Dynamics of single cell femtosecond laser printing.

In the present study, we investigated the dynamics of a femtosecond (fs) laser induced bio-printing with cell-free and cell-laden jets under the variation of laser pulse energy and focus depth, by using time-resolved imaging. By increasing the laser pulse energy or decreasing the focus depth thresholds for a first and second jet are exceeded and more laser pulse energy is converted to kinetic jet energy. With increasing jet velocity, the jet behavior changes from a well-defined laminar jet, to a curved jet and further to an undesired splashing jet. We quantified the observed jet forms with the dimensionless hydrodynamic Weber and Rayleigh numbers and identified the Rayleigh breakup regime as the preferred process window for single cell bioprinting. Herein, the best spatial printing resolution of 42 ± 3 µm and single cell positioning precision of 12.4 µm are reached, which is less than one single cell diameter about 15 µm.

Comparison of ultrashort pulse ablation of gold in air and water by time-resolved experiments.

Laser ablation in liquids is a highly interdisciplinary method at the intersection of physics and chemistry that offers the unique opportunity to generate surfactant-free and stable nanoparticles from virtually any material. Over the last decades, numerous experimental and computational studies aimed to reveal the transient processes governing laser ablation in liquids. Most experimental studies investigated the involved processes on timescales ranging from nanoseconds to microseconds. However, the ablation dynamics occurring on a sub-nanosecond timescale are of fundamental importance, as the conditions under which nanoparticles are generated are established within this timeframe. Furthermore, experimental investigations of the early timescales are required to test computational predictions. We visualize the complete spatiotemporal picosecond laser-induced ablation dynamics of gold immersed in air and water using ultrafast pump-probe microscopy. Transient reflectivity measurements reveal that the water confinement layer significantly influences the ablation dynamics on the entire investigated timescale from picoseconds to microseconds. The influence of the water confinement layer includes the electron injection and subsequent formation of a dense plasma on a picosecond timescale, the confinement of ablation products within hundreds of picoseconds, and the generation of a cavitation bubble on a nanosecond timescale. Moreover, we are able to locate the temporal appearance of secondary nanoparticles at about 600 ps after pulse impact. The results support computational predictions and provide valuable insight into the early-stage ablation dynamics governing laser ablation in liquids.

Extending Single Cell Bioprinting from Femtosecond to Picosecond Laser Pulse Durations.

Femtosecond laser pulses have been successfully used for film-free single-cell bioprinting, enabling precise and efficient selection and positioning of individual mammalian cells from a complex cell mixture (based on morphology or fluorescence) onto a 2D target substrate or a 3D pre-processed scaffold. In order to evaluate the effects of higher pulse durations on the bioprinting process, we investigated cavitation bubble and jet dynamics in the femto- and picosecond regime. By increasing the laser pulse duration from 600 fs to 14.1 ps, less energy is deposited in the hydrogel for the cavitation bubble expansion, resulting in less kinetic energy for the jet propagation with a slower jet velocity. Under appropriate conditions, single cells can be reliably transferred with a cell survival rate after transfer above 95% through the entire pulse duration range. More cost efficient and compact laser sources with pulse durations in the picosecond range could be used for film-free bioprinting and single-cell transfer.

The free vascularized lateral femoral trochlea osteochondral graft: a reliable alternative for Stage III Kienböck's disease.

Lunate reconstruction using a lateral femoral trochlea osteochondral graft was carried out in 27 patients with Stage III Kienböck's disease from 2012 to 2019. Twenty-three of these patients could be followed-up in this retrospective study. Ten were women and 13 men. Nine were Lichtman Stage IIIA, seven Stage IIIB and seven Stage IIIC. The mean follow-up was 39 months (range 12-86). Bony consolidation was found in 18 of the 23 patients, with no graft loss. The mean Disabilities of the Arm, Shoulder and Hand score (DASH score) was 11 and the Modified Mayo Wrist Score was 83. There were only two radiological deteriorations, with the same or improved Lichtman classifications in the other patients and a significant reduction in pain. Postoperative extension of the wrist (52°) and flexion (48°) were comparable with preoperative values and, respectively, 81% and 72% of the contralateral side. Grip strength and pinch grip were 32 kg and 12 kg, 88% and 94% of the other hand, respectively, and an insignificant increase compared with the preoperative values. The vascularized lateral femoral trochlea osteochondral graft yields good short- and mid-term results in Grade III Kienböck's disease.Level of evidence: IV.

