Simplicity Of Protocol Research Articles

Comparative analysis of reagent kits for DNA extraction from dry blood stains

Neonatal screening is a mandatory newborn screening procedure that detects the presence of genetic diseases. Dry blood stains are used for mass screening of children. This technology is the most affordable and convenient for the transportation and storage of biological material. DNA extraction is one of the important steps in molecular diagnostics, the accuracy of which is particularly important in genetic analysis. Different DNA extraction kits offer different protocols and reagents, which may vary in efficiency and quality of extraction.The aim of our work was to perform a comparative analysis of reagent kits for DNA extraction from dried blood spots.The materials were dried blood drop samples on Guthrie cards obtained from healthy preterm infants on day 3-4 of life as part of a newborn screening program.The study methods included spectrophotometric analysis to determine the concentration and purity of DNA, the simplicity of protocols, the duration of isolation, and the possibility of automating the process were also evaluated. The efficiency of DNA isolation using different reagent kits was additionally monitored by real-time PCR results using a test system to assess the level of TREC and KREC in peripheral blood, since quantitative analysis requires more attention to the material under study.In assessing the purity of the nucleic acid extracted using four kits analyzed, successful deproteinization of DNA samples and relative purity could be observed. The average DNA purity for the “Extra-DNA-Bio” set was 2.2±0.23, for “EXTRA-prep PS” – 1.89±0.23, for “MagnoPrime UNI” with manual and automatic isolation – 2.31±0.21 and 2.85±0.09, respectively. The average DNA concentration for the Extra-DNA-Bio kit was 15.28 μg/mL, for the EXTRA-Prep PS kit it was 16.26 μg/mL, and for the MagnoPrime UNI for manual and automated isolation it was 62.5 μg/mL and 102.28 μg/mL, respectively. According to the applied Kraskell-Wallis criterion and Dunn’s test, significant differences in both TREC and KREC parameters are present between the group of DNA samples extracted using the “MagnoPrime UNI” reagent kit for manual extraction and the groups of samples extracted by other methods (“MagnoPrime UNI” for automatic extraction) or “Extra-DNA-Bio” and “EXTRA-Prep PS” kits.In the course of the present study, four comparable reagent sets demonstrated a high level of convergence of the obtained data, satisfying all the necessary parameters for further molecular genetic analysis, can be used for neonatal screening and in other areas of research requiring DNA extraction from a dried blood spots.

Immobilization of carbonic anhydrase on poly(ionic liquid) composite membranes for CO2 absorption by gas-liquid membrane contactors

In our current climate crisis, an impressive global effort is being deployed to develop and implement technologies to mitigate CO2 emissions. For instance, CO2 capture and storage/utilization (CCUS) will be fundamental during the energy transition. In the context of CCUS, the enzyme carbonic anhydrase (CA) has gained growing attention because it acts as a green catalyst in CO2 capture by absorption. A few works investigated the preparation of membranes with immobilized CA for CO2 capture by gas-liquid membrane contactors. Although these biocatalytic membranes exhibited improvements in the CO2 capture rate, limited immobilization strategies and carriers have been investigated so far. This work describes the fabrication, characterization, and testing of novel composite membranes with immobilized CA for CO2 capture. The membranes were comprised of a hydrophilic poly(ionic liquid) (PIL) top layer coated on a porous poly(vinylidene fluoride) (PVDF) support. The layer was prepared by in-situ polymerization of an ionic liquid monomer solution cast on the PVDF. CA was easily immobilized on the PIL layer by physical adsorption. The immobilized enzyme's catalytic activity was evaluated to identify the optimum formulation of the PIL layer (monomer and cross-linker concentrations) and immobilization protocol (enzyme concentration in the buffer solution and pH). Despite the immobilization protocol's simplicity, the enzyme was strongly bonded as the enzyme-loaded membranes could be reutilized 10 times while retaining above 90% of their initial activity. These membranes were further characterized by SEM, EDX, ATR-FTIR, TGA, DSC, zeta potential, and water contact angle. Moreover, the biocatalytic composite membranes were tested in a gas absorption set-up to demonstrate their applicability. They showed an increase in the overall mass transfer coefficient with respect to the pristine support by a factor of 2.5, making them interesting candidates for promoting CO2 capture.

