Lysis Solution Research Articles

Validation of the performance of a point of care molecular test for leprosy: From a simplified DNA extraction protocol to a portable qPCR.

The study aimed to optimize qPCR reactions using oligonucleotides from the first Brazilian molecular diagnostic kit for leprosy on a portable platform (Q3-Plus). In addition, we sought to develop a simplified protocol for DNA extraction that met point-of-care criteria. During optimization on the Q3-Plus, optical parameters, thresholds, and cutoffs for the 16S rRNA and RLEP targets of M. leprae were established using synthetic DNA, purified DNA from M. leprae, and pre-characterized clinical samples. For the simplified extraction protocol, different lysis solutions were evaluated using chaotropic agents, and purification was carried out by transferring the lysed material to FTA cards. The complete protocol (simplified extraction + qPCR on the portable platform) was then evaluated with pre-characterized clinical skin biopsy samples and compared with standard equipment (QuantStudio-5). LOD95% for the optimized reactions was 113.31 genome-equivalents/μL for 16S rRNA and 17.70 genome-equivalents/μL for RLEP. Among the lysis solutions, the best-performing was composed of urea (2 M), which provided good dissolution of the skin fragment and a lower Ct value, indicating higher concentrations of DNA. The complete technological solution showed a sensitivity of 52% in reactions. Our results highlight the need for additional optimization to deal with paucibacillary samples, but also demonstrate the feasibility of the portable platform for the qPCR detection of M. leprae DNA in low infrastructure settings.

Development and validation of a rapid five-minute nucleic acid extraction method for respiratory viruses

BackgroundThe rapid transmission and high pathogenicity of respiratory viruses significantly impact the health of both children and adults. Extracting and detecting their nucleic acid is crucial for disease prevention and treatment strategies. However, current extraction methods are laborious and time-consuming and show significant variations in nucleic acid content and purity among different kits, affecting detection sensitivity and efficiency. Our aim is to develop a novel method that reduces extraction time, simplifies operational steps, and ensures high-quality acquisition of respiratory viral nucleic acid.MethodsWe extracted respiratory syncytial virus (RSV) nucleic acid using reagents with different components and analyzed cycle threshold (Ct) values via quantitative real-time polymerase chain reaction (qRT-PCR) to optimize and validate the novel lysis and washing solution. The performance of this method was compared against magnetic bead, spin column, and precipitation methods for extracting nucleic acid from various respiratory viruses. The clinical utility of this method was confirmed by comparing it to the standard magnetic bead method for extracting clinical specimens of influenza A virus (IAV).ResultsThe solution, composed of equal parts glycerin and ethanol (50% each), offers an innovative washing approach that achieved comparable efficacy to conventional methods in a single abbreviated cycle. When combined with our A Plus lysis solution, our novel five-minute nucleic acid extraction (FME) method for respiratory viruses yielded superior RNA concentrations and purity compared to traditional methods. FME, when used with a universal automatic nucleic acid extractor, demonstrated similar efficiency as various conventional methods in analyzing diverse concentrations of respiratory viruses. In detecting respiratory specimens from 525 patients suspected of IAV infection, the FME method showed an equivalent detection rate to the standard magnetic bead method, with a total coincidence rate of 95.43% and a kappa statistic of 0.901 (P < 0.001).ConclusionsThe FME developed in this study enables the rapid and efficient extraction of nucleic acid from respiratory samples, laying a crucial foundation for the implementation of expedited molecular diagnosis.

Sodium alginate hydrogel encapsulating microglia cell lysate subjected to serum starvation for mitigating glioma cells.

Glioma is the most common malignant tumor in the brain, accounting for over 80% of all primary intracranial tumors. The current clinical treatment has shown certain limitations. Although M1 type microglia can secrete various pro-inflammatory cytokines and are expected to be used for glioma treatment, direct use of microglia may lead to overactivation and trigger immune storms. Therefore, we first found that serum starvation can stimulate the transformation of microglia into M1 type. Subsequently, we found through comparative experiments that the inhibitory effect of microglial cell lysis medium on glioma cells was stronger than that of microglial cell culture medium. Finally, we successfully prepared sodium alginate hydrogel loaded with microglia lysis solution to achieve sustained inhibitory effect on the growth of glioma and avoid its proliferation.

