Triplex-hybridizing bioconjugated gold nanoparticles for specific Y-chromosome sequence targeting of bull spermatozoa.

Gold nanoparticles (AuNPs) are widely used in biomedical applications for drug targeting and bioimaging. This often neccesitates their functionalization with biomolecules carrying a defined biological function, yielding gold nanoparticle bioconjugates. The utilization of triplex-forming oligonucleotides (TFOs) as ligands gives access to nanoconjugates as tools for specific DNA-related nanotargeting via triplex hybridization. Since triplex hybridization with nanobioconjugates has to date not been shown on biologically relevant samples, sex-specific sperm marking may be an appropriate model system to demonstrate the opportunities of this targeting method in vitro. In this study, we focused on specific labeling of repetitive target sites enriched on the bovine Y-chromosome using triplex forming oligonucleotides. First, the functionality of a specific locked nucleic acid (LNA) sequence was confirmed on bovine free DNA and on demembranated sperm heads. Thereafter, the influence of AuNPs on triplex hybridization was spectrophotometrically analyzed employing synthetic dsDNA, genomic DNA and demembranated sperm heads. Results from the SPR-peak shift indicate that TFO-AuNP hybridize to bovine gDNA in a qualitative and significant manner. These results confirm successful triplex hybridization on biologically relevant target sites as well as the establishment of a method to use gold nanoparticles as a suitable tool for sex-selective hybridization.

Sex-Sorted Boar Sperm - An Update on Related Production Methods.

As in other mammals, sex sorting of pig sperm is based on quantitative flow cytometry. A major disadvantage of the technique is the relatively low efficiency to produce enough sorted sperm for artificial insemination. However, several approaches are on the way to make sexed pig sperm available for commercial application. In this context, for example, the growing field of nanotechnology may significantly contribute to these developments, as it provides highly efficient bio-nanoprobes, for example, based on plasmonic nanoparticles. Independent of the method, further development requires enormous investments and set-up of logistics to get the technology into the practical pig market. Only global players will be able to establish the necessary research projects, but in the end, a significant shift of sex ratios will be available for pig producers as it is already the case for the dairy industry.

Sex selection of sperm in farm animals: status report and developmental prospects.

Pre-selection of spermatozoa based on the relative DNA difference between X- and Y-chromosome bearing populations by flow cytometry is an established method that has been introduced into commercial cattle production. Although several important improvements have increased the sort efficiency, the fertilising ability of sexed spermatozoa based on offspring per insemination is still behind farmers' expectations. The main stress factors, especially on mitochondria, that reduce the lifespan of spermatozoa are described, and new technical as well as biological solutions to maintain the natural sperm integrity and to increase the sorting efficiency are discussed. Among these methods are the identification of Y-chromosome bearing spermatozoa by bi-functionalised gold nanoparticles and triplex hybridisation in vivo as well as new laser-controlled deflection system that replaces the deflection of spermatozoa in the electrostatic field. Additionally, as well as a new nonsurgical transfer system of spermatozoa into the oviduct of cows has been developed and allows a significant reduction of spermatozoa per transfer. Altogether, the improvements made in the recent years will allow a broader use of sex-sorted spermatozoa even in those species that require more cells than cows and sheep.

Cavitation dynamics of laser ablation of bulk and wire-shaped metals in water during nanoparticles production.

Although the first nanoseconds to microseconds rule the resulting process yield of laser ablation in liquid, a comprehensive view involving combination of time-resolved measurement techniques is still lacking. In this paper, fundamental aspects of laser ablation of metals in water during the production of nanoparticles are discussed. Three fast diagnostic methods have been applied simultaneously. These are Optical Emission Spectroscopy for the plasma characterization, fast shadowgraph for plasma and cavitation bubble dynamics and laser scattering for the mechanisms of delivery of the produced materials in the liquid. Moreover, in order to validate the discussion, the effect on cavitation dynamics of the ablation of bulk and wire-shaped targets has been investigated together with the relative nanoparticles production yield. Unusual arrow-bow ejection phenomena between the cavitation bubble and the wire result in suppressed material back-deposition, causing efficient ejection of ablated matter into the liquid. The presented nanosecond and microsecond-resolved analysis allows estimating the timescale and role of the basic mechanisms involved in laser ablation in liquids as well as the thermodynamic characteristics of the processes.

