Uorescent Dyes Research Articles

CORRELATION OF PROGNOSIS OF COVID POSITIVE PATIENTS BETWEEN REAL TIME RT-PCR AND CT-VALUES OF E. GENE, N.GENE, RDRP GENE

INTRODUCTION: COVID-19 – Corona virus disease-19 – A respiratory illness caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), colloquially referred to as coronavirus. Real-time PCR is conducted to quantify the absolute amount of target sequence or to compare relative amount of a target sequence among samples. This technique monitors amplication of the target in real time via a target specic uorescent signal emitted during amplication. During most real times PCR, a considerable amount of background uorescence occurs. Inspite of this fact PCR uorescent dyes and probes should be sequence specic. This CT value or cycle threshold value in a real time RT- PCR is dened as the number of cycles required for the uorescent signal to cross the threshold i.e., exceeds background level). This is inversely proportional to the amount of target nuclei in the sample. i.e., lower the CTlevel the greater the amount of target nuclei in the sample. AIM: To explore the association of the mutation pattern of covid-19 genetic variation with the severity and fatality of patients with covid-19 viral illness. Factors considered are Age, Sex, and Duration of hospital stay and outcome of patient. MATERIALS AND METHODS: The study was a comparative, correlation, retrospective, lab values based study. All covid+ve patients tested at Peerless hospital, Kolkata by RT-PCR on admission. It is imperative to choose reference genes whose expression levels are not expected to change during our experiment. Common housekeeping genes include actin, alpha-tubuliun, gapdh and ubiquitin. it is wise to use at least two reference gene for one study may not be suitable for another. Total 370 participants were present in this study. RESULT: In our study, 114(30.8%) COVID positive patients were in E GENE GR 11-20, 147(39.7%) COVID positive patients were in E GENE GR 21-30 and 109(29.5%) COVID positive patients were in E GENE GR 31-40. 163(44.1%) COVID positive patients were in N GENE GR 11-20, 132(35.7%) COVID positive patients were in N GENE GR 21-30 and 75(20.3%) COVID positive patients were in N GENE GR 31-40. 228(61.6%) COVID positive patients were in RdRp GENE GR 11-20, 112(30.3%) COVID positive patients were in RdRp GENE GR 21-30 and 30(8.1%) COVID positive patients were in RdRp GENE GR 31-40. We found that, 30(8.1%) COVID positive patients died and 340 (91.9%) patients have survived. CONCLUSION: The patients who died had signicantly lower RT-PCR CT-values OF E. GENE, N.GENE, RdRp GENE than patients who survived. We concluded that the time of onset of symptoms were earlier for patients with lower CTvalue, than the other group who survived. So we concluded after correlating CT-values of real time RT-PCR and E. GENE, N.GENE, RdRp GENE, that the patients with lower CTvalues had more severity and had poor prognosis than the other group with higher RT-PCR CT-values of E.GENE, N, GENE, RdRp GENE

Features of proteomic profi le of blood serum of children with bronchial asthma who underwent perinatal central nervous system damage

Objective: proteomic analysis of blood serum of children with bronchial asthma with a history of indication of the pathology of the perinatal period.Materials and methods: a comprehensive examination of children suffering from asthma and patients of the control group using proteomic analysis of blood serum was carried out. Proteomic analysis of blood serum was performed using two-dimensional high-resolution gel electrophoresis followed by staining of gels with fl uorescent dye and time-of-fl ight MALDI mass spectrometry. The proteins were identifi ed using Mascot MS peptide fi ngerprint analysis algorithm and Swiss-Prot protein database.Results: comparative analysis of the proteomic profi le of blood serum of children with pathology of the central nervous system in the perinatal period and developed in the subsequent bronchial asthma and control group patients allowed to establish that number proteins products were reduced in this disease. The proteins in the molecular mass range 16-33 kDa (p<0.05) were identifi ed: glutathione peroxidase 3, transthyretin and system components compliment С4В and C3.Conclusion: modifi cation of the production of these proteins indicates the presence of damage at the molecular level, leading to metabolic and functional disorders in the body of a patient with asthma having a history of indicating the pathology of the perinatal period.