Establishment and Optimization of an Experiment System for Flow Cytometry in Oil-Seed Camellia

Ploidy identification is a basic method for determining germplasm resources and for breeding new varieties of oil-seed camellia. In this study, flow cytometry and K-mer analysis were used to identify the ploidy of oil-seed camellia germplasms. To determine the best tissue organ type, lysis time, and dyeing time, evaluation indices such as the presence of a clear main peak, the ease of sampling, and the coefficient of variation were used. A technique was established, and the ploidies of different oil-seed camellia germplasms were identified. The results showed that pollen was the best material and that using a 400 mL PI lysis solution for 10 min lysis, followed by dyeing with a 1600 mL DAPI dyeing solution for 10 min, was the most suitable technique. According to the peak value of the control diploid Camellia azalea, 15 samples were estimated to be diploid, 24 samples were tetraploid, 18 samples were hexaploid, and 13 samples were octoploid. In addition, the K-mer analysis results showed that among the five samples, CK, C051, and C047 were diploid, while C037 and C043 were tetraploid, results that are consistent with the results of the flow cytometry identification. This study is therefore valuable for the polyploid selection and use of different ploidy germplasm resources for the cross breeding of oil-seed camellia.

Guanidinium Chloride-Induced Haemolysis Assay to Measure New Permeation Pathway Functionality in Rodent Malaria Plasmodium berghei.

Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable Plasmodium parasites to take up nutrients from the plasma to facilitate their survival. Thus, NPPs represent a potential novel therapeutic target for malaria. The putative channel component of the NPP in the human malaria parasite P. falciparum is encoded by mutually exclusively expressed clag3.1/3.2 genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three clag paralogs whose contribution to the P. falciparum channel is uncertain. Rodent malaria P. berghei contains only two clag genes, and thus studies of P. berghei clag genes could significantly aid in dissecting their overall contribution to NPP activity. Previous methods for determining NPP activity in a rodent model have utilised flux-based assays of radioisotope-labelled substrates or patch clamping. This study aimed to ratify a streamlined haemolysis assay capable of assessing the functionality of P. berghei NPPs. Several isotonic lysis solutions were tested for their ability to preferentially lyse infected RBCs (iRBCs), leaving uninfected RBCs (uRBCs) intact. The osmotic lysis assay was optimised and validated in the presence of NPP inhibitors to demonstrate the uptake of the lysis solution via the NPPs. Guanidinium chloride proved to be the most efficient reagent to use in an osmotic lysis assay to establish NPP functionality. Furthermore, following treatment with guanidinium chloride, ring-stage parasites could develop into trophozoites and schizonts, potentially enabling use of guanidinium chloride for parasite synchronisation. This haemolysis assay will be useful for further investigation of NPPs in P. berghei and could assist in validating its protein constituents.

Spectroscopic insights into multi-phase protein crystallization in complex lysate using Raman spectroscopy and a particle-free bypass.

Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity. However, monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements. Especially for capturing proteins from complex feedstock containing various impurities, establishing reliable process analytical technology (PAT) to monitor protein crystallization processes can be complicated. In heterogeneous mixtures, important product characteristics can be found by multivariate analysis and chemometrics, thus contributing to the development of a thorough process understanding. In this project, an analytical set-up is established combining offline analytics, on-line ultraviolet visible light (UV/Vis) spectroscopy, and in-line Raman spectroscopy to monitor a stirred-batch crystallization process with multiple phases and species being present. As an example process, the enzyme Lactobacillus kefir alcohol dehydrogenase (LkADH) was crystallized from clarified Escherichia coli (E. coli) lysate on a 300mL scale in five distinct experiments, with the experimental conditions changing in terms of the initial lysate solution preparation method and precipitant concentration. Since UV/Vis spectroscopy is sensitive to particles, a cross-flow filtration (cross-flow filtration)-based bypass enabled the on-line analysis of the liquid phase providing information on the lysate composition regarding the nucleic acid to protein ratio. A principal component analysis (PCA) of in situ Raman spectra supported the identification of spectra and wavenumber ranges associated with productspecific information and revealed that the experiments followed a comparable, spectral trend when crystals were present. Based on preprocessed Raman spectra, a partial least squares (PLS) regression model was optimized to monitor the target molecule concentration in real-time. The off-line sample analysis provided information on the crystal number and crystal geometry by automated image analysis as well as the concentration of LkADH and host cell proteins (HCPs) In spite of a complex lysate suspension containing scattering crystals and various impurities, it was possible to monitor the target molecule concentration in a heterogeneous, multi-phase process using spectroscopic methods. With the presented analytical set-up of off-line, particle-sensitive on-line, and in-line analyzers, a crystallization capture process can be characterized better in terms of the geometry, yield, and purity of the crystals.

