Deoxyribonucleic Acid Content Research Articles

Evaluation of In Vivo Wound Healing Potential of Schima wallichii (Korth.) Choisy

The process of delayed wound-healing and unhealed wounds can cause serious problems to the patients. In this context it is revealed that the scientists are looking for herbal treatment of wounds, as the remedy is known for having less or minimum undesired effects. Schima wallichii is a lesser known traditional medicinal plant used by certain section of people in Assam, India as a healing agent; however, record on scientific validation of its wound-healing potential is not available. Therefore, an in vivo study was undertaken to evaluate the wound-healing efficacy of the leaves of the plant using excision wound model in Swiss albino mice. The animals were divided into three groups after creating the wounds, namely control, positive control and treated groups. On application of methanolic crude extract of the leaves of Schima wallichii, it was observed that the epithelialization period was lesser and the rate of closure of wounds was faster in the extract treated group when compared with the control group indicating that the application of the extract has accelerated the healing process in the extract treated group. The concentration of protein, Schima wallichii deoxyribonucleic acid and hydroxyproline were also found to be augmented in the treated group than the control suggesting faster rate of healing as deoxyribonucleic acid and protein content in the healing tissues serve as indicator of protein synthesis and cellular proliferation. The above results were supported by the histological and ultra-structural observations as scanning electron microscopy revealed smoother surface topography, H and E staining revealed thicker epithelial layer in the treated animals. Van Gieson′s staining showed higher level of collagen in the extract treated group validating biochemical observation where higher concentration of hydroxyproline was found in treated group compared to the control. Thus, going by the results obtained in the study it can be concluded that the leaves of the plant possess wound-healing potential.

Advanced glycation end-product accumulation differs by location and sex in aged osteoarthritic human menisci

There is a clear link between increasing age and meniscus degeneration, leading to increased injury, osteoarthritis (OA) progression, and often total knee replacement. Advanced glycation end-products (AGEs) are non-enzymatic crosslinks and adducts that accumulate in collagen with age, altering tissue mechanics and cell function, ultimately leading to increased injury and inflammation. AGEs, both fluorescent and non-fluorescent, play a central role in age-related degradation of tissues throughout the body; however, little is known about their role in meniscus degeneration. The objective of this study was to characterize changes in aged OA menisci, specifically evaluating zonal AGE accumulation, to gain a better understanding of changes that may lead to age-related meniscal degeneration. Deidentified human menisci (N=48, 52-84 years old) were obtained from subjects undergoing total knee replacement. Changes in extracellular matrix (ECM) were assessed by gross morphology, confocal analysis, and biochemical assays. Deoxyribonucleic acid (DNA), glycosaminoglycan (GAG), collagen, and AGE accumulation were compared with patient age, zonal region, and patient sex. There were minimal changes in DNA, GAG, and collagen concentration with age or zone. However, collagen fraying and AGEs increased with age, with more AGEs accumulating in the meniscal horns compared to the central body and in male menisci compared to females. Overall, this work provides greater insights into regional changes that occur in human menisci with age and OA. These results suggest AGEs may play a role in the degeneration of the meniscus, with AGEs being a possible target to reduce age-related tears, degeneration, and OA progression.

Efficacy of Supercritical Fluid Decellularized Porcine Acellular Dermal Matrix in the Post-Repair of Full-Thickness Abdominal Wall Defects in the Rabbit Hernia Model