New chromosome characteristics of the monozoic tapeworm Caryophyllaeus laticeps (Cestoda, Caryophyllidea)

Summary The karyotype of a caryophyllidean tapeworm Caryophyllaeus laticeps (Pallas, 1781) from the freshwater bream Abramis brama (L.) caught in the Slovak part of the River Tisa, was described and originally inspected for amount of heterochromatin and its chromosome localization. The chromosome set comprised nine metacentric and one submetacentric (No. 3) pairs (2n = 20). The chromosomes were up to 12.0 ± 2.5 μm long and the mean total length of haploid genome (TLC) reached 80.6 μm that represents one of the highest yet recorded values among tapeworms. C-banding and staining with fl uorescent dyes DAPI and YOYO1 revealed a distinct banding pattern explicitly on chromosomes with centromeric bright heterochromatin bands present on all 10 chromosome pairs; no pair showed any interstitial heterochromatin. A complete course of spermatocyte meiosis and dynamics of nucleolus formation and degradation during meiotic division was described.

BioEssays 1/2013

Chemical biology and sensitive microscopy: How labeling and advanced microscopy techniques can synergistically drive the fi eld of biomolecular imaging. Major developments in the fi elds of protein engineering for fl uorescence labeling and advanced microscopy techniques, such as super‐resolution microscopy, or single‐molecule spectroscopy, have in the recent past allowed both areas to advance substantially. On pages 65–74, Milles and Lemke review the state of the art in both areas and evaluate how they can feedback on each other to fulfi ll the demands of precision single‐molecule studies (lower left). While the modifi cation of proteins through unnatural amino acids offers the minimal possible modifi cation to which a fl uorophore can be attached (upper left), it also has the potential of labeling within living cells. The suitability of different fl uorescent dyes for super‐resolution microscopy, as well as fl uorescence resonance energy transfer based approaches, is also reviewed with a view to multi‐parameter single molecule microscopy inside the cell (right).Cover by S. Milles and E. A. Lemke

Hydrogen Peroxide Vapor Sensing with Organic Core/Sheath Nanowire Optical Waveguides

Continuing efforts have been focused on detecting H 2 O 2 by different strategies such as infrared/Raman spectroscopy, [ 4 ] mass spectrometry, [ 5 ] electrochemical method, [ 6 ] colorimetric [ 7 ] and fl uorimetric detection. [ 8 ] Generally these methods either require expensive bulky equipment and tedious sample preparation, or have poor selectivity and limits of detection. Therefore, the development of contrast agents that allow the expedient detection of H 2 O 2 at low concentration is in demand. Peroxalate ester and its derivatives are typical chemiluminogenic compounds highly sensitive to H 2 O 2 . [ 9 ] The reaction between peroxalate ester groups and H 2 O 2 can generate a high-energy intermediate that may transfer energy to the fl uorescent dye, resulting in effi cient luminescence. This has been proved to be a promising strategy for the expedient and sensitive detecting of H 2 O 2 . [ 1 , 10 ]