Bulk lysis procedures alter target cell population counts.

To achieve high-sensitivity cell measurements (<1 in 105 cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents.

A methodological exploration of distinguishing hair quality based on hair proteomics

Hair is an advantageous biological sample due to its recordable, collectable, and storable nature. Hair's primary components are keratin and keratin-associated proteins. Owing to its abundance of cystine, keratin possesses impressive mechanical strength and chemical stability, formed by creating disulfide bonds as crosslinks within the protein peptide chain. Furthermore, keratin is cross-linked with keratin-associated proteins to create a complex network structure that provides the hair with strength and rigidity. Protein extraction serves as the foundation for hair analysis research. Bleaching hair causes damage to the structure between keratin and keratin-associated proteins, resulting in texture issues and hair breakage. This article outlines various physical treatment methods and lysate analysis that enhance the efficiency of hair protein extraction. The PLEE method achieves a three-fold increase in hair protein extraction efficiency when using a lysis solution containing SDS and combining high temperatures with intense shaking, compared to previous methods found in literature. We utilized the PLEE method to extract hair from both normal and damaged groups. Normal samples identified 156–157 proteins, including 51 keratin and keratin-associated proteins. The damaged group consisted of 155–158 identified proteins, of which 48–50 were keratin and keratin-associated proteins. Bleaching did not cause any notable difference in the protein identification of hair. However, it did reduce coverage of keratin and keratin-associated proteins significantly. Our hair protein extraction method provides extensive coverage of the hair proteome. Our findings indicate that bleaching damage results in subpar hair quality due to reduced coverage of protein primary sequences in keratin and keratin-associated proteins.

Carboxymethyl chitosan/alginate/polyethylene glycol composite hydrogel for nucleic acid lysis solution storage at the point of care

Carboxymethyl chitosan/alginate/polyethylene glycol composite hydrogel for nucleic acid lysis solution storage at the point of care

Bone Proteomics Method Optimization for Forensic Investigations.

The application of proteomic analysis to forensic skeletal remains has gained significant interest in improving biological and chronological estimations in medico-legal investigations. To enhance the applicability of these analyses to forensic casework, it is crucial to maximize throughput and proteome recovery while minimizing interoperator variability and laboratory-induced post-translational protein modifications (PTMs). This work compared different workflows for extracting, purifying, and analyzing bone proteins using liquid chromatography with tandem mass spectrometry (LC-MS)/MS including an in-StageTip protocol previously optimized for forensic applications and two protocols using novel suspension-trap technology (S-Trap) and different lysis solutions. This study also compared data-dependent acquisition (DDA) with data-independent acquisition (DIA). By testing all of the workflows on 30 human cortical tibiae samples, S-Trap workflows resulted in increased proteome recovery with both lysis solutions tested and in decreased levels of induced deamidations, and the DIA mode resulted in greater sensitivity and window of identification for the identification of lower-abundance proteins, especially when open-source software was utilized for data processing in both modes. The newly developed S-Trap protocol is, therefore, suitable for forensic bone proteomic workflows and, particularly when paired with DIA mode, can offer improved proteomic outcomes and increased reproducibility, showcasing its potential in forensic proteomics and contributing to achieving standardization in bone proteomic analyses for forensic applications.

