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.

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.

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.

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.

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.

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.

Nontarget Screening with High Resolution Mass Spectrometry in the Environment: Ready to Go?

The vast, diverse universe of organic pollutants is a formidable challenge for environmental sciences, engineering, and regulation. Nontarget screening (NTS) based on high resolution mass spectrometry (HRMS) has enormous potential to help characterize this universe, but is it ready to go for real world applications? In this Feature article we argue that development of mass spectrometers with increasingly high resolution and novel couplings to both liquid and gas chromatography, combined with the integration of high performance computing, have significantly widened our analytical window and have enabled increasingly sophisticated data processing strategies, indicating a bright future for NTS. NTS has great potential for treatment assessment and pollutant prioritization within regulatory applications, as highlighted here by the case of real-time pollutant monitoring on the River Rhine. We discuss challenges for the future, including the transition from research toward solution-centered and robust, harmonized applications.

Tubular Constructs as Artificial Urinary Conduits.

PURPOSE: A readily available artificial urinary conduit might be substituted for autologous bowel in standard urinary diversions and minimize bowel associated complications. However, the use of large constructs remains challenging as host cellular ingrowth and/or vascularization is limited. We investigated large, reinforced, collagen based tubular constructs in a urinary diversion porcine model and compared subcutaneously pre-implanted constructs to cell seeded and basic constructs. MATERIALS AND METHODS: Reinforced tubular constructs were prepared from type I collagen and biodegradable Vicryl® meshes through standard freezing, lyophilization and cross-linking techniques. Artificial urinary conduits were created in 17 female Landrace pigs, including 7 with a basic untreated construct, 5 with a construct seeded with autologous urothelial and smooth muscle cells, and 5 with a free graft formed by subcutaneous pre-implantation of a basic construct. All pigs were evaluated after 1 month. RESULTS: The survival rate was 94%. At evaluation 1 basic and 1 cell seeded conduit were occluded. Urinary flow was maintained in all conduits created with pre-implanted constructs. Pre-implantation of the basic construct resulted in a vascularized tissue tube, which could be used as a free graft to create an artificial conduit. The outcome was favorable compared to that of the other conduits. Urinary drainage was better, hydroureteronephrosis was limited and tissue regeneration was improved. CONCLUSIONS: Subcutaneous pre-implantation of a basic reinforced tubular construct resulted in a vascularized autologous tube, which may potentially replace bowel in standard urinary diversions. To our knowledge we introduce a straightforward 2-step procedure to create artificial urinary conduits in a large animal model.

Ultrafast pump-probe ellipsometry setup for the measurement of transient optical properties during laser ablation.

Ultrashort pulsed lasers offer a high potential in precise and efficient material processing and deep understanding of the fundamental laser-material interaction aspects is of great importance. The transient pulse reflectivity in conjunction with the transient absorption influences decisively the laser-material interaction. Direct measurements of the absorption properties by ultrafast time-resolved ellipsometry are missing to date. In this work, a unique pump-probe ellipsometry microscope is presented allowing the determination of the transient complex refractive index with a sub-ps temporal resolution. Measurements on molybdenum show ultrafast optical penetration depth changes of -6% to + 77% already within the first 10 ps after the laser pulse impact. This indicates a significant absorption variation of the pump pulse or subsequent pulses irradiating the sample on this timescale and paves the road towards a better understanding of pulse duration dependent laser ablation efficiency, double or burst mode laser ablation and lattice modifications in the first ps after the laser pulse impact.

Rapid Screening for Exposure to "Non-Target" Pharmaceuticals from Wastewater Effluents by Combining HRMS-Based Suspect Screening and Exposure Modeling.