LED Light Source: Major Advance in Fluorescence Microscopy

Fluorescence microscopy requires an intense light source at the specifi c wavelength that will excite fl uorescent dyes and proteins. The traditional method employs a white light, typically from a mercury or xenon arc lamp. Although such broad spectrum lamps can generate ample light at desired wavelengths, only a small percentage of the projected light is useful in any particular application. The other wavelengths need to be suppressed to avoid background noise that reduces image contrast and obscures the fl uorescent light emissions. This process of subtraction is complex, expensive, and only partially effective. Even after decades of refi nements, the best fi lters are not 100% successful at blocking the bleedthrough of non-specifi c photons. Some mitigation techniques end up not only suppressing peripheral light, but also signifi cantly diminishing the intensity of the desired wavelengths. To address the root cause of the problem—the presence of non-specifi c photons—a radically different approach is coming to light. In theory, a design that only introduces specifi c photons is preferable to one that requires subsequent mitigation. In other words, an ideal light source would start with a baseline that contributes zero peripheral light and would allow the user to precisely control the addition of only those selective wavelengths of light that match the particular excitation wavelengths of the fl uorochromes. The user should also be able to control the intensity and duration of the light to achieve the highest possible image quality while protecting the sample against bleaching and phototoxicity. Recent advances in high performance Light Emitting Diode (LED) technology have enabled the practical implementation of this theoretical model. High-intensity monochromatic LEDs are now available in a variety of colors that match the excitation bandwidth of many commonly used fl uorescent dyes and proteins. This new LED technology uses specifi c wavelength windows with much less need to suppress unwanted peripheral wavelengths from a white light arc lamp. One such system has up to four LEDs, each individually and instantly controlled by electrical current without any of the mechanical switching devices like fi lterwheels or shutters required by traditional illumination systems. LEDs of different colors can be used in combination, giving users the option of seeing multiple fl uorochromes simultaneously or rapidly capturing sequential images of each fl uorochrome.

Modern slice culture for direct observation of production and migration of brain neurons.

For the understanding of histogenetic events in the three-dimensional brain primordia, direct observation of progenitor cells and young neurons is required. Although slice culture, which is one of the tissue or organ culture methods, effectively preserves the in vivo microenvironment where normal developmental processes occur, conventional phase-contrast microscopic observation of brain slices fails to provide good visibility of single cells. However, a combination of slice culture with the use of fluorescent dyes and/or the introduction of fluorescent protein genes provides live, three-dimensional information on cytogenetic and histogenetic events at the individual cell level. Dynamic cellular behaviors can then be vividly captured without destroying tissue structures.

A Liposome-Permeating Activity From the Surface of the Carapace of the American Horseshoe Crab, Limulus polyphemus

Colonization by sessile fouling organisms (epibionts) is the usual fate for solid surfaces in the marine environment. The American horseshoe crab, Limulus polyphemus, ceases molting upon reaching maturity and lives several years as an adult in an epibiont-rich milieu, yet it typically maintains a cuticle that is largely free of macroscopic fl ora and fauna. Indeed, it is in the interest of the animal to maintain its carapace free from such organisms, as colonization of the cuticle by green and blue-green algae can be fatal (1). The mechanisms by which Limulus maintains a clean carapace are not well understood. We have investigated the antibiological properties of a potential anti-fouling system of Limulus, a viscous secretion of a system of dermal glands that discharge their product onto the surface of the carapace (2). We propose that this substance, dermal exudate (DE), contributes to maintaining the cleanliness, and thus the integrity, of the cuticle. In the past we have identifi ed and partially characterized a hemolytic activity present in DE (3, 4). It was shown that the presence of macromolecular osmolites in the hemolysis assay medium, dextran-8 and to a lesser extent dextran-4, prevented lysis of the red blood cells. This effect is attributed to the ability of these macromolecular osmolites to establish an osmotic balance between the interior and exterior of the cell so that no net water fl ow into the cell occurs, protecting the cell from cytolysis. The inhibitory effect of dextrans suggests that DE-induced lysis results from hydrophilic pore formation in the lipid bilayer of the red blood cell rather than from a detergent-like disruption of lipid packing or from phospholipase activity. To investigate the potential interactions of DE directly with lipid bilayers, we have utilized a model system in which a self-quenching fl uorescent dye has been trapped inside liposomes; an increase in the fl uorescence of the dye is a marker for membrane permeation. Here we report the ability of an acid-precipitable constituent of DE to permeabilize liposomes. The agent or agents responsible for these hemolytic and liposomepermeating activities may function as deterrents against potential colonizers of the Limulus cuticle. The secretion of DE can be stimulated by housing Limulus in stressful conditions such as a cage stocked with decaying fi sh material. Dermal exudate was collected by scraping the dorsal carapace. The exudate was stored at 20 °C or with 0.02% NaN3 to prevent bacterial growth. Liposomes were prepared with soybean type II phosphatidylcholine from Sigma. Lyophilized lipid was dissolved in chloroform and dried under a nitrogen stream to a thin fi lm. The fi lm was placed under vacuum overnight to remove any traces of solvent. The lipids were hydrated in 20 mM Tris pH 7.4, 100 mM carboxyfl uorescein (CF), a self-quenching fl uorescent dye. The resulting multilamellar liposomes were made unilamellar through fi ve successive freeze-thaw cycles. Untrapped CF was removed from the liposome suspension by passage over a Sephadex G-50 column. The release of CF from the interior of liposomes was monitored as an indicator of lipid bilayer permeation. Assays were performed with a 1:1000 dilution of liposomes in 20 mM Tris, pH 7.2. Fluorescence intensity was observed with a Photon technologies fl uorometer. Excitation and emission wavelengths were 460 nm and 550 nm respectively. The slit widths were adjusted such that the Raman scatter peak of distilled, deionized water at 397 nm gave an intensity of approximately 350,000 counts/s when excited at 350 nm. Release of entrapped CF from liposomes was seen as an increase in fl uorescence intensity. A unit of activity is arbitrarily de fi ned as an initial increase in fl uorescent intensity of 5% per minute. Intensity values corresponding to 100% lysis were obtained with the addition of Triton X-100.

