Green Synthesis and Antiproliferative Activity of Gold Nanoparticles of a Controlled Size and Shape Obtained Using Shock Wave Extracts from Amphipterygium adstringens.

In this study, green chemistry was used as a tool to obtain gold nanoparticles using Amphipterygium adstringens extracts as a synthesis medium. Green ethanolic and aqueous extracts were obtained using ultrasound and shock wave-assisted extraction. Gold nanoparticles with sizes ranging between 100 and 150 nm were obtained with ultrasound aqueous extract. Interestingly, homogeneous quasi-spherical gold nanoparticles with sizes between 50 and 100 nm were achieved with shock wave aqueous-ethanolic extracts. Furthermore, 10 nm gold nanoparticles were obtained by the traditional methanolic macerate extraction method. The physicochemical characteristics, morphology, size, stability, and Z potential of the nanoparticles were determined using microscopic and spectroscopic techniques. The viability assay in leukemia cells (Jurkat) was performed using two different sets of gold nanoparticles, with final IC50 values of 87 µM and 94.7 µM, reaching a maximum cell viability decrease of 80% The results do not indicate a significant difference between the cytotoxic effects produced by the gold nanoparticles synthesized in this study and vincristine on normal lymphoblasts (CRL-1991).

Metabolic engineering of Aspergillus niger to enhance production of ethanol.

This work describes the genetic transformation of a strain of Aspergillus niger with five different constructs containing 16 different heterologous genes, coding for four oxidoreductases, two cellobiohydrolases, one endoglucanase, one ß-glucosidase, six enzymes involved in xylose metabolism, and two enzymes involved in fermentation. The aim was to try and engineer a consolidated bioprocessing in A. niger. The fungus already contains most of these enzymes and we only enhanced endogenous activities. We recovered nine transformants containing all genes, as indicated by polymerase chain reaction (PCR). To confirm that the products of the genes were functional, we measured the activity of five different enzymes in all the strains, and they all showed enhanced activity over the wild-type (wt) strain. The strains were grown on carboxymethyl cellulose (CMC) and xylan as substrates, and they produced considerably more ethanol than the wt. The levels of ethanol production were comparable to those reported in the literature.

Effect of Shock Waves on the Growth of Aspergillus niger Conidia: Evaluation of Germination and Preliminary Study on Gene Expression.

Shock waves, as used in medicine, can induce cell permeabilization, genetically transforming filamentous fungi; however, little is known on the interaction of shock waves with the cell wall. Because of this, the selection of parameters has been empirical. We studied the influence of shock waves on the germination of Aspergillus niger, to understand their effect on the modulation of four genes related to the growth of conidia. Parameters were varied in the range reported in protocols for genetic transformation. Vials containing conidia in suspension were exposed to either 50, 100 or 200 single-pulse or tandem shock waves, with different peak pressures (approximately 42, 66 and 83 MPa). In the tandem mode, three delays were tested. To equalize the total energy, the number of tandem "events" was halved compared to the number of single-pulse shock waves. Our results demonstrate that shock waves do not generate severe cellular effects on the viability and germination of A. niger conidia. Nevertheless, increase in the aggressiveness of the treatment induced a modification in four tested genes. Scanning electron microscopy revealed significant changes to the cell wall of the conidia. Under optimized conditions, shock waves could be used for several biotechnological applications, surpassing conventional techniques.

Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.

Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

Shock wave-assisted extraction of phenolic acids and flavonoids from Eysenhardtia polystachya heartwood: A novel method and its comparison with conventional methodologies.