Biomolecules-mediated electrochemical signals of Cu2+: Y-DNA nanomachines enable homogeneous rapid one-step assay of lung cancer circulating tumor cells

This study presents a straightforward efficient technique for extracting circulating tumor cells (CTCs) and a rapid one-step electrochemical method (45 min) for detecting lung cancer A549 cells based on the specific recognition of mucin 1 using aptamers and the modulation of Cu2+ electrochemical signals by biomolecules. The CTCs separation and enrichment process can be completed within 45 min using lymphocyte separation solution (LSS), erythrocyte lysis solution (ELS), and three centrifugations. Besides, the influence of various biomolecules on Cu2+ electrochemical signals is comprehensively discussed, with DNA nanospheres selected as the medium. Three single-stranded DNA sequences were hybridized to form Y-shaped DNA (Y-DNA), creating DNA nanospheres. Upon specific capture of mucin 1 by the aptamer, most DNA nanospheres could form complexes with Cu2+ (DNA nanosphere-Cu2+), significantly reducing the concentration of free Cu2+. Our approach yielded the limit of detection (LOD) of 2 ag/mL for mucin 1 and 1 cell/mL for A549 cells. 39 clinical blood samples were used for further validation, yielding results closely correlated with pathological, computed tomography (CT) scan findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve displayed an area under the curve (AUC) value of 0.960, demonstrating 100% specificity and 93.1% sensitivity for the assay. Taken together, our findings indicate that this straightforward and efficient pretreatment and rapid, highly sensitive electrochemical assay holds great promise for liquid biopsy-based tumor detection using CTCs.

Development of a Genus Identification Method for Poisonous Plants Using Real-Time PCR

We have developed a rapid genus identification method for poisonous plants. The real-time PCR using the TaqMan® probe method was employed for detection, with the amplified targets being the "trnL (UAA)-intron" or "trnL-trnF intergenic spacer" regions of chloroplast DNA. The targeted plants were selected six genera (Aconitum, Colchicum, Veratrum, Brugmansia, Scopolia and Narcissus), which have been implicated in many instances of food poisoning in Japan. A tissue lysis solution was used for DNA extraction, which can be completed within approximate 30 min. A master mix corresponding to the tissue lysis solution was used for real-time PCR reagents. As a result, we were able to complete the entire process from DNA extraction to genus identification in 4 to 5 hr. The detection sensitivity was estimated at approximately 1 pg of DNA for all six plant genera. Remarkably, an amplification plot was discerned even with the crude cell lysates of all samples. It was also possible to obtain amplification curves for three plant samples that had been subjected to simulated cooking (boiling). This study suggests that the developed method can rapidly identify six genera of poisonous plants.

Continuous microfluidic platform combining cell lysis and protein extraction for screening overall process conditions

AbstractBACKGROUNDMicrofluidic devices have been increasingly used as tools to accelerate the development of new biomanufacturing methods, given their ability to test a large set of variables in a short amount of time with minimal reagent consumption. However, while being able to expedite a bioprocess design, the modular aspect of these devices has not been systematically explored, with most applications focusing on single unitary operations. This paper explores the modular design of these devices through the development of an integrated microfluidic chip.RESULTSTwo separate unitary operations were included into a single device for continuous operation: product release by chemical cell lysis and product concentration by aqueous two‐phase system. In this way, it is possible to screen multiple conditions for each operation and evaluate their combined effect on the final product, making this a great tool in the development and optimization of a process. A recombinant Escherichia coli (E. coli) strain producing green fluorescent protein (GFP) was used as model system, which allows for lysis efficiency and partition coefficient to be evaluated by fluorescence microscopy. A chemical lysis solutions was used in the lysis step, presenting lysis efficiencies of about 100%, while a polyethylene glycol (PEG)/phosphate system was screened for the separation stage, presenting partition coefficients of about 4.CONCLUSIONSThe integrated device allowed for the continuous screening of multiple combinations of operation conditions, highlighting, for example, conditions where improvements on the lysis efficiency subsequently impaired the separation, decreasing the partition of the target product, thus demonstrating the need for the evaluation of a process in its entirety. In this way, the microfluidic device delivered a rapid process screening and optimization, with very low reagent consumption, and using a simple fluorescence microscope for data collection. © 2023 Society of Chemical Industry (SCI).

