Effect of Iron Binding on the Ability of Crocidolite to Cause DNA Single-Strand Breaks

Fibrous carcinogens, such as crocidolite asbestos, are known to catalyze many of the same reactions as iron, namely 0_2 consumption, generation of reduced oxygen species, and damage to DNA, such as strand breaks, and modifications of bases. Upon inhalation, fibers are also known to become coated with an iron-rich material. The mechanism by which this iron is bound to fibers in the lung is not known, and the effect of this additional iron on the reactivity of the fibers is also not well understood. The studies described here were undertaken to elucidate the abilities of crocidolite asbestos, in its native, soaked, and iron depleted forms, as well as three varieties of silicon carbide whiskers, to acquire reactive iron on their surfaces. The aim has been to quantitate the amount of iron that can bind in short periods of time, and to measure any changes in biochemical reactivity toward DNA following binding of iron. All forms of the naturally occurring mineral fiber crocidolite, and the man-made mineral fibers (silicon carbide whiskers), were capable of acquiring iron, to varying degrees. Native crocidolite was able to bind up to 57 nmol Fe+ 2/mg crocidolite in one hour, while the iron-depleted form was capable of binding only 5. 5 nmol Fe+ 2/mg crocidolite, and the three varieties of silicon carbide whiskers bound from 2.9 to 29.0 nmol Fe+ 2/mg in the same time period. Following iron binding, the fibers were more capable of forming DNA single-strand breaks. The increase in the ability of the fibers to cause DNA strand breaks was greatest with the silicon carbide whiskers, less with iron depleted crocidolite, and the least with native crocidolite, which is likely because of the inherently high iron content of native crocidolite. Other investigation attempted to determine whether iron could be bound from more complex, physiologically relevant iron-containing solutions where potential iron chelators are abundant. Iron appeared to be acquired even from such complex mixtures as tissue culture media. Following incubation in media, the fibers were more active in catalyzing the formation of DNA strand breaks. An interesting correlation was noted between the abilities of the fibers to cause DNA strand breaks after incubation in tissue culture media and the cytotoxicity of crocidolite to A549 cells grown in the same media

Using Rigidity Analysis to Probe Mutation-Induced Structural Changes in Proteins

Predicting the effect of a single amino acid substitution on the stability of a protein structure is a fundamental task in macromolecular modeling. It has relevance to drug design and understanding of disease-causing protein variants. We present KINARI-Mutagen, a web server for performing in silico mutation experiments on protein structures from the Protein Data Bank. Our rigidity-theoretical approach permits fast evaluation of the effects of mutations that may not be easy to perform in vitro, because it is not always possible to express a protein with a specific amino acid substitution. We use KINARI-Mutagen to identify critical residues, and we show that our predictions correlate with destabilizing mutations to glycine. In two in-depth case studies we show that the mutated residues identified by KINARI-Mutagen as critical correlate with experimental data, and would not have been identified by other methods such as Solvent Accessible Surface Area measurements or residue ranking by contributions to stabilizing interactions. We also generate 48 mutants for 14 proteins, and compare our rigidity-based results against experimental mutation stability data. KINARI-Mutagen is available at http://kinari.cs.umass.edu. © 2012 Imperial College Press

Extrusion Production of 3D Printer Filament for Additive Manufacturing

Additive manufacturing, also known as 3D printing, is becoming a go-to production method for short production runs and rapid prototyping on a commercial scale. The growth of additive manufacturing is due to many fac­tors including development of concept modeling, product designing, prototyping, and customized parts. Pittsburg State University’s College of Technology strives to stay at the cutting edge of processing materials for additive manufacturing. The capability of making our own 3D-printer filament would allow students to work with new and different materials and would allow students to learn at the forefront of 3D printing technology. The Wayne Yellowjacket Extruder and Brabender Puller and Winder was used to produce 1.75 mm 3D-printing filament. The Yellowjacket required a lid on the hopper that was fabricated via thermoforming. The Brabender puller and winder was necessary for controlling the filament speed entering the winder and isolating the tension created by the spool from the extruder. When extruding plastic, the thickness and cross-sectional profile are dependent on the rate of cooling; therefore, a cooling trough was designed and fabricated. Polylactic acid (PLA) or acrylonitrile butadiene styrene copolymer (ABS) was used to extrude filament from this line. Successful extrusion of 3D print­ing filament will allow students to produce their own filament as needed for additive manufacturing using the existing Stratysys FDM 1600 and the student-built 3D printer

A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for thera- peutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus–host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivi- rus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B–NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluo- rescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus inter- nal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbec- co’s plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus–host interactions and facilitate future applications in antiviral drug research to manage flavivi- rus infections.International Centre for Genetic Engineering and Biotechnology Grant CRP/CRI18-02.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis

