A bio-inspired cell-free system for cannabinoid production from inexpensive inputs.

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.

Association of Neisseria gonorrhoeae Plasmids With Distinct Lineages and The Economic Status of Their Country of Origin.

Plasmids are vehicles for horizontal gene transfer between bacteria, and in Neisseria gonorrhoeae plasmids can mediate high-level antimicrobial resistance (AMR). Using genomic and phylogenetic analyses, we show that plasmids are widespread in a collection of 3724 gonococcal isolates from 56 countries, and characterized the conjugative, ß-lactamase and cryptic plasmids. We found that variants of the conjugative plasmid (which can mediate tetracycline resistance) and the ß-lactamase plasmid expressing TEM-135 are associated with distinct gonococcal lineages. Furthermore, AMR plasmids are significantly more prevalent in gonococci from less wealthy countries, highlighting the need for further studies. More than 94% of gonococci possess the cryptic plasmid, with its absence correlated with the presence of a novel chromosomal type IV secretion system. Our results reveal the extent of plasmid-mediated AMR in the gonococcus, particularly in less wealthy countries, where diagnostic and therapeutic options can be limited, and highlight the risk of their global spread.

An improved platform for functional assessment of large protein libraries in mammalian cells.

Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.

Quality assessment of supercoiled minicircle DNA by cadaverine-modified analytical chromatographic monolith.

Minicircle DNA (mcDNA) is the ultimate non-viral DNA vector, presenting higher biosafety and therapeutic effect than conventional plasmid DNA (pDNA). However, given the similarity between mcDNA and its precursor, the parental plasmid (PP), analytical methodologies established for pDNA are unable to distinguish mcDNA from PP. Thus, a new need emerged for the implementation of suitable, rapid and non-expensive analytical methodologies for the characterization of mcDNA samples. Recently, our research group was able to develop a purification strategy for the isolation of supercoiled (sc) mcDNA resorting to cadaverine-modified monolith. Considering the promising results obtained with this strategy, a cadaverine-modified analytical monolith was prepared and explored for mcDNA quantification. Thus, a strategy of three-step increasing NaCl gradient was considered to first elute RNA/protein content, then isolate sc mcDNA and finally eliminate PP and other impurities still bounded to the matrix. A calibration curve was constructed with different sc mcDNA standards within a range of 1-25 µg/mL. Linearity, accuracy, precision and selectivity of this method were validated according to the international guidelines and the limit of detection and the lower limit of quantification were determined as 1 µg/mL. For the first time, to the best of our knowledge, an analytical method for mcDNA quantification is described. Besides ensuring the safety of mcDNA application by assessing the product purity, such methodology can be used in the future to control industrial mcDNA production and purification, perhaps aiding in the establishment of optimized and less expensive biotechnological operations.

In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO-PPO-PEO polymeric micelles on cornea.

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO-PPO-PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB-DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB-DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB-DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO-PPO-PEO polymers as a vehicle for drug delivery.

Cost of Nosocomial Outbreak Caused by NDM-1-Containing Klebsiella pneumoniae in the Netherlands, October 2015-January 2016.

During October-December 2015, 29 patients in a hospital in the Netherlands acquired nosocomial infection with a multidrug-resistant, New Delhi-metallo-ß-lactamase-positive Klebsiella pneumoniae strain. Extensive infection control measures were needed to stop this outbreak. The estimated economic impact of the outbreak was $804,263; highest costs were associated with hospital bed closures.

Conjugative plasmids enable the maintenance of low cost non-transmissible plasmids.

Some plasmids can be transferred by conjugation to other bacterial hosts. But almost half of the plasmids are non-transmissible. These plasmid types can only be transmitted to the daughter cells of their host after bacterial fission. Previous studies suggest that non-transmissible plasmids become extinct in the absence of selection of their encoded traits, as plasmid-free bacteria are more competitive. Here, we aim to identify mechanisms that enable non-transmissible plasmids to persist, even if they are not beneficial. For this purpose, an individual-based model for plasmid population dynamics was set up and carefully tested for structural consistency and plausibility. Our results demonstrate that non-transmissible plasmids can be stably maintained in a population, even if they impose a substantial burden on their host cells growth. A prerequisite is the co-occurrence of an incompatible and costly conjugative plasmid type, which indirectly facilitates the preservation of the non-transmissible type. We suggest that this constellation might be considered as a potential mechanism maintaining plasmids and associated antibiotic resistances. It should be investigated in upcoming laboratory experiments.

Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS).

