Laser tissue soldering of the gastrointestinal tract: A systematic review LTS of the gastrointestinal tract.

Background: Laser Tissue Soldering (LTS) is a promising tissue bonding technique in which a solder is applied between the tissues and then irradiated by laser, causing it to solidify and form links with the tissue. Methods: A comprehensive systematic review summarizing the state of research of LTS in the gastrointestinal tract. Results: Most studies were conducted on large animal tissues, using liquid proteinaceous solder, and irradiated by a continuous wave laser at 808 nm. LTS can provide better sealing and burst pressure than conventional methods. The application of LTS on top of or in addition to sutures showed an impressive increase in burst pressures. LTS may decrease the inflammatory and foreign body reaction caused by sutures. Conclusions: LTS has strong potential to be applied in a clinical setting in leak prevention and in closure of gastrointestinal structures as an adjunct or additional anastomotic technology, decreasing leak rates, morbidity, and mortality.

Metaphase Cells Enrichment for Efficient Use in the Dicentric Chromosome Assay.

The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48 h in vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24-48 h, resulting in an overall processing time of 72-96 h for dose estimation. In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on fixed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be specific for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a flow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to ~2 h. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.

A DAPI-Based Modified C-banding Technique for a Rapid Achieving High Photographic Contrast of Centromeres on Chromosomes.

Many chromosome assays rely on the quantification of chromosome abnormalities in cells, and one important abnormality is the existence of more than one centromere for each chromosome. The quantification of such abnormalities has been studied before. However, this process is labor-intensive and time consuming. Thus, this assay is challenging for ex-laboratory applications, where speed is required. We present a visualization method that uses a cheap stain-DAPI, long (e.g., high-resolution) chromosomes and our modified C-banding method for labeling chromosomes. The labeled chromosomes can then be easily seen with a conventional and readily available fluorescence microscopy system. This method achieves an acceleration of the detection of the presence of constitutive heterochromatin in chromosomal centromeres by more than 10 times, to ~2 h, in Human lymphocyte cells and in cells of the human Jurkat line. This new procedure will ultimately provide an easier and cheaper alternative to FISH/PNA probes, or the classic Giemsa staining method. Simplification and reduction in time of the overall procedure will enable the utilization of centromere-counting assays in laboratory and ex-laboratory applications, including in emergency response.

Technical Note: Noninvasive mid-IR fiber-optic evanescent wave spectroscopy (FEWS) for early detection of skin cancers.

PURPOSE: Melanoma is the most lethal of the three primary skin cancers, including also basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), which are less lethal. The accepted diagnosis process involves manually observing a suspicious lesion through a Dermascope (i.e., a magnifying glass), followed by a biopsy. This process relies on the skill and the experience of a dermatologist. However, to the best of our knowledge, there is no accepted automatic, noninvasive, and rapid method for the early detection of the three types of skin cancer, distinguishing between them and noncancerous lesions, and identifying each of them. It is our aim to develop such a system. METHODS: We developed a fiber-optic evanescent wave spectroscopy (FEWS) system based on middle infrared (mid-IR) transmitting AgClBr fibers and a Fourier-transform infrared spectrometer (FTIR). We used the system to perform mid-IR spectral measurements on suspicious lesions in 90 patients, before biopsy, in situ, and in real time. The lesions were then biopsied and sent for pathology. The spectra were analyzed and the differences between pathological and healthy tissues were found and correlated. RESULTS: Five of the lesions measured were identified as melanomas, seven as BCC, and three as SCC. Using mathematical analyses of the spectra of these lesions we were able to tell that all were skin cancers and we found specific and easily identifiable differences between them. CONCLUSIONS: This FEWS method lends itself to rapid, automatic and noninvasive early detection and characterization of skin cancers. It will be easily implemented in community clinics and has the potential to greatly simplify the diagnosis process.

Strong bonding of corneal incisions using a noncontact fiber-optic laser soldering method.

Suturing of corneal incisions requires significant skill. We demonstrate a noncontact method that will simplify the bonding process. 5-mm-long penetrating vertical and slanted incisions were made in corneas of eyes, extracted from dead piglets. A fiber-optic laser system was used for laser soldering of the incisions, under close temperature control, using albumin solder. The burst-pressure PB immediately after the soldering was found to be PB ≈ 92 and 875 mmHg, for vertical and slanted incisions, respectively. PB = 875 mmHg is an exceptionally high figure, ≈10 times the clinically acceptable value for sutured incisions. Laser soldering was then performed on penetrating incisions made in the corneas of live healthy piglets, of weight ≈10 Kg. After a healing period, the eyes were extracted, and the corneas were examined by histopathology and by optical coherence tomography. Our method immediately generated watertight and strong bonding without noticeable corneal shape distortion. These results would be beneficial for cataract surgery and for corneal transplantations. The fiber-optic system makes it much easier to bond corneal incisions. In the future, laser soldering could be automated and efficiently used by less experienced surgeons, thereby reducing the workload on the experienced ones.