BACKGROUND: The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. METHODS: We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. RESULTS: Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. CONCLUSION: Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies

Magnetic field stabilization for high-accuracy mass measurements on exotic nuclides

The magnetic-field stability of a mass spectrometer plays a crucial role in precision mass measurements. In the case of mass determination of short-lived nuclides with a Penning trap, major causes of instabilities are temperature fluctuations in the vicinity of the trap and pressure fluctuations in the liquid helium cryostat of the superconducting magnet. Thus systems for the temperature and pressure stabilization of the Penning trap mass spectrometer ISOLTRAP at the ISOLDE facility at CERN have been installed. A reduction of the fluctuations by at least one order of magnitude downto dT=+/-5mK and dp=+/-50mtorr has been achieved, which corresponds to a relative frequency change of 2.7x10^{-9} and 1.5x10^{-10}, respectively. With this stabilization the frequency determination with the Penning trap only shows a linear temporal drift over several hours on the 10 ppb level due to the finite resistance of the superconducting magnet coils.Comment: 23 pages, 13 figure

Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors

The Effects of Static and Dynamic Culture Systems on Cell Proliferation and Conditioned Media of Umbilical Cord-derived Mesenchymal Stem Cells

Preclinical and clinical studies have demonstrated the therapeutic effects of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and secretome to cure various degenerative diseases. Thus, the mass-scale production of MSCs is necessary to ensure their availability and cost-effectiveness. In the current study, we evaluated the effect of dynamic 3D and static 2D culture systems on cell proliferation and conditioned media of UC-MSCs.  The lysate of concentrated thrombocyte was used to substitute animal-derived serum in the culture media. From two experimental sets with different UC and lysates of concentrated thrombocyte donors, it was found that the shortest PDTs for experimental set 1 were 12.3 h (2D culture) and 14.8 h (3D culture), whereas in experimental set 2, they were 17.7 h (2D culture) and 16.9 h (3D culture). Microscopic observation confirmed the formation of cell aggregates in the 3D system, particularly during the exponential phase. SDS-PAGE analysis revealed similar protein profiles of conditioned media from both culture systems. An anti-inflammatory cytokine, namely tumor necrosis factor beta (TGF-?), was analyzed using ELISA to evaluate the effect of culturing methods on TGF-? release. Interestingly, the relative TGF-? contents in the 2D culture were stagnant throughout the incubation times, whereas a higher accumulation of TGF-? was detected in the 3D culture, which was most likely caused by shear stress. Our study confirmed that a dynamic culture system with a microcarrier-supported bioreactor is a promising approach to scaling up MSC and secretome productions

Relevance of B-Lines on Lung Ultrasound in Volume Overload and Pulmonary Congestion: Clinical Correlations and Outcomes in Patients on Hemodialysis.

BACKGROUND: Volume overload in patients on hemodialysis (HD) is an independent risk factor for cardiovascular mortality. B-lines detected on lung ultrasound (BLUS) assess extravascular lung water. This raises interest in its utility for assessing volume status and cardiovascular outcomes. METHODS: End-stage renal disease patients on HD at the Island Rehab Center being older than 18 years were screened. Patients achieving their dry weight (DW) had a lung ultrasound in a supine position. Scores were classified as mild (0-14), moderate (15-30), and severe (\u3e30) for pulmonary congestion. Patients with more than 60 were further classified as very severe. Patients were followed for cardiac events and death. RESULTS: 81 patients were recruited. 58 were males, with a mean age of 59.7 years. 44 had New York Heart Association (NYHA) class 1, 24 had class 2, and 13 had class 3. In univariate analysis, NYHA class was associated with B-line classes ( CONCLUSION: At DW, BLUS is an independent risk factor for death and cardiovascular events in patients on HD

Affect Recognition using Psychophysiological Correlates in High Intensity VR Exergaming

User experience estimation of VR exergame players by recognising their affective state could enable us to personalise and optimise their experience. Affect recognition based on psychophysiological measurements has been successful for moderate intensity activities. High intensity VR exergames pose challenges as the effects of exercise and VR headsets interfere with those measurements. We present two experiments that investigate the use of different sensors for affect recognition in a VR exergame. The first experiment compares the impact of physical exertion and gamification on psychophysiological measurements during rest, conventional exercise, VR exergaming, and sedentary VR gaming. The second experiment compares underwhelming, overwhelming and optimal VR exergaming scenarios. We identify gaze fixations, eye blinks, pupil diameter and skin conductivity as psychophysiological measures suitable for affect recognition in VR exergaming and analyse their utility in determining affective valence and arousal. Our findings provide guidelines for researchers of affective VR exergames.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 665992 </p