Francisco Fernández

Tandem shock wave cavitation enhancement for extracorporeal lithotripsy

Extracorporeal shock wave lithotripsy (ESWL) has been successful for more than twenty years in treating patients with kidney stones. Hundreds of underwater shock waves are generated outside the patients body and focused on the kidney stone. Stones fracture mainly due to spalling, cavitation and layer separation. Cavitation bubbles are produced in the vicinity of the stone by the tensile phase of each shock wave. Bubbles expand, stabilize and finally collapse violently, creating stone-damaging secondary shock waves and microjets. Bubble collapse can be intensified by sending a second shock wave a few hundred microseconds after the first. A novel method of generating two piezoelectrically generated shock waves with an adjustable time delay between 50 and 950 micros is described and tested. The objective is to enhance cavitation-induced damage to kidney stones during ESWL in order to reduce treatment time. In vitro kidney stone model fragmentation efficiency and pressure measurements were compared with those for a standard ESWL system. Results indicate that fragmentation efficiency was significantly enhanced at a shock wave delay of about 400 and 250 micros using rectangular and spherical stone phantoms, respectively. The system presented here could be installed in clinical devices at relatively low cost, without the need for a second shock wave generator.

Physical methods for genetic plant transformation.

Production of transgenic plants is a routine process for many crop species. Transgenes are introduced into plants to confer novel traits such as improved nutritional qualities, tolerance to pollutants, resistance to pathogens and for studies of plant metabolism. Nowadays, it is possible to insert genes from plants evolutionary distant from the host plant, as well as from fungi, viruses, bacteria and even animals. Genetic transformation requires penetration of the transgene through the plant cell wall, facilitated by biological or physical methods. The objective of this article is to review the state of the art of the physical methods used for genetic plant transformation and to describe the basic physics behind them.

A novel and highly efficient method for genetic transformation of fungi employing shock waves.

Genetic transformation of filamentous fungi is an essential tool in many areas such as biotechnology, medicine, phytopathology and genetics. However, available protocols to transform fungi are inefficient, laborious and have low reproducibility. We report the use of underwater shock waves as a novel method to transform filamentous fungi. An experimental piezoelectric shock wave generator was designed to expose fungal conidia to heterologous DNA. The device was successfully tested in Aspergillus niger, Fusarium oxysporum, Trichoderma reesei and Phanerochaete chrysosporium. The transformation frequency per number of conidia was between two and four orders of magnitude higher in comparison to previously published methods. For example, the frequency of transformation in A. niger was improved up to 5400-fold as compared with Agrobacterium protocols. Transformation was verified by expression of the green fluorescent protein, PCR and Southern blot. Our method offers new possibilities for fast, easy and efficient genetic manipulation of diverse fungal species.

ENHANCED SHOCK WAVE-ASSISTED TRANSFORMATION OF ESCHERICHIA COLI

The objective of the study was to demonstrate that shock wave-induced transfer of DNA into bacteria can be increased by enhancing cavitation using dual-pulse (tandem) shock waves. Escherichia coli and plasmid were transferred to test vials. Competent cells were prepared at different concentrations of CaCl(2). Single pulses and tandem shock waves were compared as were three treatment temperatures: 0, 10 and 25 °C. Three delays (250, 500, 750 μs) between double pulses were tested. Characterization was achieved by using a plasmid that provided green fluorescent protein expression. At 0 °C double pulses generated at a delay of 750 μs significantly increased the number of fluorescent colonies compared with single pulses. In general, the lowest temperature enhanced the mean number of transformants compared with the two higher temperatures. A strong influence of the CaCl(2) concentration on the transformation efficiency was also found. The main conclusion is that gene transfer to target cells may be increased up to 50 times at 0 °C by enhancing cavitation using pairs of shock waves.

Physical methods for genetic transformation of fungi and yeast

The production of transgenic fungi is a routine process. Currently, it is possible to insert genes from other fungi, viruses, bacteria and even animals, albeit with low efficiency, into the genomes of a number of fungal species. Genetic transformation requires the penetration of the transgene through the fungal cell wall, a process that can be facilitated by biological or physical methods. Novel methodologies for the efficient introduction of specific genes and stronger promoters are needed to increase production levels. A possible solution to this problem is the recently discovered shock-wave-mediated transformation. The objective of this article is to review the state of the art of the physical methods used for genetic fungi transformation and to describe some of the basic physics and molecular biology behind them.

Treatment time reduction using tandem shockwaves for lithotripsy: an in vivo study.

BACKGROUND AND PURPOSE Reducing extracorporeal shockwave lithotripsy (SWL) time by increasing the shockwave rate of the lithotripter has been tested in the past; however, basic research and treatment outcomes revealed that this is not convenient. The purpose of this study was to use an animal model to demonstrate that SWL treatment time can be reduced significantly without sacrificing stone fragmentation efficiency using tandem shockwaves. MATERIALS AND METHODS A tandem research lithotripter was used to treat 50 artificial kidney stones implanted into the kidneys of 50 rabbits. Standard single-pulse and tandem shockwaves were compared in two different scenarios: Without a fluid-filled expansion chamber and with a fluid-filled expansion chamber surrounding the stone. RESULTS The presence of fluid surrounding the stone enhances fragmentation in both the standard and tandem modes. No significant difference in fragmentation efficiency was recorded between the standard and tandem SWL with stones surrounded by fluid; however, the treatment time with tandem shockwaves was reduced by 50%. CONCLUSIONS Significantly shorter SWL treatments may be possible in the future using tandem shockwaves on urinary stones that are surrounded by fluid.