Damage to abdominal wall integrity occurs in accidents, infection and herniation. Repairing the hernia remains to be one of the most recurrent common surgical techniques. Supercritical carbon dioxide (SCCO2) was used to decellularize porcine skin to manufacture acellular dermal matrix (ADM) for the reparation of full-thickness abdominal wall defects and hernia. The ADM produced by SCCO2 is chemically equivalent and biocompatible with human skin. The ADM was characterized by hematoxylin and eosin (H&E) staining, 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) staining, residual deoxyribonucleic acid (DNA) contents and alpha-galactosidase (α-gal staining), to ensure the complete decellularization of ADM. The ADM mechanical strength was tested following the repair of full-thickness abdominal wall defects (4 × 4 cm) created on the left and right sides in the anterior abdominal wall of New Zealand White rabbits. The ADM produced by SCCO2 technology revealed complete decellularization, as characterized by H&E, DAPI staining, DNA contents (average of 26.92 ng/mg) and α-gal staining. In addition, ADM exhibited excellent performance in the repair of full-thickness abdominal wall defects. Furthermore, the mechanical strength of the reconstructed abdominal wall after using ADM was significantly (p < 0.05) increased in suture retention strength (30.42 ± 1.23 N), tear strength (63.45 ± 7.64 N and 37.34 ± 11.72 N) and burst strength (153.92 ± 20.39 N) as compared to the suture retention (13.33 ± 5.05 N), tear strength (6.83 ± 0.40 N and 15.27 ± 3.46 N) and burst strength (71.77 ± 18.09 N) when the predicate device materials were concomitantly tested. However, the efficacy in hernia reconstruction of ADM is substantially equivalent to that of predicate material in both macroscopic and microscopic observations. To conclude, ADM manufactured by SCCO2 technology revealed good biocompatibility and excellent mechanical strength in post-repair of full-thickness abdominal wall defects in the rabbit hernia model.

Influence of lyophilization primary drying time and temperature on porous silk scaffold fabrication for biomedical applications.

Lyophilization of protein solutions, such as silk fibroin (silk), produces porous scaffolds useful for tissue engineering (TE). The impact of modifying lyophilization primary drying parameters on scaffold properties has not yet been explored previously. In this work, changes to primary drying duration and temperature were investigated using 3%, 6%, 9%, and 12% (w/v) silk solutions, via protocols labeled as Long Hold, Slow Ramp, and Standard. The 9% and 12% scaffolds were not successfully fabricated using the Standard protocol, while the Long Hold and Slow Ramp protocols resulted in scaffolds from all silk solution concentrations. Scaffolds fabricated using the Long Hold protocol had higher Young's moduli, smaller pore Feret diameters, and faster degradation. To investigate the utility of the different lyophilized scaffolds for in vitro cell culturing, the HepaRG liver cell line was cultured in the 3% to 12% scaffolds fabricated using the Long Hold protocol. The HepaRG cells grown in 3% scaffolds initially had greater lipid accumulation and metabolic activity than the other groups, although these differences were no longer apparent by Day 28. The deoxyribonucleic acid content of the HepaRG cells grown in 3% scaffold group was also initially significantly higher than the other groups. Significant differences in gene expression by 9% scaffolded HepaRG cells (CK19, HNFα) were seen on Day 14 while significant differences by 12% scaffolded HepaRG cells (ALB, APOA4) were seen on Day 28. Overall, modifying the primary drying parameters and silk concentration resulted in lyophilized scaffolds with tunable properties useful for TE applications.

Nanopore Probes Using Hydrogel-Filled Nanopipettes as Sensors for Chemical Imaging

Scanning ion conductance microscopy (SICM) using biological nanopores is a powerful analytical tool that can provide spatially resolved topography and chemical information. However, the low spatial resolution caused by the large tip diameter of the probe has limited the practical application of the SICM system. Although the tip-dip method has conventionally been used to form a lipid bilayer at the tip of pipettes, an alternative method to form the lipid bilayer is required because the lifetime of lipid bilayers formed by the tip-dip method is too short to map chemical information by stochastic nanopore sensing. Here, we propose a method to prepare lipid bilayers using a hydrogel-filled nanopipette. The lipid bilayer is formed at the tip of the pipette by inserting the pipette into a layered solution of an oil/lipid mixture and an aqueous electrolyte. Since the hydrogel supports the lipid bilayer, the lifetime of the lipid bilayers prepared by this method is improved over that formed by the tip-dip method. Furthermore, the hydrogel at the pore aperture reduces the translocation speed of analytes through nanopores, indicating that the hydrogel-filled nanopipette system can offer highly sensitive chemical sensing. Finally, we measure local single-strand deoxyribonucleic acid concentrations in the concentration gradient generated by a microhole using the biological nanopore probe. We believe that the SICM system using the hydrogel-filled nanopipette-based biological nanopore probe will offer a powerful analytical system for biological phenomena, including cell communication and signal transduction.