Ultrashort single-pulse laser ablation of stainless steel, aluminium, copper and its dependence on the pulse duration.

In this work, we investigate single-pulse laser ablation of bulk stainless steel (AISI304), aluminium (Al) and copper (Cu) and its dependence on the pulse duration. We measured the reflectivity, ablation thresholds and volumes under the variation of pulse duration and fluence. The known drop of efficiency with increasing pulse duration is confirmed for single-pulse ablation in all three metals. We attribute the efficiency drop to a weakened photomechanically driven ablation process and a stronger contribution of photothermal phase explosion. The highest energetic efficiency and precision is achieved for pulse durations below the mechanical expansion time of 3-5 ps, where the stress confinement condition is fulfilled.

Directed Transport of CRP Across In Vitro Models of the Blood-Saliva Barrier Strengthens the Feasibility of Salivary CRP as Biomarker for Neonatal Sepsis.

C-reactive protein (CRP) is a commonly used serum biomarker for detecting sepsis in neonates. After the onset of sepsis, serial measurements are necessary to monitor disease progression; therefore, a non-invasive detection method is beneficial for neonatal well-being. While some studies have shown a correlation between serum and salivary CRP levels in septic neonates, the causal link behind this correlation remains unclear. To investigate this relationship, CRP was examined in serum and saliva samples from 18 septic neonates and compared with saliva samples from 22 healthy neonates. While the measured blood and saliva concentrations of the septic neonates varied individually, a correlation of CRP levels between serum and saliva samples was observed over time. To clarify the presence of active transport of CRP across the blood-salivary barrier (BSB), transport studies were performed with CRP using in vitro models of oral mucosa and submandibular salivary gland epithelium. The results showed enhanced transport toward saliva in both models, supporting the clinical relevance for salivary CRP as a biomarker. Furthermore, CRP regulated the expression of the receptor for advanced glycation end products (RAGE) and the addition of soluble RAGE during the transport studies indicated a RAGE-dependent transport process for CRP from blood to saliva.

Anterior open bite - diagnostics and therapy.

An anterior open bite (AOB) is an occlusal disorder that causes the patient both an aesthetic and functional handicap. The lower third of the face is disproportionately larger. Patients are unable to properly occlude with the anterior part of dental arch and occlusion only happens in the premolar and/or molar regions. An anterior open bite may be the result of anatomical anomalies. Long term stability as well as an immediate outcome of the surgery depends on the choice of a suitable treatment strategy. In this article, we review options of AOB treatment, from classical orthodontic treatment to current combined orthodontic and surgical approach with a benefit of an anchor system.

Paradise lost? Pesticide pollution in a European region with considerable amount of traditional agriculture.

Pesticide contamination of agricultural streams has widely been analysed in regions of high intensity agriculture such as in Western Europe or North America. The situation of streams subject to low intensity agriculture relying on human and animal labour, as in parts of Romania, remains unknown. To close this gap, we determined concentrations of 244 pesticides and metabolites at 19 low-order streams, covering sites from low to high intensity agriculture in a region of Romania. Pesticides were sampled with two passive sampling methods (styrene-divinylbenzene (SDB) disks and polydimethylsiloxane (PDMS) sheets) during three rainfall events and at base flow. Using the toxic unit approach, we assessed the toxicity towards algae and invertebrates. Up to 50 pesticides were detected simultaneously, resulting in sum concentrations between 0.02 and 37 µg L-1. Both, the sum concentration as well as the toxicities were in a similar range as in high intensity agricultural streams of Western Europe. Different proxies of agricultural intensity did not relate to in-stream pesticide toxicity, contradicting the assumption of previous studies. The toxicity towards invertebrates was positively related to large scale variables such as the catchment size and the agricultural land use in the upstream catchment and small scale variables including riparian plant height, whereas the toxicity to algae showed no relationship to any of the variables. Our results suggest that streams in low intensity agriculture, despite a minor reported use of agrochemicals, exhibit similar levels of pesticide pollution as in regions of high intensity agriculture.

INDICATION AND IMPORTANCE OF RECONSTRUCTIVE SURGERIES OF FACIAL SKELETON IN MAXILLOFACIAL SURGERY: REVIEW.

