Lysis Method Research Articles

Extent of Tissue Washing Can Significantly Alter the Composition of Adipose-Derived Stromal Vascular Fraction Cell Preparations: Implications for Clinical Translation.

Stromal vascular fraction (SVF) cell preparations have recently attracted much interest as a form of autologous cell therapy. These heterogenous cell populations typically include some proportion of blood-derived cells (BDCs)-including both red blood cells (RBCs) and leukocytes (WBCs). The objectives of this paper were to evaluate the effects of tissue washing and hypotonic RBC lysis-separately and together-on BDC concentrations within SVF, and further to explore whether BDCs can confer detectable and modifiable effects on adipose-derived cell activity. Using various cell culture assays, flow cytometry and ELISA analysis of human-derived SVF preparations, we show that thorough washing of adipose tissue prior to enzymatic dissociation effectively removes RBCs from SVF preparations as well as standard lysis methods and significantly alters the type and relative quantities of WBCs. In addition, these studies demonstrate that potentially toxic RBC components are detectable for up to 1 week in cultures containing RBC lysate, but not those with intact RBCs, and, that culture-expanded cells proliferate significantly more in the presence of intact RBCs versus RBC lysis products or control media. Broadly, these data exemplify how different seemingly mundane tissue processing steps can significantly influence SVF identity/composition, purity, and potency. Based on the findings of this work, we propose that translational efforts in the field would benefit by a better understanding of the impact of RBCs, WBCs, and non-viable cells on the in vivo therapeutic activity of SVF therapies.

Selected Biomarkers of Coagulation Disturbance in Newly Diagnosed Breast Cancer Patients

Coagulation disturbance can be quite detrimental to health, whether as a primary disorder or secondary to other medical conditions. The risk of thrombosis has been previously reported in breast cancer. This case- control study purposively enrolled 50 newly diagnosed female breast cancer patients. Quick's One-stage method was used for prothrombin time (PT) and activated partial thromboplastin time (APTT) tests, while fibrin plate lysis method was used for euglobulin lysis time (ELT) assessment. Data analysis was carried out on statistical package for social sciences (SPSS) version 20.0. A p-value ≤ 0.05 was considered significant. Mean values of PT, APTT and ELT of newly diagnosed breast cancer patients were compared to values from control subjects. The ELT was significantly prolonged in the breast cancer patients compared to that of controls (p = 0.001). These measured parameters were further compared within the breast cancer group on the basis of hours of immobility per day into three categories (<3 hours, 3-6 hours and >6 hours). Subjects with more than 6 hours of immobility Subjects with more than 6 hours of immobility per day had significantly prolonged ELT compared to those with lesser hours of immobility. This study concludes that fibrinolytic challenge exists in breast cancer prior to commencement of treatment particularly among patients with increasing immobility.

Analysis of frequency and molecular genetics of Jk (a-b-) phenotype among blood donors from Jining area

To screen for Jk(a-b-) phenotype among blood donors from Jining area and explore its molecular basis to enrich the rare blood group bank for the region. The population who donated blood gratuitously at Jining Blood Center from July 2019 to January 2021 were selected as the study subjects. The Jk(a-b-) phenotype was screened with the 2 mol/L urea lysis method, and the result was confirmed by using classical serological methods. Exons 3 to 10 of the SLC14A1 gene and its flanking regions were subjected to Sanger sequencing. Among 95 500 donors, urea hemolysis test has identified three without hemolysis, which was verified by serological method as the Jk(a-b-) phenotype and demonstrated no anti-Jk3 antibody. The frequency of the Jk(a-b-) phenotype in Jining area is therefore 0.0031%. Gene sequencing and haplotype analysis showed that the genotypes of the three samples were JK*02N.01/JK*02N.01, JK*02N.01/JK-02-230A and JK*02N.20/JK-02-230A, respectively. The splicing variant of c.342-1G>A in intron 4, missense variants of c.230G>A in exon 4, and c.647_ 648delAC in exon 6 probably underlay the Jk(a-b-) phenotype in the local population, which is different from other regions in China. The c.230G>A variant was unreported previously.