Active pharmaceutical ingredients (APIs) have raised considerable concern over the past decade due to their widespread detection in water resources and their potential to affect ecosystem health. This triggered many attempts to prioritize the large number of known APIs to target monitoring efforts and testing of fate and effects. However, so far, a comprehensive approach to screen for their presence in surface waters has been missing. Here, we explore a combination of an automated suspect screening approach based on liquid chromatography coupled to high-resolution mass spectrometry and a model-based prioritization using consumption data, readily predictable fate properties and a generic mass balance model for activated sludge treatment to comprehensively detect APIs with relevant exposure in wastewater treatment plant effluents. The procedure afforded the detection of 27 APIs that had not been covered in our previous target method, which included 119 parent APIs. The newly detected APIs included seven compounds with a high potential for bioaccumulation and persistence, and also three compounds that were suspected to stem from point sources rather than from consumption as medicines. Analytical suspect screening proved to be more selective than model-based prioritization, making it the method of choice for focusing analytical method development or fate and effect testing on those APIs most relevant to the aquatic environment. However, we found that state-of-the-practice exposure modeling used to predict potential high-exposure substances can be a useful complement to point toward oversights and known or suspected detection gaps in the analytical method, most of which were related to insufficient ionization.

Characterization of energy transfer for passively Q-switched laser ignition.

Miniaturized passively Q-switched Nd:YAG/Cr(4+):YAG lasers are promising candidates as spark sources for sophisticated laser ignition. The influence of the complex spatial-temporal pulse profile of such lasers on the process of plasma breakdown and on the energy transfer is studied. The developed measurement technique is applied to an open ignition system as well as to prototypes of laser spark plugs. A detected temporal breakdown delay causes an advantageous separation of plasma building phase from energy transfer. In case of fast rising laser pulses, an advantageous reduction of the plasma breakdown delay occurs instead.

Non-target screening with high-resolution mass spectrometry: critical review using a collaborative trial on water analysis.

In this article, a dataset from a collaborative non-target screening trial organised by the NORMAN Association is used to review the state-of-the-art and discuss future perspectives of non-target screening using high-resolution mass spectrometry in water analysis. A total of 18 institutes from 12 European countries analysed an extract of the same water sample collected from the River Danube with either one or both of liquid and gas chromatography coupled with mass spectrometry detection. This article focuses mainly on the use of high resolution screening techniques with target, suspect, and non-target workflows to identify substances in environmental samples. Specific examples are given to emphasise major challenges including isobaric and co-eluting substances, dependence on target and suspect lists, formula assignment, the use of retention information, and the confidence of identification. Approaches and methods applicable to unit resolution data are also discussed. Although most substances were identified using high resolution data with target and suspect-screening approaches, some participants proposed tentative non-target identifications. This comprehensive dataset revealed that non-target analytical techniques are already substantially harmonised between the participants, but the data processing remains time-consuming. Although the objective of a "fully-automated identification workflow" remains elusive in the short term, important steps in this direction have been taken, exemplified by the growing popularity of suspect screening approaches. Major recommendations to improve non-target screening include better integration and connection of desired features into software packages, the exchange of target and suspect lists, and the contribution of more spectra from standard substances into (openly accessible) databases. Graphical Abstract Matrix of identification approach versus identification confidence.

Ion Prostate Irradiation (IPI) - a pilot study to establish the safety and feasibility of primary hypofractionated irradiation of the prostate with protons and carbon ions in a raster scan technique.

BACKGROUND: Due to physical characteristics, ions like protons or carbon ions can administer the dose to the target volume more efficiently than photons since the dose can be lowered at the surrounding normal tissue. Radiation biological considerations are based on the assumption that the α/ß value for prostate cancer cells is 1.5 Gy, so that a biologically more effective dose could be administered due to hypofractionation without increasing risks of late effects of bladder (α/ß = 4.0) and rectum (α/ß = 3.9). METHODS/DESIGN: The IPI study is a prospective randomized phase II study exploring the safety and feasibility of primary hypofractionated irradiation of the prostate with protons and carbon ions in a raster scan technique. The study is designed to enroll 92 patients with localized prostate cancer. Primary aim is the assessment of the safety and feasibility of the study treatment on the basis of incidence grade III and IV NCI-CTC-AE (v. 4.02) toxicity and/or the dropout of the patient from the planned therapy due to any reason. Secondary endpoints are PSA-progression free survival (PSA-PFS), overall survival (OS) and quality-of-life (QoL). DISCUSSION: This pilot study aims at the evaluation of the safety and feasibility of hypofractionated irradiation of the prostate with protons and carbon ions in prostate cancer patients in an active beam technique. Additionally, the safety results will be compared with Japanese results recently published for carbon ion irradiation. Due to the missing data of protons in this hypofractionated scheme, an in depth evaluation of the toxicity will be created to gain basic data for a following comparison study with carbon ion irradiation. TRIAL REGISTRATION: Clinical Trial Identifier: NCT01641185 (clinicaltrials.gov).