Impact of spacer and strand length on oligonucleotide conjugation to the surface of ligand-free laser-generated gold nanoparticles.

Gold nanoparticles conjugated to nucleic acids are widely used for biomedical targeting and sensing applications; however, little is known about the conjugation chemistry covering the impact of steric dimension and strand orientation of single-stranded oligonucleotides (ssO) on the conjugation process and binding efficiencies. In this context, we present an extensive investigation concerning the attachment of thiolated ssO to the surface of laser-generated gold nanoparticles, altering both strand length and binding orientation by the insertion of different spacer types at either the 3' or 5' ssO terminus. A significant reduction of conjugation efficiency of about 30-50% is determined for spacer-prolonged bionanoconjugates due to coiling effects of the flexible ssO strand on the particle surface which increases deflection angle of oligonucleotides and limits the number of biomolecules attached to the nanoparticles.

Toxicity of gold nanoparticles on somatic and reproductive cells.

Along with the number of potential applications for gold nanoparticles (AuNP) especially for medical and scientific purposes, the interest in possible toxic effects of such particles is rising. The general perception views nanosized gold colloids as relatively inert towards biological systems. However, a closer analysis of pertinent studies reveals a more complex picture. While the chemical compound of which the nanoparticles consists plays an important role, further biocompatibility determining aspects have been made out. The vast majority of trials concerning AuNP-toxicity were performed using somatic cell culture lines. The results show a considerable dependency of toxic effects on size, zeta potential and surface functionalisation. In vivo studies on this subject are still rare. Based on the existing data it can be assumed, that a dosage of under <400 µg Au/kg showed no untoward effects. If higher amounts were applied toxicity depended on route of administration and particle size. Since nanoparticles have been shown to cross reproduction-relevant biological barriers such as the blood-testicle and the placental barrier the question of their reprotoxicity arises. Yet data concerning this subject is far from adequate. Regarding gametes, recent experiments showed a dose-dependent sensitivity of spermatozoa towards AuNP. Oocytes have not yet been tested in that respect. Interestingly, so far no effects were detected on embryos after gold nanoparticle exposure. In conclusion, the biocompatibility of gold nanoparticles depends on a range of particle specific aspects as well as the choice of target tissue. Further clarification of such matters are subject to ongoing research.

Impact of metal nanoparticles on germ cell viability and functionality.

Metal nanoparticles play an increasing role in consumer products, biomedical applications and in the work environment. Therefore, the effects of nanomaterials need to be properly understood. This applies especially to their potential reproductive toxicology (nanoreprotoxicity), because any shortcomings in this regard would be reflected into the next generation. This review is an attempt to summarize the current knowledge regarding the effects of nanoparticles on reproductive outcomes. A comprehensive collection of significant experimental nanoreprotoxicity data is presented, which highlight how the toxic effect of nanoparticles can be influenced, not only by the particles' chemical composition, but also by particle size, surface modification, charge and to a considerable extent on the experimental set-up. The period around conception is characterized by considerable cytological and molecular restructuring and is therefore particularly sensitive to disturbances. Nanoparticles are able to penetrate through biological barriers into reproductive tissue and at least can have an impact on sperm vitality and function as well as embryo development. Particularly, further investigations are urgently needed on the repetitively shown effect of the ubiquitously used titanium dioxide nanoparticles on the development of the nervous system. It is recommended that future research focuses more on the exact mechanism behind the observed effects, because such information would facilitate the production of nanoparticles with increased biocompatibility.