Development and validation of isothermal polymerase spiral reaction assay for the specific authentication of goat (Capra hircus) meat

Goat meat (chevon) is one of the costlier meats and closely resembles other red meat species mostly the sheep meat (mutton). In order to establish authenticity of chevon and confirm mislabelling there is a need for a reliable laboratory test. Polymerase spiral reaction (PSR) assay is one of the recent DNA based isothermal tests available for molecular analysis mostly microorganisms. Present study describes development and validation of a novel PSR for the detection of chevon for the first time. The goat specific PSR makes use of a pair of chevon-specific primers targeted against the mitochondrial DNA and test could be read based on the visual colour change after the addition of SYBR Green I dye to the amplified product. The possibility of cross-amplification was ruled out by testing related meat species ( buffalo, cattle, chicken, pig, sheep, and ostrich) and results of PSR were further confirmed by fluorometry and agarose gel electrophoresis. PSR could be undertaken at isothermal temperature of 62 °C temperature for 60 min with the limit of detection of 0.5 ng DNA and detection of meat admixing of 0.1%. The PSR was found efficient in the detection of not only raw meat but also meat heated to 121 °C for 30 min. When combined with the alkaline lysis method of DNA extraction, the goat-specific PSR takes only 90 min of sample analysis time and could be used for the rapid analysis of field samples.

Engineering osmolysis susceptibility in Cupriavidus necator and Escherichia coli for recovery of intracellular products

BackgroundIntracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis. ResultsTo overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed.ConclusionsOur strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.

MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples.

Recently, urinary tract infection (UTI) triggered by bacteria carrying pan-drug-resistant genes, including carbapenem resistance gene blaNDM and blaKPC, colistin resistance gene mcr-1, and tet(X) for tigecycline resistance, have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of-care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 102 CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug-resistant genes in clinical UTI samples and provides the necessary medication guidance for UTI treatment.

Potensi Ekstrak Kasar Enzim dari Tempe Kedelai Hitam (Glycine soja (L.) Merr.) sebagai Obat Fibrinolitik Alami dengan Metode Clot Lysis In Vitro

Fibrinolytic enzymes are enzymes that can be used to break down fibrin. In thrombus therapy (cardiovascular disease), this enzyme has an activity similar to plasmin, which is able to reduce fibrin by hydrolyzing fibrin into dissolved products and inhibiting the formation of fibrin clots that can trigger cardiovascular disease. Black soybean tempeh has various benefits, one of which is to lower the risk of cardiovascular disease and reduce the risk of blood clots. Due to the content of the enzyme protease in black soybeans which is able to degrade fibrin threads. The purpose of this study is to determine the potential of black soybean tempeh (Glycine soja (L.) Merr) can produce enzyme extracts as natural fibrinolytic drugs. This study was conducted by taking ingredients, crude extraction of enzymes from black soybean tempeh by centrifugation, purification of crude extracts of enzymes using ammonium sulfate salts of 80%, determination of enzyme protein levels with the Lowry method and testing of fibrinolytic potential with the clot lysis method in vitro. The variation in the concentration of the extract used in the test was 12.5; 25; 50 and 100%. The results showed that crude extracts of black soybean tempeh enzymes were able to lyse blood clots. The protein content of the black soybean tempeh extract sample after purification using ammonium sulfate was 245.76 ?g/mL. The optimum percentage of blood clot lysis at a concentration of 100% of the sample after purification was 59%.

High prevalence of multidrug-resistant bacteria in fomites in a tertiary institution in Southwestern Nigeria