Strategies to characterize polar organic contamination in wastewater: exploring the capability of high resolution mass spectrometry.

Wastewater effluents contain a multitude of organic contaminants and transformation products, which cannot be captured by target analysis alone. High accuracy, high resolution mass spectrometric data were explored with novel untargeted data processing approaches (enviMass, nontarget, and RMassBank) to complement an extensive target analysis in initial "all in one" measurements. On average 1.2% of the detected peaks from 10 Swiss wastewater treatment plant samples were assigned to target compounds, with 376 reference standards available. Corrosion inhibitors, artificial sweeteners, and pharmaceuticals exhibited the highest concentrations. After blank and noise subtraction, 70% of the peaks remained and were grouped into components; 20% of these components had adduct and/or isotope information available. An intensity-based prioritization revealed that only 4 targets were among the top 30 most intense peaks (negative mode), while 15 of these peaks contained sulfur. Of the 26 nontarget peaks, 7 were tentatively identified via suspect screening for sulfur-containing surfactants and one peak was identified and confirmed as 1,3-benzothiazole-2-sulfonate, an oxidation product of a vulcanization accelerator. High accuracy, high resolution data combined with tailor-made nontarget processing methods (all available online) provided vital information for the identification of a wider range of heteroatom-containing compounds in the environment.

Suspect and nontarget screening approaches to identify organic contaminant records in lake sediments.

Sediment cores provide a valuable record of historical contamination, but so far, new analytical techniques such as high-resolution mass spectrometry (HRMS) have not yet been applied to extend target screening to the detection of unknown contaminants for this complex matrix. Here, a combination of target, suspect, and nontarget screening using liquid chromatography (LC)-HRMS/MS was performed on extracts from sediment cores obtained from Lake Greifensee and Lake Lugano located in the north and south of Switzerland, respectively. A suspect list was compiled from consumption data and refined using the expected method coverage and a combination of automated and manual filters on the resulting measured data. Nontarget identification efforts were focused on masses with Cl and Br isotope information available that exhibited mass defects outside the sample matrix, to reduce the effect of analytical interferences. In silico methods combining the software MOLGEN-MS/MS and MetFrag were used for direct elucidation, with additional consideration of retention time/partitioning information and the number of references for a given substance. The combination of all available information resulted in the successful identification of three suspect (chlorophene, flufenamic acid, lufenuron) and two nontarget compounds (hexachlorophene, flucofuron), confirmed with reference standards, as well as the tentative identification of two chlorophene congeners (dichlorophene, bromochlorophene) that exhibited similar time trends through the sediment cores. This study demonstrates that complementary application of target, suspect, and nontarget screening can deliver valuable information despite the matrix complexity and provide records of historical contamination in two Swiss lakes with previously unreported compounds.

Exploring the Behaviour of Emerging Contaminants in the Water Cycle using the Capabilities of High Resolution Mass Spectrometry.

To characterize a broad range of organic contaminants and their transformation products (TPs) as well as their loads, input pathways and fate in the water cycle, the Department of Environmental Chemistry (Uchem) at Eawag applies and develops high-performance liquid chromatography (LC) methods combined with high-resolution tandem mass spectrometry (HRMS/MS). In this article, the background and state-of-the-art of LC-HRMS/MS for detection of i) known targets, ii) suspected compounds like TPs, and iii) unknown emerging compounds are introduced briefly. Examples for each approach are taken from recent research projects conducted within the department. These include the detection of trace organic contaminants and their TPs in wastewater, pesticides and their TPs in surface water, identification of new TPs in laboratory degradation studies and ozonation experiments and finally the screening for unknown compounds in the catchment of the river Rhine.