The radiosensitizing effect of platinum nanoparticles in proton irradiations is not caused by an enhanced proton energy deposition at the macroscopic scale.

Objective.Due to the radiosensitizing effect of biocompatible noble metal nanoparticles (NPs), their administration is considered to potentially increase tumor control in radiotherapy. The underlying physical, chemical and biological mechanisms of the NPs' radiosensitivity especially when interacting with proton radiation is not conclusive. In the following work, the energy deposition of protons in matter containing platinum nanoparticles (PtNPs) is experimentally investigated.Approach.Surfactant-free monomodal PtNPs with a mean diameter of (40 ± 10) nm and a concentration of 300 µg ml-1, demonstrably leading to a substantial production of reactive oxygen species (ROS), were homogeneously dispersed into cubic gelatin samples serving as tissue-like phantoms. Gelatin samples without PtNPs were used as control. The samples' dimensions and contrast of the PtNPs were verified in a clinical computed tomography scanner. Fields from a clinical proton machine were used for depth dose and stopping power measurements downstream of both samples types. These experiments were performed with a variety of detectors at a pencil beam scanning beam line as well as a passive beam line with proton energies from about 56-200 MeV.Main results.The samples' water equivalent ratios in terms of proton stopping as well as the mean proton energy deposition downstream of the samples with ROS-producing PtNPs compared to the samples without PtNPs showed no differences within the experimental uncertainties of about 2%.Significance.This study serves as experimental proof that the radiosensitizing effect of biocompatible PtNPs is not due to a macroscopically increased proton energy deposition, but is more likely caused by a catalytic effect of the PtNPs. Thus, these experiments provide a contribution to the highly discussed radiobiological question of the proton therapy efficiency with noble metal NPs and facilitate initial evidence that the dose calculation in treatment planning is straightforward and not affected by the presence of sensitizing PtNPs.

Effects of metal ions on fibroblasts and spiral ganglion cells.

Degeneration of spiral ganglion cells (SGC) after deafness and fibrous tissue growth around the electrode carrier after cochlear implantation are two of the major challenges in current cochlear implant research. Metal ions are known to possess antimicrobial and antiproliferative potential. The use of metal ions could therefore provide a way to reduce tissue growth around the electrode array after cochlear implantation. Here, we report on in vitro experiments with different concentrations of metal salts with antiproliferative and toxic effects on fibroblasts, PC-12 cells, and freshly isolated spiral ganglion cells, the target cells for electrical stimulation by a cochlear implant. Standard cell lines (NIH/3T3 and L-929 fibroblasts and PC-12 cells) and freshly isolated SGC were incubated with concentrations of metal ions between 0.3 µmol/liter and 10 mmol/liter for 48 hr. Cell survival was investigated by neutral red uptake, CellQuantiBlue assay, or counting of stained surviving neurons. Silver ions exhibited distinct thresholds for proliferating and confluent cells. For zinc ions, the effective concentration was lower for fibroblasts than for PC-12 cells. SGC showed comparable thresholds for reduced cell survival not only for silver and zinc ions but also for copper(II) ions, indicating that these ions might be promising for reducing tissue growth on the surface of CI electrode arrays. These effects were also observed when combinations of two of these ions were investigated.

Golden perspective: application of laser-generated gold nanoparticle conjugates in reproductive biology.

The current demand for female calves has grown rapidly and controlling the sex of offspring provides an economically flexible management for the livestock producer. The only functioning method of efficiently producing separate populations of X and Y sperm in mammals is based on relative DNA differentiation by high-speed flow cytometry. In this context, gold nanoparticles conjugated to sex chromosome-specific moieties display promising application as novel fluorophor-alternative for the high-throughput screening, since they feature no photo bleaching, high quantum yield, good biocompatibility and the possibility of non-destructive membrane penetration. Especially, gold nanoparticles fabricated by pulsed laser ablation are in the recent focus of interest, due to excellent biocompatibility, fabrication-dependent, tuneable particle size as well as surface charge and ease of (bio)-functionalization with a remarkably strong ligand binding. For the purpose of our studies functionalized gold nanoparticles may be used as novel markers for sex-sorting of mammalian sperm and, depending on the selected probe, also for the selection of sperm with heritable DNA-sequences interesting for animal breeding.