Detection and characterization of meat adulteration in various types of meat products by using a high-efficiency multiplex polymerase chain reaction technique.

Identification of meat authenticity is a matter of increasing concerns due to religious, economical, legal, and public health reasons. However, little is known about the inspection of eight meat species in one tube reaction due to technological challenge of multiplex polymerase chain reaction (PCR) techniques. Here, a developed multiplex PCR method can simultaneously authenticate eight meat species including ostrich (753 bp), cat (564 bp), goose (391 bp), duck (347 bp), chicken (268 bp), horse (227 bp), dog (190 bp), and sheep (131 bp). The detectable deoxyribonucleic acid (DNA) contents for each target species was as low as 0.01 ng in both raw and heat-treated meat or target meat down to 0.1% (w/w) of total meat weight reflecting high stability of the assay in heat processing condition, indicating that this method is adequate for tracing meat origin in real-world meat products, which has been further validated by authenticity assays of commercial meat products. Overall, this method is a powerful tool for accurate evaluation of meat origin with a good application foreground.

Benefits of genetic and immunohistochemical markers in understanding abnormalities in aging retina.

The aim of the study was to better understand the interplay between genetic factors and the aging process in the human retina through mapping complement factor H (CFH) and related proteins. Two human eyes, from 92- and 64-year-old donors, were genotyped for the expression of CFH-related 1 (CFHR1) and CFH-related 3 (CFHR3) genes. Deoxyribonucleic acid (DNA) was extracted and analyzed for concentration and purity with a spectrophotometer, at 260 nm. The results showed a DNA concentration of 469.17 ng∕μL in the aged retina and of 399.20 ng∕μL in the younger one. Through polymerase chain reaction (PCR) genotyping, the DNA CFHR1 and CFHR3 were visible as bands of 175 bp and 181 bp. Immunohistochemistry by immunofluorescence method was used with a panel of specific antibodies for CFH, CFHR1, CFHR3 and GFAP, a marker for Müller cells. All the samples were examined, and images captured using confocal microscopy. In the younger retina, CFH was localized in the inner plexiform layer and below the outer nuclear layer, while in the aged retina, it was found in the photoreceptors. CFH was also detected in the choriocapillaris and within the end-feet of the Müller cells. Our controls showed autofluorescence of the retinal pigment epithelium shedding light on a false positive CFH immunostaining of this layer. GFAP immunoreactivity highlighted an increased gliosis within the aged retina. CFHR3 signal was found in the microglia, while CFHR1 was detected in the choriocapillaris. In summary, underpinning the expression of these components can show the potential involvement of these modulators in implementing new treatment strategies.

Rapid Nucleic Acid Extraction for Aquatic Animal DNA Virus Determination Using Chelex 100 Resin via Conventional PCR and Digital Droplet PCR Detection

Simple SummaryConvenient, fast, and high-quality nucleic acid extraction methods are urgently needed in molecular diagnostic testing for viral pathogens in aquaculture. We developed a viral DNA extraction method from diseased tissues and cells using the Chelex 100 resin solution workflow. The only extraction reagents required are the Chelex 100 resin and phosphate-buffered saline. The whole extraction process only takes about 15 min from the tissue homogenate to obtain the DNA. The concentration of extracted DNA is at least 100 ng/µL. This methodology has clear benefits in terms of cost and time saving compared to the commercial kit extraction for aquatic animal DNA virus determination by PCR in the laboratory. In addition, the simplified method using Chelex 100 resin with a pH value of 10–11 presented excellent results in PCR application and could be a standard for the DNA extraction for DNA virus testing in the future.Molecular diagnostic testing for viral pathogens is crucial in aquaculture. The efficient and convenient preparation of pathogenic microbial nucleic acids is the basis of molecular diagnosis. Here, we developed a simplified deoxyribonucleic acid (DNA) extraction method from aquatic animal DNA viruses using the Chelex 100 resin. The nucleic acid was extracted from infected tissues and cell culture for the detection of three common aquatic viral pathogens (CEV, CyHV-2, and GSIV). We compared the extraction effects of a current commercial kit extraction method and the Chelex 100 resin extraction method according to nucleic acid concentration, conventional polymerase chain reaction (PCR), and digital droplet PCR (ddPCR). The results indicated that both extraction procedures could obtain high-quality nucleotide samples. Extracting DNA using the Chelex 100 resin led to better detective efficiency for ddPCR molecular diagnostic testing. The whole process took less than 20 min, and only Chelex 100 resin solution was added to the tissues or cells without multiple tubes being transferred several times. The extracted DNA concentration and the detection sensitivity were high. These results indicated that the Chelex 100 resin solution has the advantages of speed, efficiency, and economy compared to the commercial kit. In addition, the higher pH value (10–11) of the Chelex 100 resin solution markedly improved the detection sensitivity compared to a lower pH value (9–10). In conclusion, the comparison of the Chelex 100 Resin and commercial viral DNA extraction kits revealed the good performance of the Chelex 100 resin solution at pH 10–11 in DNA extraction for PCR amplification from aquatic animal viral samples of tissues and cells in molecular diagnostic testing. It is both rapid and cost-effective.