Vibrio parahaemolyticus is a causative agent of acute hapatopancreatic necrosis syndrome (AHPNS) which causes early mortality in white shrimp. Emergence of AHPNS has caused tremendous economic loss for aquaculture industry particularly in Asia since 2010. Previous studies reported that strains causing AHPNS harbor a 69-kb plasmid with possession of virulence genes, pirA and pirB. However, genetic variation of the 69-kb plasmid among AHPNS related strains has not been investigated. This study aimed to analyze genetic composition and diversity of the 69-kb plasmid in strains isolated from shrimps affected by AHPNS. Plasmids recovered from V. parahaemolyticus strain VPE61 which represented typical AHPNS pathogenicity, strain VP2HP which did not represent AHPNS pathogenicity but was isolated from AHPNS affected shrimp and other AHPNS V. parahaemolyticus isolates in Genbank were investigated. Protein coding genes of the 69-kb plasmid from the strain VPE61 were identical to that of AHPNS strain from Vietnam except the inverted complement 3.4-kb transposon covering pirA and pirB. The strain VP2HP possessed remarkable large 183-kb plasmid which shared similar protein coding genes to those of the 69-kb plasmid from strain VPE61. However, the 3.4-kb transposon covering pirA and pirB was absent from the 183-kb plasmid in strain VP2HP. A number of protein coding genes from the 183-kb plasmid were also detected in other AHPNS strains. In summary, this study identified a novel 183-kb plasmid that is related to AHPNS causing strains. Homologous recombination of the 69-kb AHPNS plasmid and other naturally occurring plasmids together with loss and gain of AHPNS virulence genes in V. parahaemolyticus were observed. The outcome of this research enables understanding of plasmid dynamics that possibly affect variable degrees of AHPNS pathogenicity.

Targeted Intraceptor Nanoparticle for Neovascular Macular Degeneration: Preclinical Dose Optimization and Toxicology Assessment.

Neovascular age-related macular degeneration (AMD) is treated with anti-VEGF intravitreal injections, which can cause geographic atrophy, infection, and retinal fibrosis. To minimize these toxicities, we developed a nanoparticle delivery system for recombinant Flt23k intraceptor plasmid (RGD.Flt23k.NP) to suppress VEGF intracellularly within choroidal neovascular (CNV) lesions in a laser-induced CNV mouse model through intravenous administration. In the current study, we examined the efficacy and safety of RGD.Flt23k.NP in mice. The effect of various doses was determined using fluorescein angiography and optical coherence tomography to evaluate CNV leakage and volume. Efficacy was determined by the rate of inhibition of CNV volume at 2 weeks post-treatment. RGD.Flt23k.NP had peak efficacy at a dose range of 30-60 µg pFlt23k/mouse. Using the lower dose (30 µg pFlt23k/mouse), RGD.Flt23k.NP safety was determined both in single-dose groups and in repeat-dose (three times) groups by measuring body weight, organ weight, hemoglobin levels, complement C3 levels, and histological changes in vital organs. Neither toxicity nor inflammation from RGD.Flt23k.NP was detected. No side effect was detected on visual function. Thus, systemic RGD.Flt23k.NP may be an alternative to standard intravitreal anti-VEGF therapy for the treatment of neovascular AMD.

Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations.

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods.

Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software.

Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

Targeted Delivery with Imaging Assessment of siRNA Expressing Nanocassettes into Cancer.

Molecular therapy using small interfering RNA (siRNA) shows great promise in the development of novel therapeutics for cancer. Although various approaches have been developed for in vivo delivery of siRNAs into tumors, stability of siRNA in blood circulation, and low efficiency of siRNA delivery into tumor cells are the major obstacles for further translation into cancer therapeutics. In this protocol, we describe methods of the production of shRNA expressing DNA nanocassettes by PCR amplification of double-stranded DNA fragments containing a U6 promoter and a shRNA gene. Those DNA nanocassettes can be conjugated to the polymer coating of nanoparticles that are targeted to cellular receptors highly expressed in tumor cells, such as urokinase plasminogen activator receptor (uPAR), for targeted delivery and receptor mediated internalization of shRNA expressing DNA nanocassettes. Methods for in vitro and in vivo evaluation of target specificity and gene-knockdown effect are also provided.

A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.

Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

Structure-Function Assessment of Mannosylated Poly(ß-amino esters) upon Targeted Antigen Presenting Cell Gene Delivery.