Increasing the Size-Selectivity in Laser-Based g/h Liquid Flow Synthesis of Pt and PtPd Nanoparticles for CO and NO Oxidation in Industrial Automotive Exhaust Gas Treatment Benchmarking.

PtPd catalysts are state-of-the-art for automotive diesel exhaust gas treatment. Although wet-chemical preparation of PtPd nanoparticles below 3 nm and kg-scale synthesis of supported PtPd/Al2O3 are already established, the partial segregation of the bimetallic nanoparticles remains an issue that adversely affects catalytic performance. As a promising alternative, laser-based catalyst preparation allows the continuous synthesis of surfactant-free, solid-solution alloy nanoparticles at the g/h-scale. However, the required productivity of the catalytically relevant size fraction <10 nm has yet to be met. In this work, by optimization of ablation and fragmentation conditions, the continuous flow synthesis of nanoparticles with a productivity of the catalytically relevant size fraction <10 nm of >1 g/h is presented via an in-process size tuning strategy. After the laser-based preparation of hectoliters of colloid and more than 2 kg of PtPd/Al2O3 wash coat, the laser-generated catalysts were benchmarked against an industry-relevant reference catalyst. The conversion of CO by laser-generated catalysts was found to be equivalent to the reference, while improved activity during NO oxidation was achieved. Finally, the present study validates that laser-generated catalysts meet the size and productivity requirements for industrial standard operating procedures. Hence, laser-based catalyst synthesis appears to be a promising alternative to chemical-based preparation of alloy nanoparticles for developing industrial catalysts, such as those needed in the treatment of exhaust gases.

Cardiorespiratory function before and after operation for pectus excavatum: medium-term results.

OBJECTIVE: The study was undertaken in order to assess the degree of impairment of the cardiovascular system in patients with funnel chest and to investigate any changes caused by surgical correction. METHODS: Echocardiographic examinations with systolic, diastolic and ejection volume indices calculation as well as pulmonary function tests were performed before surgery and at medium-term follow-up on 34 patients who were operated on for pectus excavatum between 1987 and 1992. RESULTS: The mean age was 13.4 years. There were 70.6% males. Pulmonary function was found to be restricted preoperatively in 18 patients. Inspiratory vital capacity and forced expiratory volume were increased or did not change at follow-up (5 years) in these patients. In cases with normal or moderately restricted pulmonary function (inspiratory vital capacity, forced expiratory volume more than 75% predicted) the reduction of lung function was noted after surgery. Marked haemodynamic improvement was found with the increase of diastolic and ejection volume of both heart ventricles (mainly right one). The improvement was more evident in patients with severe deformations. CONCLUSION: Only in case of severe reduction of lung function in a patient with funnel chest can one expect improvement after surgery. Sternocostal elevation improves function of both heart ventricles at rest.

Right ventricular morphology and function after pulmonary resection.