<b>Background</b><b>: </b>The emergence and spread of multidrug-resistant (MDR) bacteria have become a major threat to public and environmental health because they limit treatment options against infections. Fomites have been implicated in the spread of pathogenic and resistant bacteria, making them an important source of disease. This study was carried out to determine the frequency and characteristics of resistant bacteria linked with environmental fomites in a tertiary institution in Nigeria as part of global efforts to provide information to contain the spread of antimicrobial resistance.<br /> <b>Methods: </b>300 swab samples, determined by factorial design, were aseptically collected from 10 fomites in 10 locations in three different facilities at Obafemi Awolowo University, Nigeria, and transported to the laboratory for processing. The isolates were identified by standard procedure and the Microbact<sup>TM</sup> identification kit. The susceptibility profile of the isolates was determined using Kirby-Bauer disc diffusion technique. MDR bacteria were examined for the presence of plasmids using the alkali lysis method, and plasmid-mediated quinolone resistance, extended-spectrum beta-lactamase, and methicillin resistance genes by polymerase chain reaction.<br /> <b>Results:</b> 89 diverse bacteria were isolated from fomites in the study environment. <i>Staphylococcus aureus </i>(49.4%) predominated among the bacteria, followed by <i>Staphylococcus saprophyticus</i> (10.1%), <i>Providencia stuartti</i> (5.6%), <i>Proteus vulgaris</i> (4.5%), and<i> Acinetobacter baumannii</i> (3.4%). The isolates were commonly (84%-100%) resistant to ceftazidime, cefuroxime, and augmentin, while the least (8%-12.5%) resistance was to ciprofloxacin, amikacin, meropenem, and streptomycin. 58 (65.2%) isolates were MDR, and the majority (27.6%) were isolated from floors. 47 (81%) MDR isolates harbored plasmids, with their molecular weights between 2.697 kbp and 4.011 kbp. <i>mecA</i>, <i>TEM,</i> and <i>OqXAB</i> resistance genes were detected in <i>S. aureus</i>, <i>P. stuarttii</i>, <i>Pseudomonas </i>sp, and <i>K. oxytoca</i>.<br /> <b>Conclusions:</b> This study has shown that fomites in the sampled locations are potential reservoirs for MDR bacteria, and may serve as sources of their transmission, thereby posing a threat to public health. Therefore, appropriate sanitary measures are required to prevent any potential cross-contamination. In addition, it is imperative to regularly survey environments for bacterial contamination to increase community awareness and education regarding hygiene standards.

Comparative of Forensic DNA Identification Using Cell Lysis Method and Magnetic Beads Method.

To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification. The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios. When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method. The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.

A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples

A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.

Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples

Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.

Phosphoproteomic analysis of human umbilical venous endothelial cells with DENV-2 infection

To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.

Isolation of DNA from plant tissues using a miniaturized matrix solid-phase dispersion approach featuring ionic liquid and magnetic ionic liquid solvents

The isolation of high-quality plant genomic DNA is a major prerequisite in many plant biomolecular analyses involving nucleic acid amplification. Conventional plant cell lysis and DNA extraction methods involve lengthy sample preparation procedures that often require large amounts of sample and chemicals, high temperatures and multiple liquid transfer steps which can introduce challenges for high throughput applications. In this study, a simple, rapid, miniaturized ionic liquid (IL)-based extraction method was developed for the isolation of genomic DNA from milligram fragments of Arabidopsis thaliana plant tissue. This method is based on a modification of vortex-assisted matrix solid-phase dispersion (VA-MSPD) in which the trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14+][NTf2-]) IL or trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P6,6,6,14+][Ni(hfacac)3-]) magnetic IL (MIL) was directly applied to treated plant tissue (∼1.5 mg) and dispersed in an agate mortar to facilitate plant cell lysis and DNA extraction, followed by recovery of the mixture with a qPCR compatible co-solvent. This study represents the first approach to use ILs and MILs in a MSPD procedure to facilitate plant cell lysis and DNA extraction. The DNA-enriched IL- and MIL-cosolvent mixtures were directly integrated into the qPCR buffer without inhibiting the reaction while also circumventing the need for additional purification steps prior to DNA amplification. Under optimum conditions, the IL and MIL yielded 2.87 ± 0.28 and 1.97 ± 0.59 ng of DNA/mg of plant tissue, respectively. Furthermore, the mild extraction conditions used in the method enabled plant DNA in IL- and MIL-cosolvent mixtures to be preserved from degradation at room temperature.

In vitro cytotoxic, thrombolytic, anthelmintic and antioxidant activities of Litsea monopetala: a medicinal plant

The present study was conducted to assess the cytotoxicity, thrombolytic, anthelmintic and antioxidant activity of mehanolic extract of Litsea monopetala (Family: Lauraceae) leaves in laboratory using in vitro methods. Cytotoxicity test was done by brine shrimp lethality bioassay where the extract concentration was 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 (µg/ml). In vitro thrombolytic activity of Litsea monopetala was performed by clot lysis method using extract concentration 2.5, 5, 10 and 20 (mg/mL) in saline water. Anthelmintic activity test was done by using adult earthworms where 10, 20, 40, 60, 80 (mg/ml) extract concentration were used. Finally antioxidant activity was determined by total phenolic content determination using Folin-Ciocalteu reagent. The Litsea monopetala extract showed cytotoxic activity against brine shrimp nauplii and LC50 value was 41.05(µg/ml) and the investigated thrombolytic activity in our research was 9.52, 9.49, 13.64 and 17.50 % respectively as % of clot lysis. The paralysis time were at 76.75 min, 60 min, 51.75 min, 44.5 min and 64.5 min and death were at 90.50min, 63.75min, 55.50min, 44.75min and 71min. respectively. The Litsea monopetala extract displayed significant antioxidant activity which was 20.75 (mg of GAE / gm) of extracts. The activities observed could be attributed to presence of some of the phytochemicals which have been related with cytotoxic, thrombolytic, anthelmintic and antioxidant property.