Solid Lipid Nanoformulation of Berberine Attenuates Doxorubicin Triggered in vitro Inflammation in H9c2 Rat Cardiomyocytes.

The aim of this study was to evaluate the efficacy of solid lipid nanoparticles of berberine against doxorubicin-induced cardiotoxicity. Berberine (Ber) is cardioprotective, but its oral bioavailability is low, and its effect on chemotherapy-induced cardiotoxicity has not been studied. Solid lipid nanoparticles (SLNs) of berberine chloride were prepared, characterized and evaluated in vitro against doxorubicin-induced cardiomyocyte injury. Berberine-loaded SLNs (Ber-SLNs) were synthesized using the water-in-oil microemulsion technique with tripalmitin, Tween 80 and poloxamer 407. Ber-SLNs were evaluated for preventive effect against toxicity of doxorubicin in H9c2 cells. The culture was pre-treated (24 h) with Ber (10 μM) and Ber-SLNs (1 and 10 μM), and 1 μM of doxorubicin (Dox) was added for 3 h. The cell viability assay (MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) and LDH (Lactate dehydrogenase)), levels of Creatine kinase-MB (CK-MB), Nitrite, MDA (Malondialdehyde), ROS (Reactive oxygen species) generation, and apoptotic DNA (Deoxyribonucleic acid) content were assessed. Ber-SLNs had a mean particle size of 13.12±1.188 nm, the zeta potential of -1.05 ± 0.08 mV, poly-dispersity index (PDI) of 0.317 ± 0.05 and entrapment efficiency of 50 ± 4.8%. Cell viability was 81 ± 0.17% for Ber-SLNs (10 μM) and 73.22 ± 0.83% for Ber (10 μM) treated cells in the MTT assay. Percentage cytotoxicity calculated from LDH release was 58.91 ± 0.54% after Dox, 40.3 ± 1.3% with Ber (10 μM) and 40.7 ± 1.3% with Ber-SLNs (1 μM) (p<0.001). Inflammation and oxidative stress markers were lower with Ber and Ber-SLNs. Attenuation of ROS generation and apoptosis of cardiomyocytes were noted on fluorescence microscopy. Ber SLNs effectively prevented doxorubicin-induced inflammation and oxidative stress in rat cardiomyocytes. The results demonstrate that microemulsion is a simple and costeffective technique to prepare Ber-SLNs, and may be considered as a drug delivery vehicle for berberine.