Phenolic compounds, obtained from plants are important in the food, biomaterial and pharmaceutical industries; however current extraction methods, such as Soxhlet (solid-liquid) extraction, liquid-liquid extraction, microwave-assisted extraction, and ultrasonic extraction (USE), have the disadvantages of large processing times, contamination by solvents, and degradation of analytes. This study demonstrates that shock wave-assisted extraction can be used as a more efficient, eco-friendly and rapid method. Extraction of powdered samples of Eysenhardtia polystachia heartwood, a plant with high concentration of phenolic compounds, exposed to different doses of underwater shock waves, was compared with the conventional methods. Our results revealed that shock wave-assisted extraction (1500 shock waves with a peak positive pressure of approximately 88 MPa) produced 34.54% and 31.95% higher contents than Soxhlet and USE, respectively. Extraction times using shock waves were much shorter than with all other methods tested, proving that it is an attractive method to obtain both phenolic acids and flavonoids without the need for organic solvents. Furthermore, shock waves produced a significantly higher content of total reducing sugars than Soxhlet extraction and less phenolic acids which gives the insight of a more selective extraction of components.

Shock Wave Application Increases the Antineoplastic Effect of Molecular Iodine Supplement in Breast Cancer Xenografts.

We evaluated the effect of oral molecular iodine supplementation and shock wave application under three different conditions on human MDA-MB231 cancer cell xenografts. After tumor volume reached 1 cm3, mice were randomly assigned to groups and treated for 3 weeks. The results revealed that high-dose shock wave treatment (150 shock waves at a pressure of 21.7 MPa, SW150/21.7) generated tissue lesions without decreasing tumor growth, canceled the antineoplastic action of iodine and promoted pro-tumor conditions (increased hypoxia-induced factor [HIF] and vascular endothelial growth factor [VEGF]). In contrast, moderate (SW35/21.7) and low (SW35/9.9) doses of shock waves had significant antineoplastic effects and, in combination with iodine supplement, attenuated the aggressiveness of these cells by decreasing expression of the markers of stem cells (CD44 and Sox2) and invasion (HIF and VEGF). These results allow us to propose the combination of shock waves and iodine as a possible adjuvant in breast cancer therapy.

Kriging model to study the dynamics of a bubble subjected to tandem shock waves as used in biomedical applications.

The purpose of this work was to develop a metamodel (Kriging model) to identify the most important input parameters of shock wave pressure profiles as used in biomedical applications without solving a large number of differential equations. Shock wave-induced cavitation is involved in several biological effects. During bubble collapse, secondary shock waves and microjets are formed. For some applications, it is desirable to enhance this phenomenon by applying a second shock wave before bubble collapse; however, the delay between the leading shock wave and the second pressure pulse has yet to be optimized. This optimization can be done using numerical analysis. A metamodel that predicts the most convenient ranges for the input variables and provides information on the joint effects between the input variables was tested. Because the metamodel is an analytical expression, running it fifty thousand times and analyzing variables, such as the pressure amplitude, delay between pulses, and pressure rise time, was fast and easy. Furthermore, this method can be a helpful tool to study the joint effect between the input variables and reduce the computation time. The metamodel can also be adapted to analyze simulations based on equations different from the Gilmore-Akulichev formulation, which was used in this study.

pMEX01, a 70kb Plasmid Isolated From Escherichia Coli That Confers Resistance to Multiple ß-Lactam Antibiotics

Abstract Multidrug-resistant microorganisms are of great concern to public health. Genetic mobile elements, such as plasmids, are among the most relevant mechanisms by which bacteria achieve this resistance. We obtained an Escherichia coli strain CM6, isolated from cattle presenting severe diarrheic symptoms in the State of Querétaro, Mexico. It was found to contain a 70 kb plasmid (pMEX01) with a high similarity to the pHK01-like plasmids that were previously identified and described in Hong Kong. Analysis of the pMEX01 sequence revealed the presence of a blaCTX-M-14 gene, which is responsible for conferring resistance to multiple β-lactam antibiotics. Several genes putatively involved in the conjugative transfer were also identified on the plasmid. The strain CM6 is of high epidemiological concern because it not only displays resistance to multiple β-lactam antibiotics but also to other kinds of antibiotics.

Shock wave-induced permeabilization of mammalian cells.