OBJECTIVE: To identify the effect of pulmonary resection on right ventricular performance and its possible contribution to mortality and morbidity. METHODS: Before and 2 days after pulmonary resection for primary lung cancer in 31 patients (21 males; ages 32-69 years), echocardiographic examinations of the right ventricle were performed. Systolic, diastolic and stroke volumes as well as right ventricular ejection fraction were estimated. Right ventricular volumes were calculated using the subtracting method. RESULTS: Right ventricular end-diastolic volume index increased significantly in patients after pneumonectomy: 80.4+/-7.2 ml/m2 versus preoperative evaluation: 66.1+/-5.2 ml/m2 (P = 0.031). In patients who underwent pneumonectomy right ventricular ejection fraction significantly decreased from 48+/-5.0% preoperatively to 39%+/-4.1% after surgery (P = 0.027). Fourteen patients after pneumonectomy had development of supraventricular arrhythmias postoperatively. These patients had much higher right ventricular end-diastolic volume index (76.3+/-6.4/82.1+/-7.4; P = 0.032) and lower right ventricular ejection fraction (42+/-4.3/37+/-3.9; P = 0.021) after surgery in comparison with patients who had normal sinus rhythm postoperatively. CONCLUSION: Pulmonary resection caused a significant dilatation and dysfunction of right ventricle in the early postoperative period. Early detection of deterioration in right ventricular function after pneumonectomy may provide the opportunity for interventional therapy.

[Heart injury caused by air shock wave trauma]. / Obrazenia serca spowodowane przez bezposrednie dzialanie powietrznej fali underzeniowej.

The aim of the study was the estimation of heart muscle in response to air shock wave trauma. The animals were divided into four groups according to the time between trauma and the examination (3, 24, 48 and 120 hours). In all groups of animals ecg was performed and plasma level of creatinine kinase (CK), cardiac isoenzymes (CK-MB), lactic dehydrogenase (LDH) and its isoenzymes (LDH1-2) and alpha hydroxybutyrate dehydrogenase (HBDH) were determined. CK-MB activity as a percent of CK activity and rate of LDH isoenzymes activity were calculated. Morphologic estimation of heart injury based on macroscopic observation, histology and coronarography of heart specimens was performed. It was revealed that pathologic changes in ecg indicating intracardial conduction disturbances occur most intensively in first ten minutes after trauma. Plasma CPK, LDH and LDH1-2 activity increased significantly after 120 hrs following trauma. Morphologic manifestation of heart damage was multifocal injury of heart muscle.

[Therapeutic management of massive hemorrhage caused by gastroduodenal ulcer]. / Conduite thérapeutique dans l'hémorragie massive de l'ulcére gastro-duodénal.

Based on 103 patients the authors discussed policy of therapeutic management in gastric and duodenal ulcer disease complicated by massive hemorrhage. In the surgical treatment of such patients the authors prefer resection procedures aimed at simultaneous control of bleeding, prevention of its relapse and radical treatment of peptic ulcer disease.

[Effect of correcting funnel chest on respiratory function]. / Wplyw korekcji lejkowatej klatki piersiowej na czynnosc ukladu oddechowego.

The authors discuss the results of pulmonary function tests performed at rest in 25 patients treated for pectus excavatum. Surgical correction of the lesion was performed by resection of the deformed costal cartilages, longitudinal and transversal sternotomy, stabilization of the anterior chest wall with Kirschner wires. Control tests carried out 6 months following the surgical correction. In cases of severe deformation of the chest wall a decrease of pulmonary function indices was evident. Following the surgical correction all spirometric indices increased.

[Use and examination of unwoven fabrics in the production of dressings, surgical and bed linens]. / Ocena wlasciwosci wlóknin stosowanych do wytwarzania srodków opatrunkowych, odziezy chirurgicznej, bielizny operacyjnej i poscielowej oraz badania uzytkowe tych wyrobów.

The results of physicochemical examination of 32 kinds of unwoven fabrics as well as the examination of 7 kinds of the three-level dressings, operation, and bed clothes are reported. The fabrics consisted of polypropylene and viscose fibres, dilana, argona, polyamides and bonding agents (fixatives). In their water extracts the content of the chloride, sulphate and alkaline ions, reductors, the presence of heavy metals, the surface active substances and the pH were determined. The practical applicability of the dressings was tested in 300 cases. The dressings were well tolerated by the patients. The operation and bed clothes as well as the surgical clothes made from unwoven fabric and sterilized by radiosterilization may facilitate, the great deal of work in the operation units and in surgical wards.