Salmon Sperm DNA As a Corrosion Inhibitor on Ductile Cast for Use in Water Distribution System Pipes

Ductile cast iron (DCI) is a high carbon iron alloy with a composition of up to 3.7% C, 2.3% Si, 0.35% Mn and various smaller constitutes. Upon casting of DCI, the high carbon content results in solidification in a secondary phase in the form of graphite nodules. These graphite nodules form around a matrix of ferrite (α-Fe) and pearlite (α-Fe + Fe3C) and impart desirable properties such as high strength properties and corrosion resistance due to graphite’s cathodic potential. DCI is commonly used for water distribution pipework and contribute to 40% of all drinking water distribution pipes in Wales. Several different corrosion inhibitors are used to prevent corrosion of DCI in drinking water distribution applications, such as sodium silicate (Na2SiO3) and ortho-polyphosphates. However, the use of deoxyribonucleic acid (DNA) could provide a novel alternative to conventional corrosion inhibitors currently in service. The DNA molecule is formed by a double helix polynucleotide strand of phosphate and sugar groups via covalent bonds and base pairs bonded by hydrogen bonds to the molecule. The main advantage of DNA as a corrosion inhibitor is the surplus of phosphate molecules available to be chemisorbed to the surface of DCI. Alongside the potential of aromatic heterocyclic compounds found in base pairs, which could also provide corrosion inhibition via physicochemical adsorption.This work aims to assess the use of deoxyribonucleic acid (DNA) on the corrosion of ductile cast iron (DCI) in a corrosive electrolyte. DNA is considered a green, non-toxic inhibitor that shows potential when used in immersion corrosion conditions. Literature has demonstrated the use of DNA as a corrosion inhibitor for other materials such as Magnesium alloys, Copper and X80 steel. However, there is little published work on the use of DNA as a corrosion inhibitor on DCI. The DNA used in this study was extracted from salmon sperm (Sigma-Aldrich) and dissolved into a 0.17 mol dm-3 sodium chloride (NaCl) solution at the concentrations of 50, 100 and 500 mg/L adjusted to pH 7 via Sodium Hydroxide (NaOH). Electrochemical analysis and surface characterisation was carried out over a range of time periods. The corrosion inhibition afforded by DNA was determined through using a variety of electrochemical techniques such as: open circuit potential (OCP), anodic and cathodic polarisation and linear polarisation resistance (LPR). In-situ microscopy was used to study the corrosion mechanisms that occur on the surface microstructure of DCI. Post corrosion, the DCI surface was characterised using scanning electron microscopy (SEM) and FTIR (Fourier transmission infrared spectroscopy).Electrochemical results, performed over a 24-hour period, showed that as concentration of DNA increased an increase in voltage potential and decrease in corrosion rate (mpy) was observed. A decrease of 60% in corrosion rate after 24 hours is provided by 500 mg/L DNA compared to the control of 1% NaCl. However, after 30 minute’s immersion followed by cathodic polarisation studies showed an increased concentration of DNA can behave as a cathodic inhibitor. A theory for this inhibitive effect is that a passivating film forms on the DCI surface and that it is most effective after 30 minutes exposure due to the high efficiency observed during the longer-term studies. This could suggest that a film formed on the surface is most efficient after 30 minutes adsorption due to the high efficiency seen during the longer-term studies. After 72 hours of immersion there is a change in the surface of DCI, with a more robust scale appearing at higher concentrations. This change in coating surface may provide the increase in corrosion inhibition as the pathway for further corrosion attack is blocked by a protective scale. The protective scale is characterised using FTIR to examine the changes in chemical structure of the corrosion layer. Whereby PO-2 and base pairs are identified on the corrosion scale surface after 24-hours immersion in DNA. The corrosion inhibition observed using DNA on DCI in neutral immersion conditions is afforded to the adsorption of inhibitive species onto the DCI surface, providing an insoluble layer.

Single Particle Inductively Coupled Plasma Mass Spectrometry-Based Homogeneous Detection of HBV DNA with Rolling Circle Amplification-Induced Gold Nanoparticle Agglomeration.

A highly sensitive and simple method based on rolling circle amplification (RCA) and single particle inductively coupled plasma mass spectrometry (spICP-MS) was proposed for the homogeneous detection of hepatitis B virus (HBV) deoxyribonucleic acid (DNA). In the presence of target DNA, long ssDNA possessing a large number of repeating sequence units was generated by RCA. DNA-labeled AuNP probes assembled into long chains based on complementary base pairing, further aggregating into large particles. Small Au NPs hardly produced pulse signals in spICP-MS; obvious pulse signals appeared in spICP-MS after the agglomeration of Au NPs caused by the addition of RCA products and spermidine. On the basis of this, the homogeneous detection of target DNA was realized by spICP-MS with high sensitivity. Under optimal conditions, the proposed method exhibited a good linear relationship between the frequency of the pulse signal of Au in spICP-MS and the concentration of target HBV DNA in the range of 10-2000 fmol L-1 (R = 0.997), the limit of detection was 5.1 fmol L-1, and the relative standard deviation was 3.7-6.8%. Recoveries of 94.2-108% were obtained for target DNA in spiked serum samples, demonstrating a good matrix tolerance ability for the method.