Malignant head and neck tumors belong among common diseases and their incidence constantly rises. In the Czech Republic, the proportional representation of orofacial tumors ranges around 2% of the total number of malignancies. Rational treatment of these tumors is complex and long. In the course of therapeutic planning, you have to consider the age of the patient and the stage of the disease including the presence of distant metastases. Removal of the tumor with a sufficient safety margin and an eventual treatment of the relevant lymphatic system according to the type of the tumor is an important prerequisite for the success of the surgical therapy. Reconstructive procedures in maxillofacial oncosurgery presume good interdisciplinary cooperation and a high professional preparedness of the surgical and nursing team. Selection of the right patient is also very important with regard to the risks of both local and systemic postoperative complications. Use of the free flap techniques is currently the gold standard, but it is also necessary to master pedicled flap techniques, whose advantages lie in simpler technique and often better aesthetic results. At the same time, we have to realize that even traditional, classical reconstructive procedures using prosthetic replacements can still represent the ideal solution in many cases.

SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication.

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.

HIRUDOTHERAPY IN RECONSTRUCTIVE SURGERY: CASE-REPORTS AND REVIEW.

Medicinal leeches (Hirudo medicinalis, Hirudo verbana) have been used in the field of medicine to treat various diseases for thousands of years. Popularity of their use changed over time and in Europe, it peaked at the beginning of the 19th century. In modern medicine, application of leeches on flaps with venous congestion was first used and described by Deganc and Zdravic in 1960. A certain renaissance of leech use is currently taking place, especially in the field of reconstructive surgery. In general, use of leeches is indicated during critical post-operative period, in which the microcirculation and veins are incapable of sufficient drainage of venous blood, which can lead to stagnation of circulation in tissues at all levels, clinically manifested as a change in color and turgor of the flap. If this venostasis is not recognized in time and treated adequately, tissue necrosis can develop. Medicinal leeches can be used in venous drainage disorders after a replantation of fingers, auricles, lips and parts of the nose. In head and neck reconstructive surgery, there are many studies that confirm the success rate of hirudotherapy in hematoma evacuation or in dealing with complications after scalp replantation and transfers of free and pedicled flaps. Leech application therapy can also be indicated as a part of non-surgical methods that improve conditions of the venous system.

Wide-scope target screening of >2000 emerging contaminants in wastewater samples with UPLC-Q-ToF-HRMS/MS and smart evaluation of its performance through the validation of 195 selected representative analytes.

This study presents the development and validation of a comprehensive quantitative target methodology for the analysis of 2316 emerging pollutants in water based on Ultra-Performance Liquid Chromatography Quadrupole-Time-Of-Flight Mass Spectrometry (UPLC-Q-ToF-HRMS/MS). Target compounds include pesticides, pharmaceuticals, drugs of abuse, industrial chemicals, doping compounds, surfactants and transformation products, among others. The method was validated for 195 analytes, chosen to be representative of the chemical space of the target list, enabling the assessment of the performance of the method. The method involves a generic sample preparation based on mixed mode solid phase extraction, a UPLC-QTOF-MS/MS screening method using Data Independent Acquisition (DIA) mode, which provides MS and MS/MS spectra simultaneously and an elaborate strong post-acquisition evaluation of the data. The processing method was optimized to provide a successful identification rate >95 % and to minimize the number of false positive results (< 5 %). Decision limit (CCα) and detection capability (CCß) were also introduced in the validation scheme to provide more realistic metrics on the performance of a HRMS-based wide-scope screening method. A new system of identification points (IPs) based on the one described in the Commission Decision 2002/657/EC was applied to communicate the confidence level in the identification of the analytes. This system considers retention time, mass accuracy, isotopic fit and fragmentation; taking full advantage of the capacities of the HRMS instruments. Finally, 398 contaminants were detected and quantified in real wastewater.

Pedicled pectoralis major flap in head and neck reconstruction - technique and overview.