High-yield production of manganese peroxidase, lignin peroxidase, and versatile peroxidase in Phanerochaete chrysosporium

The white-rot fungus Phanerochaete chrysosporium secretes extracellular oxidative enzymes during secondary metabolism, but lacks versatile peroxidase, an enzyme important in ligninolysis and diverse biotechnology processes. In this study, we report the genetic modification of a P. chrysosporium strain capable of co-expressing two endogenous genes constitutively, manganese peroxidase (mnp1) and lignin peroxidase (lipH8), and the codon-optimized vpl2 gene from Pleurotus eryngii. For this purpose, we employed a highly efficient transformation method based on the use of shock waves developed by our group. The expression of recombinant genes was verified by PCR, Southern blot, quantitative real-time PCR (qRT-PCR), and assays of enzymatic activity. The production yield of ligninolytic enzymes was up to four times higher in comparison to previously published reports. These results may represent significant progress toward the stable production of ligninolytic enzymes and the development of an effective fungal strain with promising biotechnological applications.

Modified shock waves for extracorporeal shock wave lithotripsy: a simulation based on the Gilmore formulation.

Extracorporeal shock wave lithotripsy (SWL) is a reliable therapy for the treatment of urolithiasis. Nevertheless, improvements to enhance stone fragmentation and reduce tissue damage are still needed. During SWL, cavitation is one of the most important stone fragmentation mechanisms. Bubbles with a diameter between about 7 and 55μm have been reported to expand and collapse after shock wave passage, forming liquid microjets at velocities of up to 400m/s that contribute to the pulverization of renal calculi. Several authors have reported that the fragmentation efficiency may be improved by using tandem shock waves. Tandem SWL is based on the fact that the collapse of a bubble can be intensified if a second shock wave arrives tenths or even a few hundredths of microseconds before its collapse. The object of this study is to determine if tandem pulses consisting of a conventional shock wave (estimated rise time between 1 and 20ns), followed by a slower second pressure profile (0.8μs rise time), have advantages over conventional tandem SWL. The Gilmore equation was used to simulate the influence of the modified pressure field on the dynamics of a single bubble immersed in water and compare the results with the behavior of the same bubble subjected to tandem shock waves. The influence of the delay between pulses on the dynamics of the collapsing bubble was also studied for both conventional and modified tandem waves. For a bubble of 0.07mm, our results indicate that the modified pressure profile enhances cavitation compared to conventional tandem waves at a wide range of delays (10-280μs). According to this, the proposed pressure profile could be more efficient for SWL than conventional tandem shock waves. Similar results were obtained for a ten times smaller bubble.

Recombinant expression of four oxidoreductases in Phanerochaete chrysosporium improves degradation of phenolic and non-phenolic substrates.

Phanerochaete chrysosporium belongs to a group of lignin-degrading fungi that secretes various oxidoreductive enzymes, including lignin peroxidase (LiP) and manganese peroxidase (MnP). Previously, we demonstrated that the heterologous expression of a versatile peroxidase (VP) in P. chrysosporium recombinant strains is possible. However, the production of laccases (Lac) in this fungus has not been completely demonstrated and remains controversial. In order to investigate if the co-expression of Lac and VP in P. chrysosporium would improve the degradation of phenolic and non-phenolic substrates, we tested the constitutive co-expression of the lacIIIb gene from Trametes versicolor and the vpl2 gene from Pleurotus eryngii, and also the endogenous genes mnp1 and lipH8 by shock wave mediated transformation. The co-overexpression of peroxidases and laccases was improved up to five-fold as compared with wild type species. Transformant strains showed a broad spectrum in phenolic/non-phenolic biotransformation and a high percentage in synthetic dye decolorization in comparison with the parental strain. Our results show that the four enzymes can be constitutively expressed in a single transformant of P. chrysosporium in minimal medium. These data offer new possibilities for an easy and efficient co-expression of laccases and peroxidases in suitable basidiomycete species.

Tandem shock waves to enhance genetic transformation of Aspergillus niger.

Filamentous fungi are used in several industries and in academia to produce antibiotics, metabolites, proteins and pharmaceutical compounds. The development of valuable strains usually requires the insertion of recombinant deoxyribonucleic acid; however, the protocols to transfer DNA to fungal cells are highly inefficient. Recently, underwater shock waves were successfully used to genetically transform filamentous fungi. The purpose of this research was to demonstrate that the efficiency of transformation can be improved significantly by enhancing acoustic cavitation using tandem (dual-pulse) shock waves. Results revealed that tandem pressure pulses, generated at a delay of 300 μs, increased the transformation efficiency of Aspergillus niger up to 84% in comparison with conventional (single-pulse) shock waves. This methodology may also be useful to obtain new strains required in basic research and biotechnology.