefore, it is also an appropriate tool to record 2D-chemical images of concentration variations on the sensor surface. In the present work, two differential, LAPS-based measurement principles are introduced to determine the metabolic activity of Escherichia coli (E. coli) K12 and Chinese hamster ovary (CHO) cells as test microorganisms. Hereby, we focus on i) the determination of the extracellular acidification rate (ΔpH/min) after adding glucose solutions to the cell suspensions; and ii) recording the amplitude increase of the photocurrent (Iph) related to the produced acids from E. coli K12 bacteria and CHO cells on the sensor surface by 2D-chemical imaging. For this purpose, 3D-printed multi-chamber structures were developed and mounted on the planar sensor-chip surface to define four independent compartments, enabling differential measurements with varying cell concentrations. The differential concept allows eliminating unwanted drift effects and, with the four-chamber structures, measurements on the different cell concentrations were performed simultaneously, thus reducing also the overall measuring time.

Evidence for Compression ofEscherichia coliK12 Cells under the Effect of TiO2Nanoparticles

It has been shown that treatment with titanium dioxide nanoparticles (TiO2 NPs) combined with near-ultraviolet (UV-A) irradiation or in certain dark conditions reduced the numbers of various microorganisms, but the mechanism of this effect remains unclear. In this study to further clarify the mechanism of the antibacterial effect of TiO2 NPs the physiological state of E. coli K12 cells was estimated after incubation with the NPs (0.2 g/L) for different periods of time, with or without UV-A irradiation. Cell incubation with TiO2 NPs, combined or not combined with UV-A irradiation, showed that inactive cells were located only within cell aggregates formed after incubation with TiO2 NPs and that the larger the aggregate, the greater the number of such cells. When the formation of large aggregates was prevented, exposure to NPs under UV-A irradiation failed to result in cell inactivation. A comparative analysis of fluorescence and optical microscopic images of the same aggregates showed that the location of inactivated cells coincided with the zone of increased optical density within the aggregate. After treatment with TiO2 NPs under UV-A for 30, 60, or 120 min cells within the aggregates were the first to be inactivated. Cells on which NPs irradiated more strongly (at the periphery of large aggregates and single) remained active for a longer time than cells within the aggregates. As the time of treatment increased, so did the degree of cell compaction, with some zones of the aggregates eventually transforming into an acellular mass. After UV-A irradiation the cell aggregates spontaneously moved toward each other and gradually fused into larger structures, indicating that such exposure enhanced mutual attraction of cells treated with the NPs. Present study provides evidence for hypothesis that bacterial cells covered with TiO2 NPs are inactivated due to their mutual attraction and consequent compression.

Functionalized electrospun poly(vinyl alcohol) nanofibers for on-chip concentration of E. coli cells.

Positively and negatively charged electrospun poly(vinyl alcohol) (PVA) nanofibers were incorporated into poly(methyl methacrylate) (PMMA) microchannels in order to facilitate on-chip concentration of Escherichia coli K12 cells. The effects of fiber distribution and fiber mat height on analyte retention were investigated. The 3D morphology of the mats was optimized to prevent size-related retention of the E. coli cells while also providing a large enough surface area for analyte concentration. Positively charged nanofibers produced an 87% retention and over 80-fold concentration of the bacterial cells by mere electrostatic interaction, while negatively charged nanofibers reduced nonspecific analyte retention when compared to an empty microfluidic channel. In order to take advantage of this reduction in nonspecific retention, these negatively charged nanofibers were then modified with anti-E. coli antibodies. These proof-of-principle experiments showed that antibody-functionalized negatively charged nanofiber mats were capable of the specific capture of 72% of the E. coli cells while also significantly reducing nonspecific analyte retention within the channel as expected. The ease of fabrication and immense surface area of the functionalized electrospun nanofibers make them a promising alternative for on-chip concentration of analytes. The pore size and fiber mat morphology, as well as surface functionality of the fibers, can be tailored to allow for specific capture and concentration of a wide range of analytes.

Abstract 5378: Epigenetic regugulation of osteosarcoma metastatic phenotype via histone deacetylace inhibition