Mid-IR evanescent-field fiber sensor with enhanced sensitivity for volatile organic compounds.

The increasing awareness of the harsh environmental and health risks associated with air pollution has placed volatile organic compounds (VOCs) sensor technologies in elevated demand. While the currently available VOC-monitoring technologies are either bulky and expensive, or only capable of measuring a total VOC concentration, the selective detection of VOCs in the gas-phase remains a challenge. To overcome this, a novel method and device based on mid-IR evanescent-wave fiber-optic spectroscopy, which enables enhanced detection of VOCs, is hereby proposed. This is achieved by increasing the number of analyte molecules in the proximity of the evanescent field via capillary condensation inside nano-porous microparticles coated on the fiber surface. The nano-porous structure of the coating allows the VOC analytes to rapidly diffuse into the pores and become concentrated at the surface of the fiber, thereby allowing the utilization of highly sensitive evanescent-wave spectroscopy. To ascertain the effectiveness and performance of the sensor, different VOCs are measured, and the enhanced sensitivity is analyzed using a custom-built gas cell. According to the results presented here, our VOC sensor shows a significantly increased sensitivity compared to that of an uncoated fiber.

Robot-assisted laser tissue soldering system.

Fast and reliable incision closure is critical in any surgical intervention. Common solutions are sutures and clips or adhesives, but they all present difficulties. These difficulties are especially pronounced in classical and robot-assisted minimally-invasive interventions. Laser soldering methods present a promising alternative, but their reproducibility is limited. We present a system that combines a previously reported laser soldering system with a robotic system, and demonstrate its feasibility on the incision-closure of ex-vivo mice skins. In this demonstration, we measured tearing forces of ~2.5N, 73% of the tearing force of a mouse skin without an incision. This robot-assisted laser soldering technique has the potential to make laser tissue soldering more reproducible and revolutionize surgical tissue bonding.

Stochastic Resonance in Protein Folding Dynamics.

Although protein folding reactions are usually studied under static external conditions, it is likely that proteins fold in a locally fluctuating cellular environment in vivo. To mimic such behavior in in vitro experiments, the local temperature of the solvent can be modulated either harmonically or using correlated noise. In this study, coarse-grained molecular simulations are used to investigate these possibilities, and it is found that both periodic and correlated random fluctuations of the environment can indeed accelerate folding kinetics if the characteristic frequencies of the applied fluctuations are commensurate with the internal timescale of the folding reaction; this is consistent with the phenomenon of stochastic resonance observed in many other condensed-matter processes. To test this theoretical prediction, the folding dynamics of phosphoglycerate kinase under harmonic temperature fluctuations are experimentally probed using Förster resonance energy transfer fluorescence measurements. To analyze these experiments, a combination of theoretical approaches is developed, including stochastic simulations of folding kinetics and an analytical mean-field kinetic theory. The experimental observations are consistent with the theoretical predictions of stochastic resonance in phosphoglycerate kinase folding. When combined with an alternative experiment on the protein VlsE using a power spectrum analysis, elaborated in Dave et al., ChemPhysChem 2016, 10.1002/cphc.201501041, the overall data overwhelmingly point to the experimental confirmation of stochastic resonance in protein folding dynamics.

Environmental Fluctuations and Stochastic Resonance in Protein Folding.

Stochastic resonance is a mechanism whereby a weak signal becomes detectable through the addition of noise. It is common in many macroscopic biological phenomena, but here we ask whether it can be observed in a microscopic biological phenomenon, protein folding. We investigate the folding kinetics of the protein VlsE, with a folding relaxation time of about 0.7 seconds at 38 °C in vitro. First we show that the VlsE unfolding/refolding reaction can be driven by a periodic thermal excitation above the reaction threshold. We detect the reaction by fluorescence from FRET labels on VlSE and show that accurate rate coefficients and activation barriers can be obtained from modulated kinetics. Then we weaken the periodic temperature modulation below the reaction threshold, and show that addition of artificial thermal noise speeds up the reaction from an undetectable to a detectable rate. We observe a maximum in the recovered signal as a function of thermal noise, a stochastic resonance. Simulation of a small model-protein, analysis in an accompanying theory paper, and our experimental result here all show that correlated noise is a physically and chemically plausible mechanism by which cells could modulate biomolecular dynamics during threshold processes such as signaling.

Photobleaching of fluorescein as a probe for oxidative stress in single cells.