Controlled permeabilization of mammalian cell membranes is fundamental to develop gene and cell therapies based on macromolecular cargo delivery, a process that emerged against an increasing number of health afflictions, including genetic disorders, cancer and infections. Viral vectors have been successfully used for macromolecular delivery; however, they may have unpredictable side effects and have been limited to life-threatening cases. Thus, several chemical and physical methods have been explored to introduce drugs, vaccines, and nucleic acids into cells. One of the most appealing physical methods to deliver genes into cells is shock wave-induced poration. High-speed microjets of fluid, emitted due to the collapse of microbubbles after shock wave passage, represent the most significant mechanism that contributes to cell membrane poration by this technique. Herein, progress in shock wave-induced permeabilization of mammalian cells is presented. After covering the main concepts related to molecular strategies whose applications depend on safer drug delivery methods, the physics behind shock wave phenomena is described. Insights into the use of shock waves for cell membrane permeation are discussed, along with an overview of the two major biomedical applications thereof-i.e., genetic modification and anti-cancer shock wave-assisted chemotherapy. The aim of this review is to summarize 30 years of data showing underwater shock waves as a safe, noninvasive method for macromolecular delivery into mammalian cells, encouraging the development of further research, which is still required before the introduction of this promising tool into clinical practice.

pMEX01, a 70kb plasmid isolated from Escherichia coli that confers resistance to multiple ß-lactam antibiotics.

Multidrug-resistant microorganisms are of great concern to public health. Genetic mobile elements, such as plasmids, are among the most relevant mechanisms by which bacteria achieve this resistance. We obtained an Escherichia coli strain CM6, isolated from cattle presenting severe diarrheic symptoms in the State of Querétaro, Mexico. It was found to contain a 70kb plasmid (pMEX01) with a high similarity to the pHK01-like plasmids that were previously identified and described in Hong Kong. Analysis of the pMEX01 sequence revealed the presence of a blaCTX-M-14 gene, which is responsible for conferring resistance to multiple ß-lactam antibiotics. Several genes putatively involved in the conjugative transfer were also identified on the plasmid. The strain CM6 is of high epidemiological concern because it not only displays resistance to multiple ß-lactam antibiotics but also to other kinds of antibiotics.

Enhanced Delignification of Lignocellulosic Biomass by Recombinant Fungus Phanerochaete chrysosporium Overexpressing Laccases and Peroxidases.

Ligninolytic enzyme production and lignin degradation are typically the rate-limiting steps in the biofuel industry. To improve the efficiency of simultaneous bio-delignification and enzyme production, Phanerochaete chrysosporium was transformed by shock wave-induced acoustic cavitation to co-overexpress 3 peroxidases and 1 laccase and test it on the degradation of sugarcane bagasse and wheat bran. Lignin depolymerization was enhanced by up to 25% in the presence of recombinant fungi in comparison with the wild-type strain. Sugar release on lignocellulose was 2- to 6-fold higher by recombinant fungi as compared with the control. Wheat bran ostensibly stimulated the production of ligninolytic enzymes. The highest peroxidase activity from the recombinant strains was 2.6-fold higher, whereas the increase in laccase activity was 4-fold higher in comparison to the control. The improvement of lignin degradation was directly proportional to the highest peroxidase and laccase activity. Because various phenolic compounds released during lignocellulose degradation have proven to be toxic to cells and to inhibit enzyme activity, a significant reduction (over 40%) of the total phenolic content in the samples treated with recombinant strains was observed. To our knowledge, this is the first report that engineering P. chrysosporium enhances biodegradation of lignocellulosic biomass.

Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104 kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

Dynamic light scattering: A fast and reliable method to analyze bacterial growth during the lag phase.

A comparison between plate counting (PC) and dynamic light scattering (DLS) is reported. PC is the standard technique to determine bacterial population as a function of time; however, this method has drawbacks, such as the cumbersome preparation and handling of samples, as well as the long time required to obtain results. Alternative methods based on optical density are faster, but do not distinguish viable from non-viable cells. These inconveniences are overcome by using DLS. Two different bacteria strains were considered: Escherichia coli and Staphylococcus aureus. DLS was performed at two different illuminating conditions: continuous and intermittent. By the increment of particle size as a function of time, it was possible to observe cell division and the formation of aggregates containing very few bacteria. The scattered intensity profiles showed the lag phase and the transition to the exponential phase of growth, providing a quantity proportional to viable bacteria concentration. The results revealed a clear and linear correlation in both lag and exponential phase, between the Log10(colony-forming units/mL) from PC and the Log10 of the scattered intensity Is from DLS. These correlations provide a good support to use DLS as an alternative technique to determine bacterial population.