Electrolysis of Bacteria Based on Microfluidic Technology.

Cell lysis is a key step for studying the structure and function of proteins in cells and an important intermediate step in drug screening, cancer diagnosis, and genome analysis. The current cell lysis methods still suffer from limitations, such as the need for large instruments, a long and time-consuming process, a large sample volume, chemical reagent contamination, and their unsuitability for the small amount of bacteria lysis required for point-of-care testing (POCT) devices. Therefore, a fast, chemical-free, portable, and non-invasive device needs to be developed. In the present study, we designed an integrated microfluidic chip to achieve E. coli lysis by applying an alternating current (AC) electric field and investigated the effects of voltage, frequency, and flow rate on the lysis. The results showed that the lysis efficiency of the bacteria was increased with a higher voltage, lower frequency, and lower flow rate. When the voltage was at 10 Vp-p, the lysis efficiency was close to 100%. The study provided a simple, rapid, reagent-free, and high-efficiency cleavage method for biology and biomedical applications involving bacteria lysis.

In-droplet cell lysis of AC16 human cardiomyocyte cells via surface acoustic waves.

Although several lysis methods are available, biomedical applications are pushing the demand for miniaturised systems and thus for new ways to lyse cells in small volumes. In this work, we demonstrate in-droplet cell lysis of AC16 human cardiomyocyte cells in 20 μL droplets using high frequency surface acoustic waves. The acoustic streaming leads to high shear flow creating porous or breaking the cell membrane and releasing intracellular material. Contrary to previous work where the lysis efficiency is measured by a cell-permeant dye that can be used to determine cell viability, here we propose to quantify the DNA extracted from the cells as a measure of the lysis efficiency. This reagent-free method provides a valuable cell lysis alternative for many biological and biomedical applications, particularly for the development of point-of-care platforms.

HighPressure Homogenization for Inclusion Body Isolation.

Highpressure homogenization (HPH) is a commonly used method for cell lysis of Escherichia coli in order to release intracellularly produced recombinant proteins. For misfolded proteins in E. coli, focus is often put on the development of a suitable solubilization and refolding protocol. However, HPH can be a critical unit operation influencing inclusion body (IB) quality and, subsequently, refolding yields. Here, a protocol for homogenization and IB washing is presented in combination with analytical methods suitable to evaluate these unit operations. The protocol is based on a multivariate approach to identify suitable conditions during HPH. Furthermore, the described workflow is easily scalable and can, therefore, also be used if fixed homogenization conditions are already established.

Pharmacodynamics of frigid zone plant Taxus cuspidata S. et Z. against skin melanin deposition, oxidation, inflammation and allergy