Modified phenol chloroform protocol for genomic DNA extraction from keratinous tissue

DNA extraction enables the isolation and purification of genetic material by physical-chemical methods. There are specific protocols for different types of samples as animal and plant tissue. However, there are variations and particularities according to the nature of the fabric, such as the high presence of keratin. Thus, the present work aimed to develop and optimize a DNA extraction protocol from feather based on the Phenol-Chloroform method. Feather samples werw collected from 9 birds native to the Amazon region for the genetic material extraction. The DNA was precipitated with isopropanol and washed with 70% ethanol. Subsequently, the pellet was disolved in the TE buffer. The results indicated satisfactory concentrations for post-extraction molecular biology techniques such as PCR. The protocol showed a significant reduction in the incubation time to 1 hour compared to other protocols aimed at feather samples. Addition proteinase K to the lysis solution was the main factor that led to the optimization of the extraction time. The A260/230 and A260/280 absorbance ratios indicated the quality of the extracted DNA being close to what expected. Therefore, the results demonstrate the potential protocol use for molecular biology studies in birds from feather samples. However volume of phenol-chloroform and 70% ethanol can still be adjusted to suit the type sample.

An integrated microfluidic device for continuous bioprocessing

Microfluidic devices have been increasingly used as tools to accelerate the development of new biomanufacturing methods, given their ability to test a large set of variables in a short time, with minimal reagent consumption. However, while being able to expedite a bioprocess design, the modular aspect of these microfluidic devices has not been systematically explored, with most applications focusing on single unitary operations. Integrated continuous bioprocessing has the potential to reduce cost, optimize yield and reduce footprint as compared to batch operation. In this work an integrated microfluidic device for continuous processing is developed and qualitatively shows connected different unit operations able to produce GFP in microbioreactor, extract out of cells in lysis module and finally concentrate in ATPs module.The integrated system developed herein consists of a micro-chemostat module to produce a target protein in continuous mode, a chemical cell lysis module for protein release and a module to purify and concentrate the protein using aqueous two-phase systems (ATPS). In this way, it is possible to screen multiple conditions for each operation and evaluate their combined effect on the final product. A recombinant Escherichia coli (E. coli) strain producing green fluorescent protein (GFP) was used as a model system, which allows for GFP production, lysis efficiency and partition coefficient to be evaluated by fluorescence microscopy. Experiments were done for over a week by continuously supplying medium and monitoring the GFP production by fluorescence intensity. A chemical lysis solution B-PER® was used for continuous lysis and afterwards the extracted GFP was clarified and concentrated in the PEG-rich phase of a PEG/phosphate ATPS with a partition coefficient of 2. As the integrated device is working in continuous mode it could be integrated with further downstream processing modules.

Four Decades of the Comet Assay: pH Optimum of Lysis Buffer Still Needs to be Elucidated.

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer. Comet assay is broadly used for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25°C and 8.69 at 4°C. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.

Bone marrow concentration in combination with hyaluronan and fibrin for the treatment of primary and non-primary in orthopaedic cases of osteochondral defects of the ankle.

This research examined the effects of varying amounts of fibrinogen (the fibrin precursor) and HA-MA on mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro. In order to start a culture, fibrinogen, aprotinin, doxycycline, and HA-MA were well mixed in 200 L of AMEM containing 1% (w/v) photoinitiator. In order to produce a fibrin gel, thrombin was used at 1, 3,5, and 7 days after implantation, live/dead staining and a metabolic-activity test were used to examine the viability and proliferation of BMSCs inside fibrin/HA-MA. The cell lysis solution from the Real Time ready Cell Lysis Kit was added to each gel. Both primer and probe mixes, as well as DNA polymerase, deoxyribonucleotide triphosphates, an activator, and an enhancer, were mixed before the lysis process began to asses mRNA expression. The mechanical strength of fibrin hydrogels was shown to be proportional to the quantity of HA-MA utilised in the reinforcement. Quantitative polymerase chain reaction demonstrated a reduction in the expression of collagen type 1 alpha 1 mRNA in BMSCs after they were treated in a fibrin/HA-MA hydrogel. Using fibrin/HA-MA hydrogel as a delivery technique for bone marrow stem cells may induce BMSC differentiation into chondrocytes and perhaps aid in articular cartilage repair for OA therapy (BMSCs). We concluded that the utilisation of bone marrow concentration in conjunction with a combination of fibrin and Hyaluronan treatment is safe for patients suffering from OCD of the ankle and is well tolerated by these patients as both a primary treatment and a non-primary treatment option.