BACKGROUND: ROS are involved in the regulation of many physiological and pathological processes. Apoptosis and necrosis are processes that are induced by changes in concentrations of Reactive Oxygen Species (ROS). This study aims to detect and quantify the cellular response to changing ROS concentrations in the scope of apoptosis and necrosis. METHODS: Photobleaching of the fluorescent substrate fluorescein is used as a probe to detect the response of individual Jurkat-T-lymphocytes and Prostate-Cancer-3(PC-3) cells to oxidative stress, induced by hydrogen peroxide (H2O2). A kinetic model is proposed to describe changes in intracellular dye quantities due to photobleaching, dye hydrolysis, influx and leakage, yielding a single time-dependent decaying exponent+constant. RESULTS: Fluorescein photobleaching is controlled and used to detect intracellular ROS. An increase in the decay time of fluorescence of intracellular fluorescein (slow photobleaching) was measured from cells incubated with H2O2 at 50 µM. At higher H2O2 concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H2O2 in phosphate buffer saline (PBS), where the addition of H2O2 decreases the decay time, regardless of the irradiation dose used. CONCLUSIONS: The anomalous, ROS-concentration dependent reduction of the photobleaching rate in cells, as opposed to solutions, might indicate on the regulation of the activity of intracellular oxidative-stress protective mechanisms, as seen earlier with other methods. SIGNIFICANCE: Assessing photobleaching via the time decay of the fluorescence intensity of an ROS-sensitive fluorophore may be adapted to monitor oxidative stress or ROS-related processes in cells.

Periodic and stochastic thermal modulation of protein folding kinetics.

Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

Temporal variation of a protein folding energy landscape in the cell.

Chemical reaction rate coefficients and free energies are usually time-independent quantities. Protein folding in vitro is one such reaction with a fixed energy landscape. However, in the milieu of the cell, the energy landscape can be modulated in space and time by fluctuations in the intracellular environment such as cytoskeletal rearrangements, changes in biomolecule concentrations, and large scale cellular reorganization. We studied the time dependence of the folding landscape of a FRET-labeled enzyme, yeast phosphoglycerate kinase (PGK-FRET). Living U2OS cells served as our test tube, and the mammalian cell cycle, a process strictly regulated in time, served as our clock. We found that both the rate of folding and the thermodynamic stability of PGK-FRET are cell cycle-dependent. We also assayed folding rates of PGK-FRET in spatial proximity to and far away from mitotic chromosomes. Our results show that expedited folding in DNA-rich regions cannot account for the faster rate of PGK-FRET folding in mitotic cells.

Rapid perturbation of free-energy landscapes: from in vitro to in vivo.

Biological systems are often studied under the most "physiological" conditions possible. However, purposeful perturbation of biological systems can provide much information about their dynamics, robustness, and function. Such perturbations are particularly easy to apply at the interface of molecular biophysics and cellular biology, at which complex and highly regulated networks emerge from the behavior of individual biomolecules. Due to the size of diffusion coefficients and the length scale of biomolecules, the fastest timescales at this interface extend to below a microsecond. Thus perturbations must be induced and detected rapidly. We focus on examples of proteins and RNAs interacting with themselves (folding) or one another (binding, signaling). Beginning with general principles that have been learned from simple models and perturbation experiments in vitro, we progress to more complex environments that mimic aspects of the living cell, and finally rapid perturbation experiments in living cells. On the experimental side we highlight in particular two classes of rapid perturbation methods (nanoseconds to seconds) that have been traditionally employed in biophysical chemistry, but that will become increasingly important in cell biology and in vivo: fast relaxation techniques and phase-sensitive modulation techniques. These techniques are now increasingly married with imaging to produce a spatiotemporal map of biomolecular stability, dynamics and, in the near future, interaction networks inside cells. Many important chemical processes occur on biologically fast timescales, and yet have important ramifications for slower biological networks.

Comparative analysis of cell parameter groups for breast cancer detection.

We present a method for the comparative analysis of parameter groups according to their correlation to disease. The theoretical basis of the proposed method is Information Theory and Nonparametric Statistics. Normalized mutual information is used as the measure of correlation between parameters, and statistical conclusions are based on ranking. The fluorescence polarization (FP) parameter is considered as the principal diagnostic characteristic. The FP was measured in fluorescein diacetate (FDA)-stained individual peripheral blood mononuclear cells (PBMC), derived from healthy subjects and breast cancer (BC) patients, under different stimulation conditions: by tumor tissue, the mitogen phytohemagglutinin (PHA) or without the stimulants. The FP parameters were grouped according to their correlation with breast cancer. It was established that the greatest difference between cells of BC patients and healthy subjects is found in the PHA test (parameter P1).