The development of real-time polymerase chain reaction for identification of beef meatball

Real-time polymerase chain reaction (PCR), also known as quantitative PCR (qPCR), is a method of choice for the confirmation of halal meats such as beef from non-halal meats such as pork through analysis of their deoxyribonucleic acid (DNA). The objective of this study was to apply qPCR in combination with species-specific primer (SSP) targeting in D-loop mitochondria genes for the identification of DNA extracted from beef and beef meatballs. DNA in raw meats and meatballs was isolated using Genomic DNA Mini Kit and the obtained DNAs were subjected to SSP specificity, amplification efficiency (E), determination of limit of detection (LoD), and repeatability test. The results showed that SSP designed was specific to beef DNA with LoD value of 100 ng beef DNA. The coefficient of determination (R2) for the relationship between log DNA concentration and quantification cycle (Cq) along with efficiency values were 0.884 and 97.1% respectively. The developed method is precise enough as indicated by the low value of relative standard deviation (RSD) value of 0.77%. The qPCR method using SSP is also successfully applied for analysis of beef meatballs commercially available. It can be concluded that qPCR using SSP targeting on D-loop mitochondria could be proposed as a standard method for the confirmation beef meatballs.

Comparison of Bioinformatics Approaches for Fetal Microdeletions and Monogenic Variations Estimation in Non-invasive Prenatal Testing.

Prenatal testing provides crucial information about the health status of fetuses as well as recommending better treatment. For the past decades, prenatal testing using chorionic villus sampling and amniocentesis were the two majorly used forms of invasive prenatal diagnostic approaches. However, to facilitate prenatal testing without causing any danger to the fetus, the noninvasive prenatal diagnostic method, which uses circulating cell-free deoxyribonucleic acid (DNA), has become a suitable method of prenatal diagnosis. This review discusses the recent bioinformatics approaches used for analyzing fetal DNA concentration.

Experimental and theoretical investigation on the nonlinear optical properties of LDS 821 dye in different solvents and DNA

Linear and nonlinear optical properties of near-infrared laser grade dye LDS 821 in different solvents and Salmon Deoxyribonucleic acid (DNA) were studied using spectroscopic and Z-scan techniques. UV–Vis absorption spectrum of the dye shows a bathochromic shift with a decrease in the solvent polarity parameter, and in DNA, the dye exhibits a hypochromic shift. The fluorescence spectrum of the dye does not show any notable correlation with the solvent polarity parameter, but in DNA, the fluorescence intensity of the dye decreases with the incremental addition of DNA. Molecular docking studies reveal that the dye intercalates on the major grooves of DNA. Nonlinear optical properties of the dye in different solvents and phosphate buffer solution with varying DNA concentrations were studied using the Z-scan technique using a Q-switched Nd: YAG laser operating at fundamental and second harmonics. A closed and open aperture Z-scan of dye in different solvents was carried out to estimate the nonlinear refractive index, excited-state absorption cross-section, and two-photon absorption coefficient (TPA). The variation in nonlinear optical properties of the dye in different solvents was due to solvent-induced structural modifications. Theoretical investigation on nonlinear optical properties of the dye in different solvents was carried out using density function theory. The theoretical first and second-order hyperpolarizability was calculated using B3LYP functional. The predicated nonlinear optical parameters of the dye in different solvents does not show any direct correlation with solvent polarity. Nonlinear absorption of the dye in phosphate buffer solution (PBS) and DNA were estimated. The nonlinear absorption of the dye in PBS decreases with the addition of DNA. Molecular docking studies were carried out to determine the structural changes induced in dye due to the intercalation with DNA.