Antigen presenting cell (APC) gene delivery is a promising avenue for modulating immunological outcomes toward a desired state. Recently, our group developed a delivery methodology to elicit targeted and elevated levels of APC-mediated gene delivery. During these initial studies, we observed APC-specific structure-function relationships with the vectors used during gene delivery that differ from current non-APC cell lines, thus, emphasizing a need to re-evaluate vector-associated parameters in the context of APC gene transfer. Thus, we describe the synthesis and characterization of a second-generation mannosylated poly(ß-amino ester) library stratified by molecular weight. To better understand the APC-specific structure-function relationships governing polymeric gene delivery, the library was systematically characterized by (1) polymer molecular weight, (2) relative mannose content, (3) polyplex biophysical properties, and (4) gene delivery efficacy. In this library, polymers with the lowest molecular weight and highest relative mannose content possessed gene delivery transfection efficiencies as good as or better than commercial controls. Among this group, the most effective polymers formed the smallest polymer-plasmid DNA complexes (∼300 nm) with moderate charge densities (<10 mV). This convergence in polymer structure and polyplex biophysical properties suggests a unique mode of action and provides a framework within which future APC-targeting polymers can be designed.

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

Influence of pH and temperature on the activity of SnO2-bound α-amylase: a genotoxicity assessment of SnO2 nanoparticles.

Immobilization of biologically important molecules on a myriad of nanosized materials has attracted great attention due to their small size, biocompatibility, higher surface-to-volume ratio, and lower toxicity. These properties make nanoparticles (NPs) a superior matrix over bulk material for the immobilization of enzymes and proteins. In the present study, Bacillus amyloliquefaciens α-amylase was immobilized on SnO2 nanoparticles by a simple adsorption mechanism. Nanoparticle-adsorbed enzyme retained 90% of the original enzyme activity. Thermal stability of nanosupport was investigated by thermogravimetric and differential thermal analysis. Scanning electron microscopic studies showed that NPs have porous structure for the high-yield immobilization of α-amylase. The genotoxicity of SnO2-NPs was analyzed by pUC(19) plasmid nicking and comet assay and revealed that no remarkable DNA damage occurred in lymphocytes. The pH-optima was found to be the same for both free and SnO2-NPs bound enzyme, while the temperature-optimum for NPs-adsorbed α-amylase was 5°C higher than its free counterpart. Immobilized enzyme retained more than 70% enzyme activity even after its eight repeated uses.

A multiplexed nucleic acid microsystem for point-of-care detection of HIV co-infection with MTB and PCP.

Many individuals infected with the human immunodeficiency virus (HIV), especially children in African countries, die of co-infections with Mycobacterium tuberculosis (MTB) (coinfection rate: 50%) or Pneumocystis carinii pneumonia (PCP) (coinfection rate: 81%). The present proposal describes a rapid, portable, low-cost, multiplexed point-of-care diagnostic technique for simultaneously detecting HIV, MTB, and PCP. This technique incorporates a creative micro-device (hardware) and a loop-mediated isothermal amplification strategy (software).

A ternary conjugation system for the construction of DNA libraries for Geobacillus kaustophilus HTA426.

This report describes efficient plasmid uptake by the thermophile Geobacillus kaustophilus HTA426 by means of a ternary conjugation system, which was used to construct thermophile DNA libraries for G. kaustophilus and to identify the genes for orotidine-5'-phosphate decarboxylase by in vivo functional screening. The results indicate that the conjugation system is useful in constructing G. kaustophilus libraries, which are practical in identifying thermophile genes.

Assessment of intravenous pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) in yucatan swine.

PDX1 (pancreatic and duodenal homeobox 1) is overexpressed in pancreatic cancer, and its reduction results in tumor regression. Bi-functional pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) utilizes the endogenous micro-RNA biogenesis pathway to effect cleavage- and non-cleavage-dependent degradation of PDX1 mRNA. We have shown that OFHIRNA-PDX1 reduces pancreatic tumor volume in xenograft models. Thus, we are now exploring biorelevant large animal safety of OFHIRNA-PDX1. Mini pigs were chosen as the biorelevant species based on the similarity of human and pig PDX1 target sequence. In the initial study, animals developed fever, lethargy, hyporexia and cutaneous hyperemia following administration of OFHIRNA-PDX1. Twenty-one days later, the same animals demonstrated less toxicity with a second OFHIRNA-PDX1 infusion in conjunction with a prophylactic regimen involving dexamethasone, diphenhydramine, Indocin and ranitidine. In a new group of animals, PDX1 protein (31 kDa) expression in the pancreas was significantly repressed at 48 and 72 h (85%, P=0.018 and 88%, P=0.013; respectively) following a single infusion of OFHIRNA-PDX1 but recovered to normal state within 7 days. In conclusion, a single intravenous infusion of OFHIRNA-PDX1 in conjunction with premedication in pigs was well tolerated and demonstrated significant PDX1 knockdown.