11 research outputs found
Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli
Objectives: The context of antibiotic resistance genes can provide valuable information about the epidemiology of mobile genetic elements. This study examined the distribution of the closely related blaTEM transposons Tn1, Tn2 and Tn3, or blaTEM-containing fragments of them, in ampicillin-resistant human commensal Escherichia coli isolates.
Methods: A PCR mapping protocol was used to detect different segments of the transposons or to link partial copies to the insertion sequence IS26. Restriction digestion of one amplicon was used to assign transposons to Tn1, Tn2 or Tn3 groups and sequencing validated this approach. Restriction digestion and sequencing were used to determine how much of the transposon remained when blaTEM was linked to IS26. Sequences were compared with those in GenBank.
Results: Of 25 ampicillin-resistant E. coli strains recovered from the faecal flora of healthy humans that carried the blaTEM gene, 15 carried a complete copy of Tn2 or a Tn2 variant; one was interrupted by IS4. A further isolate carried Tn3. Tn2 was also most abundant in sequences available in GenBank. Two isolates carried Tn2 and an IS26-blaTEM fragment. The remaining 10 isolates carried only the blaTEM end of the transposon and 9 of these partial copies were flanked by IS26 at varying distances upstream of blaTEM. One configuration corresponded to that in Tn6029B and the complete transposon was shown to be present.
Conclusions: Tn1, Tn2 and Tn3 can be simply and rapidly identified. Tn2 appears to be the most widely distributed. However, the blaTEM-containing end associated with an IS26 is also widely distributed
Electroporation System for Controlled Localized Therapeutics Delivery
Patent: Embodiments of the present invention provide an electroporation system comprising an electroporation probe having at least two contiguous electrodes configured to be inserted into biological tissue for electroporation treatment, and a pulse generator electrically connected to the probe and configured to drive the electroporation probe using a sequence of one or more electric pulses to cause current transmission through the probe and induce a non-uniform electric field in the biological tissue proximate the probe electrodes. Treatment tissue can be targeted by controlling the probe configuration, carrier solution characteristics and parameters of the electroporation pulse sequence to achieve predictable electroporation outcomes. This electroporation control method can also reduce potentially toxic effects of electroporation treatment
Electroporation System for Controlled Localized Therapeutics Delivery
Patent: Embodiments of the present invention provide an electroporation system comprising an electroporation probe having at least two contiguous electrodes configured to be inserted into biological tissue for electroporation treatment, and a pulse generator electrically connected to the probe and configured to drive the electroporation probe using a sequence of one or more electric pulses to cause current transmission through the probe and induce a non-uniform electric field in the biological tissue proximate the probe electrodes. Treatment tissue can be targeted by controlling the probe configuration, carrier solution characteristics and parameters of the electroporation pulse sequence to achieve predictable electroporation outcomes. This electroporation control method can also reduce potentially toxic effects of electroporation treatment
Australian scorpion Hormurus waigiensis venom fractions show broad bioactivity through modulation of bio-impedance and cytosolic calcium
Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da—13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome
Computational simulation expands understanding of electrotransfer-based gene augmentation for enhancement of neural interfaces
The neural interface is a critical factor in governing efficient and safe charge transfer between a stimulating electrode and biological tissue. The interface plays a crucial role in the efficacy of electric stimulation in chronic implants and both electromechanical properties and biological properties shape this. In the case of cochlear implants, it has long been recognized that neurotrophins can stimulate growth of the target auditory nerve fibers into a favorable apposition with the electrode array, and recently such arrays have been re-purposed to enable electrotransfer (electroporation)-based neurotrophin gene augmentation to improve the "bionic ear." For both this acute bionic array-directed electroporation and for chronic conventional cochlear implant arrays, the electric fields generated in target tissue during pulse delivery are central to efficacy, but are challenging to map. We present a computational model for predicting electric fields generated by array-driven DNA electrotransfer in the cochlea. The anatomically realistic model geometry was reconstructed from magnetic resonance images of the guinea pig cochlea and an eight-channel electrode array embedded within this geometry. The model incorporates a description of both Faradaic and non-Faradaic mechanisms occurring at the electrode-electrolyte interface with frequency dependency optimized to match experimental impedance spectrometry measurements. Our simulations predict that a tandem electrode configuration with four ganged cathodes and four ganged anodes produces three to fourfold larger area in target tissue where the electric field is within the range for successful gene transfer compared to an alternate paired anode-cathode electrode configuration. These findings matched in vivo transfection efficacy of a green fluorescent protein (GFP) reporter following array-driven electrotransfer of the reporter-encoding plasmid DNA. This confirms utility of the developed model as a tool to optimize the safety and efficacy of electrotransfer protocols for delivery of neurotrophin growth factors, with the ultimate aim of using gene augmentation approaches to improve the characteristics of the electrode-neural interfaces in chronically implanted neurostimulation devices
Mapping of bionic array electric field focusing in plasmid DNA-based gene electrotransfer
Molecular medicine through gene therapy is challenged to achieve targeted action. This is now possible utilizing bionic electrode arrays for focal delivery of naked (plasmid) DNA via gene electrotransfer. Here we establish the properties of array-based electroporation affecting targeted gene delivery. An array with eight 300 μm platinum ring electrodes configured as a cochlear implant bionic interface was used to transduce HEK293 cell monolayers with a plasmid-DNA green fluorescent protein (GFP) reporter gene construct. Electroporation parameters were: pulse intensity, number, duration, separation and electrode configuration. The latter determined the shape of the electric fields, which were mapped using a voltage probe. Electrode array-based electroporation was found to require~100x lower applied voltages for cell transduction than conventional electroporation where the target cells were located between the electrodes. This was found to be due to compression of the field lines orthogonal to the array. A circular area of GFP-positive cells was created when the electrodes were ganged together as four adjacent anodes and four cathodes, whereas alternating electrode polarity created a linear area of GFP-positive cells. The refinement of gene delivery parameters was validated in vivo in the guinea pig cochlea. These findings have significant clinical ramifications, where spatiotemporal control of gene expression can be predicted by manipulation of the electric field via current steering at a cellular level
Presentation_1_Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice.pdf
In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the ‘cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, “contralateral suppression” of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24–32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.</p