Biosensing with a scanning planar Yagi-Uda antenna.

We investigate a model bioassay in a liquid environment using a z-scanning planar Yagi-Uda antenna, focusing on the fluorescence collection enhancement of ATTO-647N dye conjugated to DNA (deoxyribonucleic acid) molecules. The antenna changes the excitation and the decay rates and, more importantly, the emission pattern of ATTO-647N, resulting in a narrow emission angle (41°) and improved collection efficiency. We efficiently detect immobilized fluorescently-labeled DNA molecules, originating from solutions with DNA concentrations down to 1 nM. In practice, this corresponds to an ensemble of fewer than 10 ATTO-647N labeled DNA molecules in the focal area. Even though we use only one type of biomolecule and one immobilization technique to establish the procedure, our method is versatile and applicable to any immobilized, dye-labeled biomolecule in a transparent solid, air, or liquid environment.

Optimization of Complete Rat Heart Decellularization Using Artificial Neural Networks.

Whole organ decellularization techniques have facilitated the fabrication of extracellular matrices (ECMs) for engineering new organs. Unfortunately, there is no objective gold standard evaluation of the scaffold without applying a destructive method such as histological analysis or DNA removal quantification of the dry tissue. Our proposal is a software application using deep convolutional neural networks (DCNN) to distinguish between different stages of decellularization, determining the exact moment of completion. Hearts from male Sprague Dawley rats (n = 10) were decellularized using 1% sodium dodecyl sulfate (SDS) in a modified Langendorff device in the presence of an alternating rectangular electric field. Spectrophotometric measurements of deoxyribonucleic acid (DNA) and total proteins concentration from the decellularization solution were taken every 30 min. A monitoring system supervised the sessions, collecting a large number of photos saved in corresponding folders. This system aimed to prove a strong correlation between the data gathered by spectrophotometry and the state of the heart that could be visualized with an OpenCV-based spectrometer. A decellularization completion metric was built using a DCNN based classifier model trained using an image set comprising thousands of photos. Optimizing the decellularization process using a machine learning approach launches exponential progress in tissue bioengineering research.

Novel Field-Effect Transistor Sensor for DNA Storage Monitoring

This article presents a novel open gate junction field-effect transistor (OG-JFET)-based sensor that can be used for various life science applications including deoxyribonucleic acid (DNA) storage monitoring. We put forward the design, modeling, implementation, and characterization of OG-JFET sensor using a foundry process through CMC Microsystems. We also demonstrate and discuss the functionality and applicability of the proposed sensor for monitoring DNA samples suitable for DNA storage applications. Synthetic storage has emerged as an intriguing data storage solution with high density and long-term preservation potential. Most common modalities include the conversion of digital data into synthesized nucleotides, the physical storage of DNA materials, and reading out the data via sequencing and other computational processes. Among these, this article tackles the challenge of physical storage monitoring of DNA materials by developing a sensor for measurement of DNA samples in dry conditions. The proposed sensor reveals a linear response of sensor toward DNA concentration in ultra-pure water. Across a 0.7 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> sensing area, a DNA mass concentration from approximately 100–400 ng/ <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu \text{L}$ </tex-math></inline-formula> has been detected using OG-JFET demonstrating a sensitivity of 30 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu \text{A}$ </tex-math></inline-formula> /(ng/ <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu \text{L}$ </tex-math></inline-formula> ). These performance quantities imply a promising role for OG-JFETs in emerging biotechnology applications including DNA storage monitoring.

An image-to-answer algorithm for fully automated digital PCR image processing.

The digital polymerase chain reaction (dPCR) is an irreplaceable variant of PCR techniques due to its capacity for absolute quantification and detection of rare deoxyribonucleic acid (DNA) sequences in clinical samples. Image processing methods, including micro-chamber positioning and fluorescence analysis, determine the reliability of the dPCR results. However, typical methods demand high requirements for the chip structure, chip filling, and light intensity uniformity. This research developed an image-to-answer algorithm with single fluorescence image capture and known image-related error removal. We applied the Hough transform to identify partitions in the images of dPCR chips, the 2D Fourier transform to rotate the image, and the 3D projection transformation to locate and correct the positions of all partitions. We then calculated each partition's average fluorescence amplitudes and generated a 3D fluorescence intensity distribution map of the image. We subsequently corrected the fluorescence non-uniformity between partitions based on the map and achieved statistical results of partition fluorescence intensities. We validated the proposed algorithms using different contents of the target DNA. The proposed algorithm is independent of the dPCR chip structure damage and light intensity non-uniformity. It also provides a reliable alternative to analyze the results of chip-based dPCR systems.