Genus-level macroinvertebrate methods limit the conservation coverage afforded aquatic species

Benthic macroinvertebrate protocols, in the United States, have been integral in the implementation of the Clean Water Act (33 U.S.C. §1251 et seq. (1972)). The methods in these protocols are designed to describe patterns of biological water quality and ecological condition relevant to legislative oversight, and require a tradeoff between taxonomic precision and protocol simplicity. The global similarity of ecological and evolutionary responses to the challenges of life in flowing waters, by stream organisms, has greatly facilitated the development and implementation of biological data from freshwater mussels, fishes and aquatic insects into streamlined regulatory frameworks. Unfortunately, the factors that expedite the use of benthic data into ecological descriptions sufficient for regulatory processes (taxonomic generalizations, standardization of sampling procedures to focus on a narrow subset of available habitats) also limit the utility of these methods in the conservation of benthic macroinvertebrate species (the large majority of which are insects). Using simple hypothetical simulations, some examples from a taxonomic and ecological database of one state in the USA (Illinois), and data from a large-scale inventory of aquatic insect species in a network of US National Parks, I illustrate how these methods are limited in their ability to effectively describe ecological patterns relevant to the conservation of aquatic insect species. I identify, challenge, and explore three basic assumptions implicit to the genus- and family-level identification methods widely used in freshwater benthic science, then discuss how the failure of these assumptions may create empirical, theoretical and philosophical obstacles to the interpretation of data and certainly hinder the conservation of species. I offer some hopeful suggestions for improving aquatic insect species conservation that are within the reach of all benthic freshwater ecologists.

High-Speed Lens-Free Holographic Sensing of Protein Molecules Using Quantitative Agglutination Assays.

Accurate, cost-effective, easy-to-use, and point-of-care sensors for protein biomarker levels are important for disease diagnostics. A cost-effective and compact readout approach that has been used for several diagnostic applications is lens-free holographic microscopy, which provides an ultralarge field of view and submicron resolution when it is coupled with pixel super-resolution techniques. Despite its potential as a diagnostic technique, lens-free microscopy has not previously been applied to quantitative protein molecule sensing in solution, which can simplify sensing protocols and ultimately enable measurements of binding kinetics in physiological conditions. Here, we sense interferon-γ (an immune system biomarker) and NeutrAvidin molecules in solution by combining lens-free microscopy with a one-step bead-based agglutination assay, enabled by a custom high-speed light-emitting diode (LED) array and automated image processing routines. We call this a quantitative large-area binding (QLAB) sensor. The high-speed light source provides, for the first time, pixel super-resolved imaging of >104 2 μm beads in solution undergoing Brownian motion, without significant motion blur. The automated image processing routines enable the counting of individual beads and clusters, providing a quantitative sensor readout that depends on both bead and analyte concentrations. Fits to the chemical binding theory are provided. For NeutrAvidin, we find a limit of detection (LOD) of <27 ng/mL (450 pM) and a dynamic range of 2-4 orders of magnitude. For mouse interferon-γ, the LOD is <3 ng/mL (200 pM) and the dynamic range is at least 4 orders of magnitude. The QLAB sensor holds promise for point-of-care applications in low-resource communities and where protocol simplicity is important.

Solubilization of poorly water‐soluble bioactive molecules in neutral aqueous media by complexation with renatured β‐1,3‐1,6‐glucan nanoparticles

The design of scaffolds for solubilizing/dispersing poorly water-soluble bioactive molecules in neutral aqueous media is a major challenge of functional food, pharmaceuticals, and cosmetics development, as highlighted by the plethora of corresponding solubilization/dispersion strategies. Herein, renatured β-1,3-1,6-glucan (r-glucan) nanoparticles prepared by neutralization of alkali-denatured β-1,3-1,6-glucan and subsequent centrifugation are used as a host to disperse water-insoluble bioactive molecules (curcumin, all-trans-retinoic acid, and rebamipide) by simple mixing of host and guest solutions. Curcumin in the r-glucan cavity is found to be stacked in the form of J-aggregates and twisted along the helix, and is demonstrated to be retained for significantly longer than curcumin in the corresponding γ-cyclodextrin (γ-CD) complex. Specifically, curcumin incorporated in γ-CD is released within 5.5 hours, whereas that in the r-glucan complex is released very slowly, with 12% of curcumin in the latter complex retained after 31-day incubation at 37°C. Thus, inclusion protocol simplicity and slow release ability make r-glucan nanoparticles a potential carrier scaffold for various applications.