Shock Wave-Induced Damage and Poration in Eukaryotic Cell Membranes.

Shock waves are known to permeabilize eukaryotic cell membranes, which may be a powerful tool for a variety of drug delivery applications. However, the mechanisms involved in shock wave-mediated membrane permeabilization are still poorly understood. In this study, the effects on both the permeability and the ultrastructural features of two human cell lineages were investigated after the application of underwater shock waves in vitro. Scanning Electron Microscopy of cells derived from a human embryo kidney (HEK)-293 and Michigan Cancer Foundation (MCF)-7 cells, an immortalized culture derived from human breast adenocarcinoma, showed a small amount of microvilli (as compared to control cells), the presence of hole-like structures, and a decrease in cell size after shock wave exposure. Interestingly, these effects were accompanied by the permeabilization of acid and macromolecular dyes and gene transfection. Trypan blue exclusion assays indicated that cell membranes were porated during shock wave treatment but resealed after a few seconds. Deformations of the cell membrane lasted for at least 5 min, allowing their observation in fixed cells. For each cell line, different shock wave parameters were needed to achieve cell membrane poration. This difference was correlated to successful gene transfection by shock waves. Our results demonstrate, for the first time, that shock waves induce transient micro- and submicrosized deformations at the cell membrane, leading to cell transfection and cell survival. They also indicate that ultrastructural analyses of cell surfaces may constitute a useful way to match the use of shock waves to different cells and settings.

Efficient transformation of Mycosphaerella fijiensis by underwater shock waves.

Black leaf streak disease, also known as black Sigatoka, causes dramatic losses in production of banana and plantains fruits. The disease is caused by the pathogenic fungus Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis; Mycosphaerellaceae). Genetic transformation of M. fijiensis would allow a better understanding of molecular basis of pathogenicity and design novel approaches to control the infection caused by this pathogen. However, transformation of this fungus has not been easy. We report here a protocol for genetic transformation of M. fijiensis employing underwater shock waves and intact conidia. The recombinant strains recovered showed genetic stability over >10 generations. The frequency of transformation obtained was between 75 and 150 times higher than the efficiency reported in the only article published on transformation of M. fijiensis using spheroplasts. This improvement allowed the use of a thousand times less cells than the amount employed before, avoiding the need for cumbersome successive batch cultures. Our protocol is simple, highly efficient, fast and reproducible and together with the available genomes of M. fijiensis and Musa acuminata, it offers new possibilities to study the diverse mechanisms of pathogenesis of the fungus.

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.

Combined short and long-delay tandem shock waves to improve shock wave lithotripsy according to the Gilmore-Akulichev theory.

Extracorporeal shock wave lithotripsy is a common non-invasive treatment for urinary stones whose fragmentation is achieved mainly by acoustic cavitation and mechanical stress. A few years ago, in vitro and in vivo experimentation demonstrated that such fragmentation can be improved, without increasing tissue damage, by sending a second shock wave hundreds of microseconds after the previous wave. Later, numerical simulations revealed that if the second pulse had a longer full width at half maximum than a standard shock wave, cavitation could be enhanced significantly. On the other side, a theoretical study showed that stress inside the stone can be increased if two lithotripter shock waves hit the stone with a delay of only 20 µs. We used the Gilmore-Akulichev formulation to show that, in principle, both effects can be combined, that is, stress and cavitation could be increased using a pressure pulse with long full width at half maximum, which reaches the stone within hundreds of microseconds after two 20 µs-delayed initial shock waves. Implementing the suggested pressure profile into clinical devices could be feasible, especially with piezoelectric shock wave sources.

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.