Velocimetry of GHz elastic surface waves in quartz and fused silica based on full-field imaging of pump-probe reflectometry.

This study reports an imaging method for gigahertz surface acoustic waves in transparent layers using infrared subpicosecond laser pulses in the ablation regime and an optical pump-probe technique. The reflectivity modulations due to the photoelastic effect of generated multimodal surface acoustic waves were imaged by an sCMOS camera illuminated by the time-delayed, frequency-doubled probe pulses. Moving the delay time between 6 . 0 n s to 11 . 5 n s , image stacks of wave field propagation were created. Two representative samples were investigated: wafers of isotropic fused silica and anisotropic x-cut quartz. Rayleigh (SAW) and longitudinal dominant high-velocity pseudo-surface acoustic wave (HVPSAW) modes could be observed and tracked along a circular grid around the excitation center, allowing the extraction of angular profiles of the propagation velocity. In quartz, the folding of a PSAW was observed. A finite element simulation was developed to predict the measurement results. The simulation and measurement were in good agreement with a relative error of 2 % to 5 %. These results show the potential for fast and full-field imaging of laser-generated ultrasonic surface wave modes, which can be utilized for the characterization of thin transparent samples such as semiconductor wafers or optical crystals in the gigahertz frequency range.

TRIF is a critical survival factor in viral cardiomyopathy.

TRIF is a member of the innate immune system known to be involved in viral recognition and type I IFN activation. Because IFNs are thought to play an important role in viral myocarditis, we investigated the role of TRIF in induced myocarditis in mice. Whereas C57BL/6 (wild-type) mice showed only mild myocarditis, including normal survival postinfection with coxsackievirus group B serotype 3 (CVB3), infection of TRIF(-/-) mice led to the induction of cardiac remodeling, severe heart failure, and 100% mortality (p < 0.0001). These mice showed markedly reduced virus control in cardiac tissues and cardiomyocytes. This was accompained with dynamic cardiac cytokine activation in the heart, including a suppression of the antiviral cytokine IFN-ß in the early viremic phase. TRIF(-/-) myocytes displayed a TLR4-dependent suppression of IFN-ß, and pharmacological treatment of CVB3-infected TRIF(-/-) mice with murine IFN-ß led to improved virus control and reduced cardiac inflammation. Additionally, this treatment within the viremic phase of myocarditis showed a significant long-term outcome indexed by reduced mortality (20 versus 100%; p < 0.001). TRIF is essential toward a cardioprotection against CVB3 infection.

Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia.

Congenital hypothyroidism occurs in one of every three to four thousand newborns, owing to complete or partial failure of thyroid gland development. Although thyroid hypoplasia has recently been associated with mutations in the thyrotropin (TSH) receptor, the cause of thyroid agenesis is unknown. Proteins including thyroid transcription factors 1 (TTF-1; refs 4,5) and 2 (TTF-2; refs 6,7) and Pax8 (refs 8,9) are abundant in the developing mouse thyroid and are known to regulate genes expressed during its differentiation (for example, thyroid peroxidase and thyroglobulin genes). TTF-2 is a member of the forkhead/winged-helix domain transcription factor family, many of which are key regulators of embryogenesis. Here we report that the transcription factor FKHL15 (ref. 11) is the human homologue of mouse TTF-2 (encoded by the Titf2 gene) and that two siblings with thyroid agenesis, cleft palate and choanal atresia are homozygous for a missense mutation (Ala65Val) within its forkhead domain. The mutant protein exhibits impaired DNA binding and loss of transcriptional function. Our observations represent the first description of a genetic cause for thyroid agenesis.