Achim M. Loske

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.

Bactericidal effect of underwater shock waves on Escherichia coli ATCC 10536 suspensions

An electrohydraulic shock wave generator was used to study the bactericidal action of shock waves on Escherichia coli ATCC 10536 suspensions in 0.9% (w/v) NaCl solution (initial cell population: 8.2 log10 CFU/ml). The influence of treatment temperature, shock wave energy, number of applied shock waves, the acoustic cavitation produced by the shock wave, and the spark gap-generated light (UV and visible) were analyzed using E. coli cultures in the exponential phase. Part of the experiment was repeated in the stationary phase. Results indicate that light, number of shock waves, cavitation and interactions between them, influence bactericidal activity (P<0.05). The best viability reduction of 4.06 log10 CFU/ml was achieved at 350 shock waves, administered during approximately 14.5 min, with bacteria in the stationary phase by enhancing acoustic cavitation inside the vial, and without protecting the samples from the visible and UV radiation produced by the shock wave-generating spark.

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.

The role of energy density and acoustic cavitation in shock wave lithotripsy

Today a high percentage of urinary stones are successfully treated by extracorporeal shockwave lithotripsy (SWL); however, misconceptions regarding fragmentation mechanisms, as well as treatment parameters like dose, applied energy and focal area are still common. A main stone comminution mechanism during SWL is acoustic cavitation. The objective of this study was to analyze the influence of cavitation and energy density on stone fragmentation. A research lithotripter was used to expose a large set of artificial kidney stones to shock waves varying different parameters. Hundreds of pressure records were used to calculate the energy density of the lithotripter at different settings. Results indicate that energy density is a crucial parameter and that better SWL treatment outcomes could be obtained placing the calculus at a prefocal position.

An underwater shock wave research device

It is the purpose of this article to describe the design, construction, and operation of a highly versatile, low cost experimental facility to produce, by electrical breakdown of water, weak underwater shock waves in the 50 to 2000 bar range. The device as a whole is described and then the electrical circuit used to generate the shock waves is explained in some detail. The semi‐ellipsoidal or parabolic focusing mirrors, used to improve the efficiency in the transfer to the energy generated in an underwater shock wave, are also discussed. The measurement and control systems for operation of the device are described. Finally, a discussion of some of the experimental results is presented. As far as we know, this is the only apparatus of its kind with such a wide capability.

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.