Tailoring Sensitivity in Electrochemical Nucleic Acid Hybridization Biosensing: Role of Surface Chemistry and Labeling Strategies

AbstractElectrochemical nucleic acid hybridization biosensors have become a mainstay to detect DNA or RNA targets of interest in clinical diagnostics, environmental monitoring and food quality control. Despite the great progress they demonstrated during the last years, there is a constant demand to improve their performance, mainly in terms of sensitivity, simplicity of protocols and easy implementation in routine and decentralized determinations. Within this context, the tremendous possibilities offered by both, a judicious interfacing of the electrode surface, and the use of innovative labeling strategies not requiring nanomaterials or nucleic acid amplification, is discussed critically in this review.

Simplified dengue virus microwell plaque assay using an automated quantification program

The plaque assay is essential for virion quantitation but the classic protocol requires considerable efforts. A simplified dengue 96-well plaque assay with automated quantitation program is an alternative to access the level of infectious virus. Dengue plaque assay was simplified using LLC/MK2 cells and virus mixing simultaneously before semisolid addition. Results were obtained using a flatbed scanner and analysis by the self-written program optimized to manual reads. The newly developed microwell system was accurate to the standard assay because 19 independent titrations from all subtypes obtained from both systems differed less than a log10 p.f.u./ml with no significance (p>0.05) with good correlation (R2=0.9058). Coefficient of variations within and between assays, indicating assay reliability and repeatability, were 19.29%, and 12.50%, respectively. This method serves various experimental designs in drug discovery that requires viral titers assessment. Effective concentrations (EC90) results showed no significant difference between 24- and 96-well assays (p>0.05). Compound screening for potential antivirals and clinical isolate titrations were successfully arranged. The method contains distinguished features including protocol simplicity, less reagent consumption in microwell format, convenient and affordable data acquisition and analysis system.

Micro-TLC Approach for Fast Screening of Environmental Samples Derived from Surface and Sewage Waters

In this work we demonstrated analytical capability of micro-planar (micro-TLC) technique comprising one and two-dimensional (2D) separation modes to generate fingerprints of environmental samples originated from sewage and ecosystems waters. We showed that elaborated separation and detection protocols are complementary to previously invented HPLC method based on temperature-dependent inclusion chromatography and UV-DAD detection. Presented 1D and 2D micro-TLC chromatograms of SPE (solid-phase extraction) extracts were optimized for fast and low-cost screening of water samples collected from lakes and rivers located in the area of Middle Pomerania in northern part of Poland. Moreover, we studied highly organic compounds loaded in the treated and untreated sewage waters obtained from municipal wastewater treatment plant “Jamno” near Koszalin City (Poland). Analyzed environmental samples contained number of substances characterized by polarity range from estetrol to progesterone as well as chlorophyll-related dyes previously isolated and pre-purified by simple SPE protocol involving C18 cartridges. Optimization of micro-TLC separation and quantification protocols of such samples were discussed from the practical point of view using simple separation efficiency criteria including total peaks number, log(product ΔhR F), signal intensity and peak asymmetry. Outcomes of the presented analytical approach, especially using detection involving direct fluorescence (UV366/Vis) and phosphomolybdic acid (PMA) visualization are compared with UV-DAD HPLC-generated data reported previously. Chemometric investigation based on principal components analysis revealed that SPE extracts separated by micro-TLC and detected under fluorescence and PMA visualization modes can be used for robust sample fingerprinting even after long-term storage of the extracts (up to 4 years) at subambient temperature (−20 °C). Such approach allows characterization of wide range of sample components that are present in given extract in high and middle concentration range. Due to protocol simplicity and low cost of analysis this method can be useful for preliminary sample screening.