Precise Gene Expression Measurement with Outlier Detection

Many approaches of Gene Expression Pro ling (GEP) measurements were suggested. However, mostmethods exhibit signi cant gross errors. There is a need to study and improve precision of measure-ment methods. This work did it for a speci c method, i.e., the multiplex PCR with color-taggedmodule-shuing primers [3, 1, 2]. It was designed for the precise measurement of small di erences ofgene expression. However, this method performs the single measurement of each gene in each sample.Such data cannot be used to assess precision. The original method used three dyes each measuringdi erent sample. We used this three dyes for the same sample to assess measurement quality. Thisrevealed many outliers (wrong measurements), i.e., measurements di ering more than twice from themeasured value. This paper analyses this three-sample GEP measuring system from a statisticalviewpoint. The aim is (1) to determine the precision of measurement and the sources of errors, (2) todevelop the method which identi es outliers, and (3) suggests modi cations to improve precision andreproducibility. The data processing was optimized for the given technology. However, the principleslike the transposition of uorescent dyes for outlier detection, data projection for its normalization,and clustering (EM algorithm, Gaussian statistical model) can be applied to other technologies too.

QUANTITATIVE IMAGE ANALYSIS OF LUNG CONNECTIVE TISSUE IN MURINE SILICOSIS

Pulmonary brosis is a disabling consequence of many lung diseases but is difficult to quantify. Lucifer yellow CH uorescent dye (LY) appears to stain connective tissue matrix macromolecules selectively. Laser scanning confocal microscopy can quantify the intensity of uorescence and determine the area of uorescent material. We hypothesized that the abundance of lucifer yellow stained matrix macromolecules in lung tissue sections could be measured by laser scanning confocal microscopy, would reflect differences between varying degrees of pulmonary brosis, and could be compared directly to biochemical measurements of lung collagen. We exposed C57B1/6 and 129 strains of mice by aerosol to cristobalite silica (70 mg/m3, 12 days, 5 hours/day) or sham-air and examined them 2 and 16 weeks after exposure. The area of LY-stained matrix in tissue sections was quantitated by laser scanning confocal microscopy, and total lung collagen was measured biochemically as hydroxyproline (OH-proline). The LY-stained connective tissue matrix appeared as bright linear bands in the alveolar septae, and was increased significantly by image analysis in C57B1/6 and 129 mice with silicosis 16 weeks after exposure. Total lung OH-proline was significantly increased in silica-exposed mice from both stains at both time points. Comparing all 8 groups, there was a signicant linear correlation between the average area of connective tissue measured by LY stain and the total OH-proline per lung measured by chemical analysis (r =. 72, P =. 042). LY staining and confocal microscopy with image analysis offers a rapid technique for quantitative measurements of the extent of pulmonary brosis.