Molecular Level Study and Characterization of Plasmid Deoxyribonucleic Acid Profile of Acinetobacter baumannii Isolated from Diabetic Foot Ulcers

The main objective is to study the genotypic and characterization of plasmid deoxyribonucleic acid content of resistant isolates in Erbil city. A total of 39 Acinetobacter baumannii were isolated from 102 diabetic foot ulcer patients, whose ages ranged from 37 to 88 y, attending various medical wards of hospitals in Erbil city, Iraq, between May 2020 and March 2021. Isolates were tested for the presence of extended-spectrum beta-lactamases and metallo beta-lactamases. The presence and prevalence of multidrug-resistant isolates namely blaOXA-51- like, blaOXA-23-like, ISAba1 and outer membrane protein A expression of oxacillinase carbapenemases gene and biofilm formation respectively were screened on the molecular level. Finally, plasmid deoxyribonucleic acid profile was determined and the biofilm-forming capacity was measured. Most isolates showed high resistance to different antimicrobial agents tested, the most effective agents were colistin and polymyxin B. 12 (30.76 %) isolates were tested positive for extended-spectrum beta-lactamases, while all 39 (100 %) were metallo beta-lactamase producers. 27 (69.23 %) of isolates were biofilm producers, while 12 (30.76 %) were nonbiofilm producers. The blaOXA-51-like, blaOXA-23-like, ISAba1 and outer membrane protein A genes were detected in all 39 (100 %) isolates. The genes encoding resistance to certain antibiotics, in addition to genes that coding for extended-spectrum beta-lactamase and biofilm formation were located on plasmids. Analysis of plasmid deoxyribonucleic acid content revealed that 25 (64.10 %) isolates harbored from one to several plasmid bands ranging from 1 kilo base pairs to more than 10 kilo base pairs. No thermo-sensitive plasmids were detected. Due to the emergence of multidrug-resistance, Acinetobacter baumannii is evolving into a global health threat. Carbapenems are effective therapeutic agents for Acinetobacter baumannii nosocomial infections. Before deciding the course of treatment, it is important to consider the aforementioned interaction since the presence of multiple species may worsen the infection.

A simple and cost-effective method for DNA extraction suitable for PCR in “sucupira branca”

“Sucupira branca” is a plant found in the Brazilian Cerrado and is adapted to low fertility soils, and its fruit extract has anti-inflammatory, healing, antiulcerogenic, antimicrobial, cercaricidal, leishmanicidal and antioxidant activities. Furthermore, it provides protection against oxidative stress, is a natural biocontrol agent of Aedes aegypti, has very resistant wood, is a melliferous plant and has been used in reforestation programs. The development of conservation strategies is important for maintaining diversity in natural populations of “sucupira branca” since these populations are in the process of genetic erosion. Molecular biology techniques, which are important for characterizing the genetic diversity of plants to develop conservation strategies, require sufficient high-quality genomic deoxyribonucleic acid (DNA). This study aimed to compare five methods to extract DNA from “sucupira branca”. The quality and concentration of DNA were revealed by agarose gel electrophoresis, and only the protocols of Dellaporta, Wood and Hicks et al. (1983) and Khanuja et al. (1999) did not result in satisfactory quantities of DNA. When PCR (Polymerase Chain Reaction) was performed with three inter-simple sequence repeat (ISSR) primers, DNA was successfully amplified from extractions performed with the protocols proposed by Doyle and Doyle (1987), Romano and Brasileiro (1998) and Ferreira and Grattapaglia (1995), which are less expensive than commercial purification kits. These protocols resulted in DNA of sufficient quality and quantity after the amplification reactions were performed.