The pedicled pectoralis major flap was the original workhorse flap for head and neck reconstruction. Over time, it became the secondary choice for oropharyngeal reconstruction with the implementation of free-soft tissue transfers. Nowadays, a polymorbid patient is primarily indicated for pedicled pectoralis major flap reconstruction, other indications include combinations of pedicled pectoralis major flap with free microvascular flap, salvage reconstruction due to complications, salvage reconstruction due to free flap failure and salvage reconstruction due to recurrent or extended primary disease. Pedicled pectoralis major flap can be successfully used for specific oropharyngeal defects, even primary resections, especially for less cooperative patients and patients after extensive neck dissection. Improving the flap harvesting techniques can reduce undesired complications in specific cases of oropharyngeal reconstruction. Flap morbidity in these cases remains comparable to morbidity of patients who had undergone free flap reconstruction. Pedicled pectoralis major flap remains valid reconstruction tool that should be included in the armamentarium of each surgeon dealing with reconstruction of the head and neck.

Pedicled pectoralis major flap in head and neck reconstruction - our experience.

INTRODUCTION: The pedicled pectoralis major flaps are still harvested and mainly indicated for reconstruction in the polymorbid patient. Other indications are combinations of pedicled pectoralis major flaps with free microvascular flap, salvage reconstruction following complications, free flap failure and recurrent or extended primary disease. MATERIALS AND METHODS: We describe the pedicled pectoralis major flap in 18 patients operated on at the Department of Oral and Maxillofacial Surgery, Olomouc from 1st January 2014 to 1st December 2016. RESULTS: Fifteen oropharyngeal defect reconstructions were performed using pedicled pectoralis major flap (including 1 submandibular defect of the neck). Indications were primary resection in polymorbid patients in 10 cases and recurrent diseases after previous neck dissection and radiotherapy in 5 cases. Pedicled pectoralis major flap was used for secondary reconstruction in 3 cases. Complications occurred in 50 % of patients, 28 % were major and 22 % minor. Major complications included a total flap failure (defect was successfully treated with free tissue transfer of latissimus dorsi myocutaneous flap) in 1 case, plate exposure in 2 cases, large dehiscence and large hemorrhage 1 case each. Minor complications included only small dehiscences (22 %). One was associated with fluidothorax after rib harvesting (6%). There were no cases of neck contracture or supraclavicular bulge. CONCLUSION: Even today, usage the pedicled pectoralis major flap in head and neck reconstruction surgery cannot be considered as an obsolete reconstructive procedure that has been completely replaced by a free microvascular flap. Innovations of flap harvesting techniques and high rate of flap survival are the main reasons why pedicled pectoralis major flap can still be primarily indicated for high-risk patients, non-cooperative patients and also for patients with extensive neck dissection. The pedicled pectoralis major flap has been the first choice in salvage surgery, in cases of a complication or free flap failure or a recurrence of a primary disease.

Reconstruction Strategies of the Ureter and Urinary Diversion Using Tissue Engineering Approaches.

IMPACT STATEMENT: Tissue Engineering (TE) approaches are needed to advance the field of reconstructive urology. We indicate that regeneration of ureteral tissue and the formation of a urinary diversion using TE approaches are possible, although it is currently very time-consuming and complex to achieve well-developed neotissue. Faster regeneration approaches using novel scaffolds are desirable. The findings of this review may help to develop smart hybrid scaffolds and enhance the design of future studies, which may ultimately lead to improved care for patients with ureteral defects as well as to curb complications associated with urinary diversion.

Wastewater treatment plant resistomes are shaped by bacterial composition, genetic exchange, and upregulated expression in the effluent microbiomes.

Wastewater treatment plants (WWTPs) are implicated as hotspots for the dissemination of antibacterial resistance into the environment. However, the in situ processes governing removal, persistence, and evolution of resistance genes during wastewater treatment remain poorly understood. Here, we used quantitative metagenomic and metatranscriptomic approaches to achieve a broad-spectrum view of the flow and expression of genes related to antibacterial resistance to over 20 classes of antibiotics, 65 biocides, and 22 metals. All compartments of 12 WWTPs share persistent resistance genes with detectable transcriptional activities that were comparatively higher in the secondary effluent, where mobility genes also show higher relative abundance and expression ratios. The richness and abundance of resistance genes vary greatly across metagenomes from different treatment compartments, and their relative and absolute abundances correlate with bacterial community composition and biomass concentration. No strong drivers of resistome composition could be identified among the chemical stressors analyzed, although the sub-inhibitory concentration (hundreds of ng/L) of macrolide antibiotics in wastewater correlates with macrolide and vancomycin resistance genes. Contig-based analysis shows considerable co-localization between resistance and mobility genes and implies a history of substantial horizontal resistance transfer involving human bacterial pathogens. Based on these findings, we propose future inclusion of mobility incidence (M%) and host pathogenicity of antibiotic resistance genes in their quantitative health risk ranking models with an ultimate goal to assess the biological significance of wastewater resistomes with regard to disease control in humans or domestic livestock.