T 2 relaxometry measurements in low spatial frequency brain regions differ between fast spin-echo and multiple-echo spin-echo sequences

Dual-echo fast spin-echo (FSE) sequences are used in T2 relaxometry studies of neurological disorders because of shorter clinical scanning times and protocol simplicity. However, FSE sequences have possible spatial frequency-dependent effects, and derived T2 values may include errors that depend on the spatial frequency characteristics of the brain region of interest. Dual-echo FSE and multi-echo spin-echo (MESE) sequences were acquired in nine subjects. The T2 decay curves for FSE and MESE sequences were estimated and percent error maps were generated. T2 error values were obtained along each patient's corticospinal tract (CST). Whole-brain white matter (WM) and gray matter (GM) T2 error values were also obtained. The paired t test was performed to evaluate differences in T2 values in the CST between FSE and MESE sequences. Histograms of error values in CST and in whole-brain WM and GM structures revealed systematic errors in FSE sequences. Significant differences (P < 0.001) in CST T2 values were also observed between FSE and MESE sequences. Our findings indicate that T2 values derived from FSE sequences are prone to large errors, even in low spatial frequency regions such as the CST, when compared to MESE sequences. Future studies should be aware of this limitation of FSE sequences.

인터넷 키 관리 프로토콜에 관한 연구

IKE, which is the standard key management protocol for IPSEC, is said to have several known problems. To resolve the problems of the IKE, two protocol proposals are being discussed in the IETF. the IKE version 2 and Just Fast Keying protocols. They should satisfy several protocol design requirements such as the protocol simplicity, the endurability against DOS attacks, the degree of the PFS, the identity protection, the cryptographic negotiation, and the authentication methods. In this paper, we summarize the characteristics of these two protocols and try to analyze their implications according to the protocol design requirements.

Development of a Fast and Simple Method for Determination of β-Agonists in Urine by Extraction on Empore Membranes and Detection by a Test Strip Immunoassay

A rapid screening method for β-agonists in cattle urine was developed that offers speed and protocol simplicity and that could be performed on the spot. The method consists of an extraction on Empore membranes with strong cation exchange properties and a test strip immunoassay. The Empore extraction provides a fast and simple cleanup of the urines and a concentration step with recoveries ranging from 50-80% for the β-agonists of the aniline type and around 10% for salbutamol and terbutaline. A test strip immunoassay is performed on the diluted extract. With the use of an antiperoxidase antibody, an internal negative control was included, to which all color reductions are compared. Although a portable colorimeter increased the sensitivity of the test, a visual interpretation was sufficient to give a correct yes/no answer. The whole test takes about 30 min and has a visual detection limit for clenbuterol of 1 ng/mL of urine. The test also shows good sensitivity for salbutamol, despite the low recovery with the Empore extraction.

An alternative solution to the electro-optic and service bottleneck problems in integrated multi-Gbit/s LANs: the SUPERLAN architecture

Abstract This paper examines the two main bottleneck problems in the design of integrated multi-Gbit/s local area networks (LAN), one from the network perspective—the electro-optic bottleneck problem—and other from the user application perspective —the service bottleneck problem. The major design challenge is to develop viable network architectures which effectively deal with these problems, and allow sharing of the vast amount of bandwidth available on optical fiber among distributed users offering traffic with different and conflicting performance requirements whose peak rates are constrained by the limited speed of electronic interfaces. A novel architectural concept is proposed here to address these issues. It makes use of the wavelength division multiaccess (WDMA) architecture where time-synchronous channels placed in different wavelengths are dedicated to different applications, e.g., continuous or variable bit-rate video services, high speed computer data transfer, 64 kbit/s voice application, application-dependent control mechanisms for media access, error supervision/recovery and flow control, network clock distribution, etc. In the architecture proposed in this paper, the total user traffic on the fiber is separated into two classes—isochronous and nonisochronous—each carried on separate wavelengths at multi-Gbit/s rates. The principal features of this architecture, called the SUPERLAN architecture, are: extreme flexibility in providing services to diverse network and traffic conditions, low processing rates that are commensurable with the node electronic speeds, simplicity of protocols, a minimal number of readily available Gbit/s components at the physical layer (PHY) and network performance restricted only by optics and not by electronics or protocols.

Screening recommendations for gestational diabetes mellitus

As a result of extensive experiences in multiple centers and a review of the current literature, we conclude that a plasma glucose level obtained 1 hour after a 50 gm oral glucose challenge is the &#x201C;best” gestational diabetes mellitus screening test. This universal screening is performed at least once during pregnancy. The screening threshold should be no higher than 140 mg/dl, or an unacceptable loss in sensitivity occurs. Universal screening for gestational diabetes mellitus is justified by morbidity reduction, cost, and protocol simplicity and ease. (Am J Obstet Gynecol 1991;165:493-6.)