Abstract Background Taxus cuspidata S. et Z. is a precious species of frigid zone plant belonging to the Taxaceae family, which possesses anticancer, anti-inflammatory, hypoglycemic, and antibacterial pharmacological properties. While taxane extracted from Taxus chinensis has been reported to elicit antioxidant activities, whether Taxus cuspidata S. et Z. has skin-protective actions against injuries remained unknown. This study aims to explore the pharmacological effects of three Taxus extracts on skin melanin deposition, oxidation, inflammation, and allergy so as to provide new ideas for the prevention and treatment of various diseases related to skin damage. Methods Skin melanin deposition was evaluated by measuring melanin content in the skin of guinea pigs by alkali lysis method. Antioxidant capacity was evaluated by measuring superoxide dismutase (SOD) concentration and glutathione (GSH) content in skin tissue homogenates of Kunming mice by SOD assay kit and micro reduced GSH assay kit. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to examine the levels of both SOD and recombinant glutathione peroxidase 4 (GPX4). Skin inflammation was evaluated by xylene-induced ear swelling test and egg-white-induced paw swelling test in mice. In a mouse model of skin allergy induced by 4-aminopyridine (4-AP), allergy was determined by licking body counts and histamine concentrations in tissue homogenates using enzyme-linked immunosorbent assay (ELISA) kits. Two proinflammatory factors tumor necrosis factor (TNF)-α and interleukin (IL)-1β were measured by qRT-PCR. Hematoxylin and eosin (HE) staining was conducted to assess the degree of skin lesion. Results All three Taxus extracts including Taxus chinensis essential oil, Taxus chinensis extract and Taxus chinensis extract compound reduced the melanin deposits in the back skin relative to the non-treated control animals, of which Taxus chinensis essential oil produced the greatest effect. In contrast, the three Taxus extracts elevated SOD and GSH levels in the skin tissues, and the highest increase was seen with Taxus chinensis essential oil. Three Taxus extracts, especially Taxus chinensis essential oil, effectively reduce the rate of ear and paw swelling. All three Taxus extracts reduced the number of body licks, the levels of TNF-α and IL-1β, and the histamine content in tissue homogenates of mice and alleviated skin damage. Consistently, Taxus chinensis essential oil yielded the greatest magnitude of decreases. Conclusion While all three Taxus extracts possessed the anti-skin melanin deposition, oxidation, and allergy properties, Taxus chinensis essential oil produced the superior effects.

Optimization of High Concentration Plasmid DNA for Use in COVID-19 mRNA Vaccine Development: Comparison of Between Alkaline Lysis Method and Commercial Kit Results

While forming the stable IVT mRNA molecule, high concentration and purity plasmid DNA must be obtained to ligase the ORF antigen sequence initially copied from the plasmid DNA with the UTR regions. In this study, in the stage of creating the mRNA molecule, which is the first step of the COVID-19 mRNA vaccine, comparison and optimization of the pDNA containing the ORF target antigen sequence were performed as a result of isolation with alkaline lysis method and commercial kit. Plasmid DNA bacteria containing the target antigen ORF sequence were grown under appropriate conditions. Plasmid DNA was isolated by commercial kit and alkaline lysis method from bacterial cultures stopped at different OD600 nm values (0.02-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5). After the obtained pDNAs were visualized on an agarose gel, their purity and concentration were measured by spectroscopic measurement. After the stab culture is resuscitated in SOC medium, bands are formed in a single form after isolation with the kit, and in multiple forms (linear, supercoiled, circular) after pDNA isolation by alkaline lysis method. The ideal OD600 nm for both methods was 0.3-0.4. As a result of isolation with the kit, higher purity on the contrary low concentration pDNA was obtained. The ideal OD600 nm value is a critical parameter that affects the concentration and purity of pDNA. The alkaline lysis method is a cheap and powerful technique that can be used as an alternative for mRNA vaccine development compared to kit isolation.

Optimization and validation of a protein phosphatase inhibition assay for accessible microcystin detection

The presence of cyanobacterial toxins in freshwater constitutes an increasing public health concern, especially affecting developing countries where the high cost of available methods makes monitoring programs difficult. The phosphatase inhibition assay (PPIA) is a sensitive method with low instrument requirements that allows the quantification of the most frequent cyanotoxins, microcystins (MCs). In this work, we implemented a PPIA, starting from Protein Phosphatase 1 (PP1) expression up to the validation with samples of algal blooms from Argentina. To do this, we optimized the expression and lyophilization of PP1, and the assay conditions. Also, we included robustness and possible interference analysis. We evaluated the most widely used cyanobacterial lysis methods and determined that heating for 15 min at 95 °C is simple and adequate for this assay. Then, we performed MC spikes recovery assays on water samples from three dams from Argentina, resulting in a recovery ranging from 77 to 115%. The limit of detection (LOD) was 0.4 μg/L and the linear range is 0.4 μg/L - 5 μg/L. Finally, we evaluated 65 environmental samples where MCs was measured by ELISA test containing from 0 μg/L to 625 μg/L. The PPIA showed excellent correlation (Pearson correlation coefficient = 0.967), no false negative and no false positives above the 1 μg/L WHO guideline (0.11 false positive rate). In conclusion, we optimized and validated a PPIA to be an effective and accessible alternative to available commercial tests.