The Impact of γ-Irradiation and EtO Degassing on Tissue Remodeling of Collagen-based Hybrid Tubular Templates.

Clinical implementation of novel products for tissue engineering and regenerative medicine requires a validated sterilization method. In this study, we investigated the effect of γ-irradiation and EtO degassing on material characteristics in vitro and the effect on template remodeling of hybrid tubular constructs in a large animal model. Hybrid tubular templates were prepared from type I collagen and Vicryl polymers and sterilized by 25 kGray of γ-irradiation or EtO degassing. The in vitro characteristics were extensively studied, including tensile strength analysis and degradation studies. For in vivo evaluation, constructs were subcutaneously implanted in goats for 1 month to form vascularized neo-tissue. Macroscopic and microscopic appearances of the γ- and EtO-sterilized constructs slightly differed due to additional processing required for the COL-Vicryl-EtO constructs. Regardless of the sterilization method, incubation in urine resulted in fast degradation of the Vicryl polymer and decreased strength (<7 days). Incubation in SBF was less invasive, and strength was maintained for at least 14 days. The difference between the two sterilization methods was otherwise limited. In contrast, subcutaneous implantation showed that the effect of sterilization was considerable. A well-vascularized tube was formed in both cases, but the γ-irradiated construct showed an organized architecture of vasculature and was mechanically more comparable to the native ureter. Moreover, the γ-irradiated construct showed advanced tissue remodeling as shown by enhanced ECM production. This study shows that the effect of sterilization on tissue remodeling cannot be predicted by in vitro analyses alone. Thus, validated sterilization methods should be incorporated early in the development of tissue engineered products, and this requires both in vitro and in vivo analyses.

Labeling of Collagen Type I Templates with a Naturally Derived Contrast Agent for Noninvasive MR Imaging in Soft Tissue Engineering.

In vivo monitoring of tissue-engineered constructs is important to assess their integrity, remodeling, and degradation. However, this is challenging when the contrast with neighboring tissues is low, necessitating labeling with contrast agents (CAs), but current CAs have limitations (i.e., toxicity, negative contrast, label instability, and/or inappropriate size). Therefore, a naturally derived hemin-L-lysine (HL) complex is used as a potential CA to label collagen-based templates for magnetic resonance imaging (MRI). Labeling does not change the basic characteristics of the collagen templates. When hybrid templates composed of collagen type I reinforced with degradable polymers are subcutaneously implanted in mice, longitudinal visualization by MRI is possible with good contrast and in correlation with template remodeling. In contrast, unlabeled collagen templates are hardly detectable and the fate of these templates cannot be monitored by MRI. Interestingly, tissue remodeling and vascularization are enhanced within HL-labeled templates. Thus, HL labeling is presented as a promising universal imaging marker to label tissue-engineered implants for MRI, which additionally seems to accelerate tissue regeneration.

Ultrafast dynamics of hard tissue ablation using femtosecond-lasers.

Several studies on hard tissue laser ablation demonstrated that ultrafast lasers enable precise material removal without thermal side effects. Although the principle ablation mechanisms have been thoroughly investigated, there are still open questions regarding the influence of material properties on transient dynamics. In this investigation, we applied pump-probe microscopy to record ablation dynamics of biomaterials with different tensile strengths (dentin, chicken bone, gallstone and kidney stones) at delay times between 1 picosecond and 10 microseconds. Transient reflectivity changes, pressure and shock wave velocities and elastic constants were determined. The result revealed that absorption and excitation show the typical well-known transient behavior of dielectric materials. We observed for all samples a photomechanical laser ablation process, where ultrafast expansion of the excited volume generates pressure waves leading to fragmentation around the excited region. In addition, we identified tensile-strength-related differences in the size of ablated craters and ejected particles. The elastic constants derived were in agreement with literature values. In conclusion, pressure-wave-assisted material removal seems to be a general mechanism for hard tissue ablation with ultrafast lasers. This photomechanical process increases ablation efficiency and removes heated material, thus ultrafast laser ablation is of interest for clinical application where heating of the tissue must be avoided.