Smart Droplet Microfluidic System for Single-Cell Selective Lysis and Real-Time Sorting Based on Microinjection and Image Recognition.

Single-cell analysis has important implications for understanding the specificity of cells. To analyze the specificity of rare cells in complex blood and biopsy samples, selective lysis of target single cells is pivotal but difficult. Microfluidics, particularly droplet microfluidics, has emerged as a promising tool for single-cell analysis. In this paper, we present a smart droplet microfluidic system that allows for single-cell selective lysis and real-time sorting, aided by the techniques of microinjection and image recognition. A custom program evolved from Python is proposed for recognizing target droplets and single cells, which also coordinates the operation of various parts in a whole microfluidic system. We have systematically investigated the effects of voltage and injection pressure applied to the oil-water interface on droplet microinjection. An efficient and selective droplet injection scheme with image feedback has been demonstrated, with an efficiency increased dramatically from 2.5% to about 100%. Furthermore, we have proven that the cell lysis solution can be selectively injected into target single-cell droplets. Then these droplets are shifted into the sorting area, with an efficiency for single K562 cells reaching up to 73%. The system function is finally explored by introducing complex cell samples, namely, K562 cells and HUVECs, with a success rate of 75.2% in treating K562 cells as targets. This system enables automated single-cell selective lysis without the need for manual handling and sheds new light on the cooperation with other detection techniques for a broad range of single-cell analysis.

Inhibition of Hepatitis B Virus (HBV) replication and antigen expression by Brucea javanica (L.) Merr. oil emulsion.

The seeds of Brucea javanica (L.) Merr. (BJ) have been traditionally used to treat various types of cancers for many years in China. In this study, we systematically investigated a BJ oil emulsion (BJOE) produced from BJ seeds with the purpose of evaluating its antiviral effect against hepatitis B virus (HBV). HepG2.215 (a wild-type HBV cell line), HepG2, and Huh7, transfected with wildtype (WT) or lamivudine-resistance mutant (LMV-MT) HBV replicon plasmids, were treated with different doses of BJOE and then used for pharmacodynamic evaluation. Cell viability was determined using CCK8 assay. The levels of HBsAg/HBeAg in cell cultured supernatant, HBcAg in cell lysis solution, and HBV DNA in both were evaluated. BJOE at ≤5 mg/ml was nontoxic to carcinoma cell lines, but could significantly inhibit WT/LMV-MT HBV replication and HBs/e/c antigen expression in a dose-dependent manner by upregulating interleukin-6 (IL-6), demonstrating that it possesses moderate anti-HBV activity. As one of the major components of BJOE, bruceine B was found to play a dominant role in IL-6 induction and HBV inhibition. Our results demonstrated that BJOE suppressed HBV replication by stimulating IL-6, indicating that it has promising clinical therapeutic potential for both WT and LMV-MT HBV.

Application of aptamer-based rapid screening test piece integrated face mask for wearer infected with virus

Coronavirus disease 2019 (COVID-19) has significantly affected human life, and preventing the spread of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has become an important issue. Nowadays, rapid screening tests are primarily used to identify potentially infected patients and prevent viral transmission. At present, commercially available antibody-based rapid screening tests require the collection of respiratory tract samples. Sample collection is performed using methods that create discomfort. Therefore, we developed a lysis-free rapid screening test platform that can be fitted onto a medical mask. The mask can be worn for one day, after which the virus accumulates in the sampling pad. Finally, the wearer can directly activate the rapid screening test for self-monitoring by using their saliva. In this study, the structure of the novel lysis-free rapid screening test was similar to that of a commercial one. However, a lysis solution is added to the sample pad in advance and a hydrogel is added as a buffering material for viral detection. Our design improved the efficiency of combining the sample and aptamer as well as the detection efficiency. The H1N1 hemagglutinin and SARS-CoV-2 nucleocapsid protein were detected using the test platform which had a detection limit of 0.1 ng/ml. In addition, our design was validated via actual usage by patients with COVID-19 who had been ill for more than 7 d as well as healthcare workers who were close contacts and at high-risk of infection. The novel test platform can collect and accumulate viruses from patients with low viral load to achieve early viral detection and prevention of spread.