Abstract Introduction: The epigenetic modification of cancer cells regulates tumor development and metastasis. Epigenetic changes include DNA methylation and histone modification, both of which regulate gene expression but do not alter the genetic code. We have demonstrated differential DNA methylation status of tumor suppressor genes between highly metastatic K7M2 osteosarcoma (OS) cells and less metastatic K12 OS cells. The demethylation of tumor suppressor genes repressed the metastatic phenotype of K7M2 cells. Histone deacetylase inhibitors (HDAC inhibitors, HDIs) have been evaluated for epigenetic regulation of various malignancies, but the role of HDAC inhibitors in OS is unclear. Wehave investigated the effect of the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), on highly metastatic K7M2 OS cells. Methods: 1. Osteosarcoma cells: K7M2 and K12 are murineOS cell populations with differing metastatic potentials. K7M2 is highly metastatic to the lung but K12 is much less metastatic. 2. SAHA treatment: SAHA (25, 50, or 100 μM) in proliferation medium (10% FBS in DMEM) was used to culture the K7M2 cells. Cells were incubated with SAHA for 48 hours prior to fixation for observation, harvesting for mRNA isolation, or plating for invasion assays. Results: 1. SAHA treatment (25, 50, or 100 μM for 48 hours) significantly decreased cell proliferation compared with untreated cells. 2. SAHA treatment of K7M2 cells modified cell morphology. K7M2 cells were cultured with or without SAHA (25, 50, 100 μM) for 48 hours. Staining of the actin cytoskeleton revealed structural differences in SAHA-treated cells: treated cells displayed fewer invadopodia and their shape was more polygonal, resembling less metastatic K12 cells. 3. SAHA treatment of K7M2 cells modified the expression tumorigenesis and metastasis-related genes. Semi-quantitative polymerase chain reaction showed that the expressions of mammalian target of rapamycin (mTOR), aldehyde dehydrogenase-1 (ALDH-1), and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) were down-regulated in SAHA-treated K7M2 cells. The expression of microtubule-associated protein 1A/1B-light chain (LC3) was up-regulated. 4. K7M2 cells were cultured with or without SAHA (50 μM) for 2 days, and their invasion capacity through 2.5% matrigel was analyzed. SAHA treatment significantly reduced the cells’ invasion capacity. Conclusion: These data demonstrate that highly metastatic OS cells can be epigenetically modified with HDAC inhibition. SAHA treatment of K7M2 cells reduced both their proliferation and invasiveness, indicating that HDAC inhibition may potentially reduce tumor development and metastasis. The effect of SAHA on OS cells could be related to cytoskeletal changes and/or diminished expression of genes regulating tumor development and metastasis. In the future we will test the ability of SAHA to affect OS metastatic potential in vivo. Citation Format: Kurt R. Weiss, Xiaodong Mu, Daniel Brynien. Epigenetic regugulation of osteosarcoma metastatic phenotype via histone deacetylace inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5378. doi:10.1158/1538-7445.AM2015-5378

Molecular cloning and characterization of gloverin from the diamondback moth, Plutella xylostella L. and its interaction with bacterial membrane.

Gloverin restricted to Lepidoptera is known to be a glycine-rich and heat stable antimicrobial protein. The current research reports a 650 bp full-length cDNA encoding gloverin from Plutella xylostella (PxGlo) by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. PxGlo transcript was detected in both developmental stages and several tissues of 4th instar naïve larvae of P. xylostella with higher levels in the fat bodies. The mRNA levels of PxGlo increased appreciably in fat bodies after injection of Escherichia coli K12. The recombinant PxGlo expressed in S2 cells was purified by Anti-V5 M2 agarose beads which showed high activity against E. coli K12, while low activity against Bacillus thuringiensis, Staphylococcus aureus and E. coli D31. The analysis of transmission electron microscope and scan electron microscope showed PxGlo to cause significant morphological alteration in the E. coli K12 cell surface. Knockdown of PxGlo expression by RNAi increased the larval susceptibility towards the pathogenic bacteria i.e., Serratia marcescens and B. thuringiensis. Our results showed that PxGlo is an inducible antibacterial peptide which exhibits high activity mainly against E. coli K12, and PxGlo performs vital roles against the infection of pathogenic bacteria.

Induction of HER2 Immunity in Outbred Domestic Cats by DNA Electrovaccination.

Domestic cats share human living environments and genetic traits. They develop spontaneous feline mammary carcinoma (FMC) with similar histopathology to human breast cancer. HER2 and AKT phosphorylation was demonstrated in primary FMC by immunoblot analysis, indicating HER2 as a therapeutic target. FMC lines K12 and K248 expressing HER1, HER2, and HER3 were sensitive to receptor tyrosine kinase (RTK) inhibitors gefitinib and lapatinib. To test HER2 vaccine response in cats, purpose-bred, healthy cats were electrovaccinated with heterologous (xenogeneic) or point-mutated feline HER2 DNA. T-cell reactivity to feline self-HER2 was detected in 4 of 10 cats that received bear HER2, human-rat fusion HER2 (E2Neu) or mutant feline HER2 (feHER2-K), which contains a single amino acid substitution. The variable T-cell responses may resemble that in the genetically heterogeneous human population. All immune sera to heterologous HER2 recognized feline HER2 expressed in 3T3 cells (3T3/HER2), but not that in FMC K12 or K248. Immune sera to mutant pfeHER2-K bound 3T3/HER2 cells weakly, but they showed better recognition of K12 and K248 cells that also express HER1 and HER3, suggesting distinct HER2 epitopes displayed by FMC that may be simulated by feHER2-K. In summary, HER2 DNA electroporation overcomes T-cell immune tolerance in approximately 40% of healthy cats and induces antibodies with distinct specificity. Vaccination studies in domestic cats can expedite vaccine iteration to guide human vaccine design and better predict outcome, with the added benefit of helping feline mammary tumor patients.

An Escherichia coli strain, PGB01, isolated from feral pigeon Faeces, thermally fit to survive in pigeon, shows high level resistance to trimethoprim.

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon’s gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr.

Detection and classification of live and dead Escherichia coli by laser-induced breakdown spectroscopy.

A common goal for astrobiology is to detect organic materials that may indicate the presence of life. However, organic materials alone may not be representative of currently living systems. Thus, it would be valuable to have a method with which to determine the health of living materials. Here, we present progress toward this goal by reporting on the application of laser-induced breakdown spectroscopy (LIBS) to study characteristics of live and dead cells using Escherichia coli (E. coli) strain K12 cells as a model organism since its growth and death in the laboratory are well understood. Our goal is to determine whether LIBS, in its femto- and/or nanosecond forms, could ascertain the state of a living organism. E. coli strain K12 cells were grown, collected, and exposed to one of two types of inactivation treatments: autoclaving and sonication. Cells were also kept alive as a control. We found that LIBS yields key information that allows for the discrimination of live and dead E. coli bacteria based on ionic shifts reflective of cell membrane integrity.